bluuebunny/crossref_metadata_2025 huggingface dataset:
Adjuvant arthritis is associated with changes in the glycosylation of serum IgG1 and IgG2b
Authors: YAGEV H , FRENKEL A , COHEN I R , FRIEDMAN A
Published: July 2010

The increased amounts of agalactosyl IgG (N-linked oligosaccharides terminating with N-acetylglucoseamine (GlcNAe) in the serum of patients with rheumatoid arthritis (RA) and other chronic inflammatory diseases have suggested that agalactosyl IgG may be involved in the pathogenesis of RA. We have now evaluated the incidence of agalactosyl IgG in the Lewis rat during the course of adjuvant arthritis (AA). The modification in glycosylation of IgG was measured by means of polyclonal and monoclonal anti GlcNAe antibodies as well as by the lectin concanavalin A (Con A). The results show that Lewis rats undergo a change in serum IgG glycosylation during the course of AA. As in human RA patients, rats with AA lack terminal galactose on igG heavy chain oligosaccharides, and the terminal GlcNAe or mannose residues arc thus exposed. The degree of agalactosyl IgG was positively correlated with the incidence of disease, peaked 20 days after disease induction, and the igG gradually reverted to the fully glycosylated form thereafter. The post-arthritic glycosylation profile was very similar to that characteristic of the naive animal- Purified IgG was shown to contain two IgG subclasses, IgGI and IgG2b. which underwent changes in glycosylation. Western blot analysis revealed that IgGl expressed a higher degree of terminal mannose, whereas IgG2b expressed a higher degree of terminal GleN.Ac, These findings raise the question of the possible involvement of agalactosyl igG in immune complex-mediated inflammation.
Extracts of wheat gluten activate complement via the alternative pathway
Authors: UNSWORTH D J , WÜRZNER R , BROWN D L , LACHMANN P J
Published: July 2010

We studied the ability of wheat gluten and its subfractions to activate complement directly. A sensitive sandwich ELISA employing a monoclonal antibody (MoAb) to a C9 neoepitope exposed in the terminal complement complex (TCC), a functional haemolytic assay for C5b6 generation, and LaurelFs electrophoretic method of estimating C3 conversion to C3bi were used. On a weight-for-weight basis, enzyme solubilized Frazer's fraction three of gluten (Fill) produced approximately 75% of the complement activation seen with the potent activator zymosan. By contrast, activation with whole insoluble undigested gluten was very weak and similar to that seen with ovalbumin or β-lactoglobulin. The results were the same using normal human serum or sera from patients with coeliac disease, dermatitis herpetiformis, or hypogammaglobulinaemia as the complement source. Activation by both zymosan and Fill was blocked in 0.01 M EDTA, but not in 0.01 M EGTA with 0.0025 m magnesium chloride. Zymosan and Fill activated complement in a serum from a patient with an intact alternative pathway but classical pathway haemolytic activity (CH50) of zero. Preferential heat inactivation of the alternative pathway inhibited both zymosan- and Fill-induced activation. Our results confirm that Fill is a strong activator of the alternative pathway. We discuss how gluten enteropathy might be initiated by complement.
Antibody to mycobacterial 65-kD heat shock protein in commercial antisera
Authors: HAJEER A H , BERNSTEIN R M
Published: July 2010

Inhibition ELISA and immunoblotting were used to examine the antigenic cross-reactivity claimed to exist between mycobacterial 65-kD heat shock protein (hsp65) and human lactoferrin. Commercially available anti-lactoferrin antibodies produced using either Freund's complete (FCA) or Freund's incomplete adjuvant were tested for binding to recombinant mycobaeterial hsp65. Both antibody preparations showed reactivity with hsp65, this being greater with the antibody produced using FCA. However, we found no evidence of a cross-reaction. Lactoferrin failed to inhibit anti-hsp65 reactivity, while hsp65 itself did. Affinity purified anti-lactoferrin antibody showed no reaction with hsp65 by ELISA or immunoblotting. These data suggest that commercial anti-lactoferrin preparations are contaminated with antibodies to hsp65. A commercial anti-albumin antibody also bound to hsp65 in ELISA, so this may be a more general phenomenon.
Therapeutic advances in immunosuppression
Authors: THOMSON A W , FORRESTER J V
Published: July 2010

Immunosuppressive therapy is appropriate for the prevention or reversal of allograft rejection, and for the treatment of autoimmune disorders and allergic disease. Recent advances in our understanding of the cellular and molecular mechanisms that regulate immune responses have paralleled elucidation of the modes of action of a variety of therapeutic immunosuppressive agents, both ‘old’ and new. These developments have identified potential targets for more refined and specific intervention strategies that are now being tested in the clinic.
Cytomegalovirus hepatitis: characterization of the inflammatory infiltrate in resistant and susceptible mice
Authors: OLVER S D , PRICE P , SHELLAM G R
Published: July 2010

Mice susceptible and resistant to murine cytomegalovirus (MCMV) were infected with this virus and livers were harvested after 2–231 days. Cryostat sections were stained to visualize cells bearing CD4, CDS8 or Mac-1 antigens. Mac-1+ cells were prevalent in inflammatory foci after 2 days. These cells persisted in susceptible BALB/c and A/J mice, but disappeared from livers of resistant C57BI/6 and CBA/CaH mice by day 28. T cell inflammation peaked on days 7–11. This declined by day 56 in C57BI/6 and CBA/CaH mice, but persisted in BALB/c and A/J mice for at least 231 days. Persistent CD8+ cells were dispersed throughout the parenchyma. More CD8+ cells were observed 7–14 days after infection in the livers of bg/bg (natural killer (NK) cell-deficient) C57B1/6 and CBA mice, and in C57BI/6 mice depleted of NK 1.1 cells by MoAb. Thus, mice of strains susceptible to MCMV exhibit hepatitis characterized by persistence of dispersed CD8+ cells. This phenomenon may be limited by NK ceils in resistant strains.
Intranasal inoculation ofMycoplasma pulmonisin mice with severe combined immunodeficiency (SCID) causes a wasting disease with grave arthritis
Authors: EVENGÅRD B , SANDSTEDT K , BÖLSKE G , FEINSTEIN R , RIESENEELT-OURN I , SMITH C I E
Published: July 2010

Mycoplasma pulmonis or Myc. pneumoniae were inoculated intranasally to C.B-17 scid/scid mice (severe combined immunodeficient (SCID) mice). Immunocompetent C.B-17 mice were inoculated as controls. During the observation period of 5 weeks the mice were killed and necropsied. Mycoplasma pulmonis was recovered from all of the inoculated mice, and dissemination to various tissues increased with time. SCID mice, unlike immunocompetent mice, did not show lung lesions but exhibited severe inflammatory changes of the joints. Mycoplasma pulmonis, however, was isolated both from the lungs and the articular lesions. In addition, SCID mice infected for more than 3 weeks suffered from a pronounced loss of weight and emaciation. In the experiment with Myc. pneumoniae the agent could be reisolated, but lesions were not found in any of the infected mice. Mycoplasma pulmonis infection in SCID mice may be useful as a model of arthritis in immunodeficient humans.
Sequential autoantigenic determinants of the small nuclear ribonucleoprotein Sm D shared by human lupus autoantibodies and MRLIpr/lprantibodies
Authors: JAMES J A , MAMULA M J , HARLEY J B
Published: July 2010

Autoantibodies directed against the Sm proteins of the spliceosome complex are found in approximately 25% of systemic lupus erythematosus (SLE) patient sera. To determine which regions of the Sm D polypeptide are involved in the lupus autoimmune response, binding to overlapping octapeptides of Sm D has been evaluated with sera from nine Sm D-positive patients, six patients with other autoimmune serology, and five normal human sera. Lupus patient sera which are Sm precipitin-positive bind various combinations of five regions of the peptide. The major antigenie region. Epitope 5 (REAVA(GR)10 GGPRR), is bound by eight of nine Sm precipitin-positive sera tested. This region of Sm D shows significant sequence homology with Epstein-Barr nuclear antigen-1. To determine the fine specificity of the murine Sm response, four unique Sm D MoAbs derived from MRL lpr/lpr mice and three adult anti-Sm-positive MRL lpr/lpr mouse sera have been analysed. Two of these monoclonals, KSm4and Y12, as well as the MRL lpr/lpr sera tested, show binding with Epitope 5. Another of these monoclonals, KSm 2, binds octapeptides 84–91, DVEPKVKSKKREAVAG, which corresponds to Epitope 4 of this study. Antibodies from SLE patients with autoimmune serology other than anti-Sm bind the carboxyl glycine-arginine repeat (GR)10 peptides of Sm D. However, none of the antibodies tested from patients who do not have lupus and who have different autoimmune serology binds any of the Sm D octapeptides. Normal controls did not significantly bind any of the Sm D octapeptides. These results describe two major regions of shared antigenicity of Sm D between sera from SLE patients and MRL lpr/lpr mice, thereby establishing a basis for the cross-species similarity of autoimmunity to the Sm autoantigen in SLE.
Enhanced synthesis of cytokines by peripheral blood monocytes cultured in the presence of autoantibodies against U1-ribonucleoprotein and/or negatively charged molecules: implication in the pathogenesis of pulmonary hypertension in mixed connective tissue disease (MCTD)
Authors: OKAWA-TAKATSUJI M , AOTSUKA S , UWATOKO S , SUMIYA M , YOKOHARI R
Published: July 2010

An attempt was made to determine whether addition of purified autoantibodies against Ulribonucleoprotein (RNP) and negatively charged molecules (cardiolipin and double-stranded (ds) DNA) to cultures of peripheral blood monoeytes could enhance the synthesis of eytokines in patients with MCTD and normal healthy volunteers. It was found that: (i) at the baseline, levels of cytokines such as IL-1α, IL-β and IL-6 extracellularly released by or associated with monocytes were significantly higher in MCTD patients than in normal subjects; (ii) addition of antibodies against U1-RNP to cultures of MCTD monocytes resulted in a significant overall increase of the released and cell-associated IL-1α, IL-β and IL-6. On the other hand, addition of antibodies against cardiolipin or dsDNA to cultures of MCTD monocytes resulted in a significant increase of released and/or cell-associated IL-1α and IL-1β; (iii) addition of these autoantibodies to cultures of normal monocytes resulted in a significant overall increase of released and cell-associated IL-1α, IL-1β and IL-6. The extent of enhancement of cytokines released by or associated with monocytes was greater in normal subjects than in MCTD patients; (iv) a F(ab′)2 preparation of autoantibodies against U1-RNP also enhanced the level of released and cell-associated IL-1α. Our findings that both autoantibodies against U1-RNP and negatively charged molecules were able to enhance the synthesis of cytokines by monocytes suggest that these autoantibodies might cause derangement of endothelial cells and lead to proliferative vaseulopathy, which is a characteristic of pulmonary hypertension in MCTD.
Tryptic peptides of human thyroglobulin: II. Immunoreactivity with sera from patients with thyroid diseases
Authors: SABOORI A M , CATUREGLI P , ROSE N R , MARIOTTI S , PINCHERA A , BUREK C L
Published: July 2010

Tryptic peptides of human thyroglobulin (Tg) were analysed by Western immunoblot for their reactivity to circulating autoantibodies from patients with Hashimoto's thyroiditis (HT), Graves’ disease (GD) and thyroid carcinoma, and from normal human controls. Low molecular weight peptides were released after 4h incubation of Tg with trypsin. The sera of thyroid disease patients reacted with several peptides, but predominantly bound three peptides with apparent molecular weights (MWap) of 25 kD, 20 kD, and 15kD; the sera of normal individuals did not bind these fragments of Tg. The pattern of Tryptic peptides recognized by the majority of sera from GD patients differed from that recognized by sera from most patients with HT. Autoantibodies from both groups of patients recognized a 15-kD peptide with a high frequency, but the sera from 26/43 (60%) GD patients also recognized a peptide with MWap of 25 kD, whereas the sera from 22/35 (63%) of HT patients recognized a 20-kD peptide. A few sera from patients with thyroid carcinoma reacted with peptides with MWap of 15 and 20-kD, and none bound the 25-kD peptide. The immunoreactivity of autoantibodies in HT sera to the 20-kD peptide paralleled the competitive inhibition of the MoAb 137CI by these sera. In addition, MoAb 137CI and Hashimoto's sera showed the same Western immunoblot-binding pattern to Tg tryptic peptides, suggesting that a Hashimoto-associated epitope and the 137Cl-binding site are found on the same peptide. These findings suggest that distinct peptides are recognized by Tg autoantibodies from patients with different thyroid diseases.
Characterization of naturally occurring autoantibodies against tumour necrosis factor-alpha (TNF-α):in vitrofunction and precise epitope mapping by phage epitope library
Authors: SIOUD M , DYBWAD A , JESPERSEN L , SULEYMAN S , NATVIG J B , FØRRE Ø
Published: July 2010

Naturally occurring autoantibodies against cytokines exist in the sera of patients with autoimmune diseases as well as in the sera of normal individuals. We report here that affinity-purified autoantibodies against human TNF-α from one rheumatoid arthritis (RA) patient inhibited the cytotoxic effect of TNF-α on the mouse fibrosarcoma cell line WEHI 164, by 50%. In an attempt lo predict the autoantibodies” recognition site on TNF-α protein we screened a random nanopeptide phage library with the affinity-purified TNF-α autoantibodies. Among 63 random selected clones, 46 clones carried the sequence ASSLLASSP, NSSPYLNTK or PQSPGSSFP. Frequency analysis of the relative occurrence of the 20 amino acids in the nanopeptides displayed by 50 random bacteriophages picked before selection and 63 after selection to bind to TNF-α autoantibodies indicated that proline (P < 0·0003) and serine (P < 0·04) are involved in the binding of the autoantibodies to the phages. Furthermore, we demonstrated that three synthetic peptides ASSLLASSP, NSSPYLNTK and PPLFCPVTDE displayed by the selected phages reduced the binding of the autoantibodies to TNF-α protein by 50%. Interestingly, the sera of mice (BALB/c) immunized with phages displaying ASSLLASSP and NSSPYLNTK peptide showed an anti-TNF-d response as detected by ELISA. This response was not found in mice immunized with the wild type phage. Thus, the recombinant phages selected from the phage libraries could be used as carrier for immunization, and therefore as a tool for vaccine development. This work sets the stage for experiments designed to isolate ligands for protective antibodies.
Flow cytometry analysis of OKT4 epitope deficiency in South African black children
Authors: HUGHES E J , GODDARD F A , BOUIC P , BEATTY D W
Published: July 2010

A case of OKT4 epitope deficiency referred for investigation with suspected immunodeficiency is described. Flow cytometry analysis of OKT4 epitope deficiency in a study group of healthy black children showed different manifestations of the lack of OKT4 epitope; a complete lack of OKT4+CD4+ peripheral blood lymphocytes (PBL) with normal numbers of OKT4A+ and Leu-3a+CD4+ PBL, decreased percentage OKT4+CD4+ compared with OKT4A+ and Leu-3a+CD4+ PBL, decreased fluorescent staining intensity with OKT4 and a biphasic OKT4 staining pattern associated with a reduced OKT4/Leu-3a ratio. The percentage and fluorescent intensity of OKT4+CD4+ PBL in the study group were significantly lower (P < 00001) than Leu-3ab+CD4+ and OKT4A+CD4+ PBL. There is thus considerable risk of under-estimating the number of CD4+ cells in black South Africans if the OKT4 MoAb is used.
Immunopathological features of palatine tonsil characteristic of IgA nephropathy: IgA1 localization in follicular dendritic cells
Authors: KUSAKARI C , NOSE M , TAKASAKA T , YUASA R , KATO M , MIYAZONOU K , FUJITAIJ T , KYOGOKU M
Published: July 2010

IgA nephropathy (IgAN) is generally thought to be mediated by the glomerular deposition of circulating immune complexes containing IgA as the major antibody component. Upper respiratory infections and tonsillitis often precede IgAN. and in some cases tonsillectomy is affective for the (treatment of IgAN. Thus, the tonsil seems to be a unique organ causing initial and/or progressive events to generate nephritogenic immune complexes in IgAN. in this study we focused on the analysis of immunopathological features of the palatine tonsil characteristic of IgAN patients by using an immunohistochemical technique. The IgAl subclass was demonstrated in follicular dendritic cells (FDC) of the tonsil of IgAN patients, but not in FDC of non-IgAN controls. On the other hand, IgA2, IgG, IgM and C3 did not show any differences in distribution between the two groups. Moreover, the expression of decay-accelerating factor (DAF), an inhibitor of homologous complement activation, and transforming growth factor-beta I (TGF-/β1). an inducer of antibody-producing ceils to IgA class switching, in FDC and interdigitating dendritic cells of the tonsil, respectively, which was also clarified in this study for the first time, was found to be identically distributed in the two groups. These findings may support the idea that IgA1. possibly in an immune complex form, is trapped by FDC and plays an important role in the persistent activation of particular B cell repertoires responsible for ihe onset and/or progression of IgAN.
T cell receptor gene rearrangements and cytotoxic activities of clones isolated from tumour-infiltrating lymphocytes (TIL) from melanoma patients
Authors: PANDOLFI F , BOYLE L A , TRENTIN L , OLIVA A , KURNICK J T
Published: July 2010

The lymphocytes which infiltrate tumours and are grown in vitro to be used in adoptive immunotherapy are often characterized by dominant rearrangement of their T cell receptor (TCR) genes. To investigate the frequency and function of cells contributing to the ‘dominant’ rearrangement, we have cloned two bulk cell lines of TIL derived from melanoma patients (TIL-1 and TlL-5). These IL-2-propagaled TIL cell lines had a CD8+ phenotype and exerted strong cytotoxic activity against autologous melanoma cells, but not against the natural killer (NK)-sensitive K-562 cell line or LAK targets such as Daudi cells. We derived 40 clones from TIL-1 and 23 from TIL-5. All tested clones were CD3+, CD4−, CD8+ and expressed the α/β TCR. From TIL-1.27 of 40 clones, and 13/19 of the TIL-5 clones lysed autologous tumour cells. In contrast to the NK, -negative bulk cultures, K-562 killing was detected in 21 of the TIL-1 clones and 17 of the TIL-5 clones. TIL-1 contained eight clones and TiL-5 two clones with lytic capacity against neither autologous tumour cells nor the K562 cell line, although these clones possessed lytic potential as evidenced in a lectimediated lysis assay. LAK activity was not detected in most clones. Cytotoxic activity against autologous tumour could be inhibited by preincubation with anti-CD3 or anti-HLA class I MoAbs, Of the 34 TlL-1 clones analysed, 15 shared a rearranged TCRβ EcoR1 restriction fragment of approximately 9 5 kb with the bulk culture. Clones sharing the EcoR1 10 5-kb dominant band present in TIL-5 bulk culture were also isolated. When the pattern of TCRβ rearrangement was compared with the cytotoxic functions, the following conclusions could be drawn: (i) clones contributing to the dominant band had heterogeneous functions. Most killed autologous tumour cells, but clones with no cytotoxic activity or even with no proliferative capacity in response lo autologous tumour cells were also detected among those contributing to the dominant rearrangement; (ii) some clones that share an apparently identical rearranged band different from the “dominant” rearrangement, may demonstrate the same cytotoxic function. In addition, our data suggest that many of the clones that share the dominant rearrangement originated from diverse progenitors. The high frequency of clonally diverse anti-tumour reactive TIL is likely to be a reflection of the in vivo selection of the TCR repertoire at the site of tumour. Further study of the TCR gene rearrangements should help to clarify how selection at this level can benefit future immunotherapeutic approaches.
Soluble intercellular adhesion molecule-1 (ICAM-1) in sera and bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis and pulmonary sarcoidosis
Authors: SHIJUBO N , IMAI K , SHIGEHARA K , HONDA Y , KOBA H , TSUJISAKI M , HINODA Y , YACHI A , OHMICHI M , HIRAGA Y , ABE S
Published: July 2010

ICAM-1 plays an important role in inflammatory diseases. To assess level of soluble ICAM-1 in the circulation and inflamed lesions, we measured levels of soluble ICAM-1 in the circulation and bronchial veolar lavage fluid (BALF) of patients with idiopathic pulmonary fibrosis (IPF) and with pulmonary sarcoidosis (PS) and of healthy volunteers (HV), and we also analysed ICAM-I expression of BALF cells in some patients and HV. IPF patients had significantly higher levels of circulating ICAM-1 than HV, while PS patients did not. By contrast, significantly increased levels of BALF soluble ICAM-1 were found in PS patients compared with those of HV, but not in IPF patients. There were no significant differences in the proportions of ICAM-1+ BALF lymphocytes in IPF patients, PS patients and HV. whereas significantly increased proportions of ICAM-1 pulmonary alveolar macrophages were found in PS patients compared with those of HV, but not in IPF patients. There was a significant positive correlation of BALF soluble ICAM-1 levels to BALF lymphocyte proportions in PS patients. Although the source of BALF soluble ICAM-1 is unclear. BALF soluble ICAM-1 appears to reflect the grade of local activity of sarcoidosis. An interesting discrepancy between soluble ICAM-1 levels in the circulation and BALF was found in IPF patients, and this might be an important clue to an understanding of this disorder.
Regulation of C3 and factor H synthesis of human glomerular mesangial cells by IL-1 and interferon-gamma
Authors: VAN DEN DOBBELSTEEN M E A , VERHASSELT V , KAASHOEK J G J , TIMMERMAN J J , SCHROEIJERS W E M , VERWEIJ C L , VAN DER WOUDE F J , VAN ES L A , DAHA M R
Published: July 2010

Previous reports have shown production of complement components C4. C2 and factor B by renal tissue. We have shown recently that human proximal tubular epithelial cells (PTEC) synthesize C3 in vitro, and that IL-2 enhances this production. In the present study we demonstrate that human mesangial cells (MC) in culture produce factor H and that supernatants of activated peripheral blood mononuclear cells (T cell growth factor (TCGF)) induce C3 production and enhance factor H synthesis in both a time- and dose-dependent manner. To investigate whether certain defined cytokines from TCGE were responsible for the observed effect., we tested various cytokines for their effect on complement production by MC. It is shown that IL-1 induces C3 synthesis whereas factor H production is up-regulated by TFN-γ, in both a dose- and time-dependent manner. Antibody blocking experiments revealed that C3 synthesis induced by both TCGF and IL-I could be blocked with antibodies specific for IL-I, and also that TCGF and IFN-γ enhanced factor H synthesis could both be blocked with antibodies specific for IFN-γ. Cycloheximide was able to inhibit C3 and factor H production, suggesting de novo synthesis of the proteins. mRNA-polymerase chain reaction (PCR) analysis revealed mRNA encoding for C3 after stimulation with TCG Fand IL-I. Factor H genes are constitutively expressed in cultured mesangial cells and its expression is up-regulated by TCGF and IFN-γ. Northern blot analysis with specific probes for C3 and factor H revealed bands which support the results obtained by PCR analysis.
Peripheral T lymphocyte depletion by apoptosis after CD4 ligationin vivo: selective loss of CD44− and ‘activating’ memory T cells
Authors: HOWIE S E M , SOMMERFIELD A J , GRAY E , HARRISON D J
Published: July 2010

We have demonstrated that a single intravenous bolus of rat anti-CD4 MoAb caused a small but prolonged increase in apoptosis in murine lymph nodes. We have quantified this process using the novel Highly Optimized Microscope Environment (HOME) interactive image analysis system and shown that the increase in apoptosis was sufficient to account for the observed depiction of the peripheral CD4+ T cell subset. This occurred in the absence of any other exogenous signal. Furthermore, there was no evidence of an inflammatory or necrotic response in the tissues, indicating that this was unlikely to be He or complement-mediated antibody killing. The anti-CD4-induced depiction selectively removed CD44− T cells. Using mice previously immunized with yeast-derived HIV-1 p24 recombinant protein there was sparing of memory T cell function after in vivo anti-CD4 treatment, except during a window of less than 24 h duration, when simultaneous exposure to antigen and anti-CD4 antibody resulted in the depiction of specific memory T lymphocyte function. This indicated that a very minor alteration in the frequency of apoptosis had a marked effect on cell number over time, and suggested that opportunistic infection associated with CD4+ T cell depletion may be explained by loss of memory cells when there is antigenic stimulation at the same time as CD4 ligation. These results have implications for the pathology of HIV-associated disease which is associated with ligation of CD4 molecules in vivo.
Anti-endothelial cell antibodies in sera of patients with autoimmune diseases: comparison between ELISA and FACS analysis
Authors: WESTPHAL J R , BOERBOOMS A M T H , SCHALKWUK C J M , KWAST H , DE WEIJERT M , JACOBS C , VIERWINDEN G , RUITER D J , VAN DE PUTTE I B A , DE WAAL R M W
Published: July 2010

In some patients suffering from rheumatoid arthritis (RA), vasculitis is a clear clinical manifestation, mentioned as rheumatoid vasculitis (RV). Autoantibodies directed against endothelial cells (AEA) have been implicated in the pathogenesis of this disorder, and it has been suggested in a number of studies that testing for AEA should be included in diagnosing RV. To test this hypothesis, we have evaluated the presence of AEA in sera of patients suffering from various autoimmune diseases, employing an ELISA with fixed cultured endothelial cells (EC). In ail the groups of patients ELISA-positive sera were present. A significant difference in percentage of positivity was found between the RA and RV group (P <0.05). In addition, our results indicated that not only antibodies directed against antigens on the EC membrane were detected, but also antibodies directed against intracellular components like DNA. histones and cytoskeletal components. Therefore, we also tested all these patient sera on unfixed intact EC using indirect immunofluorescence followed by FACS analysis. Whereas in the total patient population 34 out of 65 patients were AEA-positive as determined in the ELISA, only seven patients were weakly positive when examined by flow cytometry. We conclude that: (i) an ELISA on fixed EC does not specifically detect AEA, A positive test result is. however, to some extent correlated with the occurrence of vasculitis, and may therefore be helpful in diagnosing this disease: (ii) EACS analysis is a more suitable method than ELISA to measure the presence of membrane-specific AEA in patient sera; (iii) specific IgG-AEA are less common in patients suffering from autoimmune disorders than was assumed previously.
Precursor frequencies for DNA-specific B lymphocytes in patients with systemic lupus erythematosus (SLE)
Authors: SCHWAB J , LUKOWSKY A , VOLKK H-D , PETER H H , MELCHERS I
Published: July 2010

Precursor frequencies for anti-DNA-secreting B cells were estimated in six healthy donors and 18 SLE patients with active and inactive disease. Precursors for IgG anti-dsDNA-secreting B cells were exclusively detected in SLE patients (73% of active patients and one inactive patient, 0.01 – 0.99% of IgG-producing B cells). These frequencies were in the same order of magnitude as frequencies of precursors for IgG anti-tetanus toxoid, which were detectable in three healthy volunteers after booster vaccination (0.07–0.8% of IgG-producing B cells), but not before (<001%). Precursors for IgG anti-ss-DNA secreting B cells were observed in 33% of healthy donors and in 78% of SLE patients (0.01 – 0.32% of IgG-producing B cells). Only patient-derived IgG anti-DNA clones cross-reacted with (33%) or were monoreactive to dsDNA (12%). Precursors for IgM anti-DNA-secreting B cells were observed in healthy donors and SLE patients in comparable frequencies and with similar reactivities with ssDNA and dsDNA. Segregation analyses and sorting experiments showed that >94% of clones secreting IgG anti-DNA were derived from in vivo sIgG + B cells. slgM + B cells were induced to switch in vitro; however, only twice were cultures containing IgM and IgG anti-DNA antibodies observed under clonal conditions, In conclusion, our results indicate that precursor B cells for IgG anti-dsDNA in SLE patients are similarly selected and expanded as are precursor B cells specific for foreign antigens such as tetanus toxoid.
Monoclonal IgM from patients with peripheral demyelinating neuropathies cross-react with bacterial polypeptides
Authors: BROUET J-C , MARIETTE X , GENDRON M-C , DUBREUIL M-L
Published: July 2010

Human monoclonal IgM associated with a demyelinating peripheral neuropathy often feature a distinct antibody activity directed against a glucuronyl sulphate epitope shared by myelin-associated glycoprotein (MAG), nerve glycolipids and low molecular weight peripheral nerve polypeptides- Earlier studies showed that these IgM use a diverse repertoire of VH, and VL genes which exhibit somatic mutations, possibly indicative of an antigen-driven process. Here, we investigated whether such monoclonal IgM may react with environmental bacterial antigens. We found that six patients' sera and purified monoclonal IgM, as well as IgM from supernatants of three clonal anti-MAG-secreting cell lines reacted with unique 90 -100kD polypeptides from extracts of two out of 10 bacterial species. Purified MAG was able to inhibit this reactivity. These results indicate molecular mimicry as a possible mechanism of this immunomediated neuropathy and associated clonal lymphoid disease.
Growth and major histocompatibility antigen expression regulation by IL-4, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) on human renal cell carcinoma
Authors: HILLMAN G G , PURI R K , KUKURUGA M A , PONTES J E , HAAS G P
Published: July 2010

We have recently shown that human renal cell carcinoma (RCC) tumour lines express high-affinity IL-4 receptors. Binding of IL-4 to RCC cells induced a growth inhibition in the range of 20 68%. To enhance the growth inhibitory effect of IL-4. we have tested the effects of two additional cytokines capable of directly affecting tumour cell growth. IFN-γ caused a significant inhibition of RCC tumour cell growth (up to 70%) in a dose-dependent manner, whereas the effect of TNF-α was more limited (0 20% inhibition). The addition of IL-4 to IFN-γ on RCC cells sensitive to lL-4 induced a greater inhibition of cell growth than that seen with each cytokine alone. IL-4 and IFN-γ rendered RCC cells more responsive to the inhibitory effect mediated by TNF-α, The combination of TNF-Q with IL-4 and IFN-γ induced an optimal growth inhibition (up lo 90 98%) of RCC cells. In addition to a direct anti-proliferative effect, we have demonstrated that these cytokines can also enhance the expression of MHC antigens on the surface of RCC tumour cell lines which may render the cells more immunogenic, All RCC lines tested expressed class 1 antigens, but not class II antigens. IFN-γ induced class II expression and up-regulated the expression of class I antigens on RCC cells. Class II antigen expression was detectable following 48 h incubation, and greater after 72 h with IFN-7. lL-4 minimally affected class I expression, whereas TNF-(v up-regulated class I antigen expression. IL-4 or TNF-α did not induce class II expression. The combination of The three cytokines slightly augmented the up-regulation of class I and class II antigens observed with IFN-γ alone. These observations confirm the direct interaction of IL-4, IFN-γ and TNF-a with RCC tumour cells. both at the level of growth regulation and MHC antigen expression, and suggest a therapeutic potential of the combination of the three cytokines for renal ceil carcinoma.
Fine specificity of the murine immune response to SV40 large tumour antigen utilizing synthetic peptides that define selected epitopes
Authors: BRIGHT R K , SHEARER M H , KENNEDY R C
Published: July 2010

Baculovirus-derived recombinant simian virus 40 large tumour antigen (SV40 T-ag) was used to immunize BALB/c, C57Bl/6 and CB6/F, mice and their anti-SV40 T-ag antibody responses were examined for the ability to bind synthetic peptides representing six predicted B cell epitopes on SV40 T-ag. In C57B1/6 mice, anti-SV40 T-ag antibodies failed to bind any of the six SV40 T-ag peptides. However, the antibody responses induced in both BALB/c and CB6/F| mice recognized synthetic peptides corresponding to two distinct epitopes (amino acids 690 708 and 660 679, respectively) associated with the carboxyl-terminal half of SV40 T-ag, In addition, murine MoAbs (BALB/c) generated to native SV40 T-ag, and previously characterized as recognizing the carboxyl-terminus of SV40 T-ag by deletion mutant analysis, also bound the synthetic peptide (residues 690–708) defining the carboxyl-terminus of SV40 T-ag. These data indicate that the antibody responses induced in BALB/c and CB6/r-| mice by immunization with baculovirus-derived recombinant SV40 T-ag are capable of recognizing sequential carboxyl-terminal epitopes on SV40 T-ag defined by peptides 690 708 and 660-679. respectively. No statistically significant differences in anti-SV40 T-ag antibody titres were observed between the three inbred mouse strains. These data suggested that the fine specificities of the anti-SV40 T-ag responses as assessed by synthetic peptide binding were different in the three inbred strains of mice examined. Finally, in vivo tumour challenge studies comparing recombinant SV40 T-ag with the two carboxyl-terminus peptide epitopes indicated that some tumour immunity was induced in BALB/c, but not CB6/F1 mice, by immunization with peptide 690 708 conjugated to a carrier protein. These studies suggest that the carboxyl-terminal region of SV40 T-ag represents A continuous sequential epitope involved in both the antibody response to SV40 T-ag and tumour immunity in BALB/c mice.
Effects of cyclosporine and rapamycin on immunoglobulin production by preactivated human B cells
Authors: KIM H-S , RASKOVA J , DEGIANNIS D , RASKA K
Published: July 2010

In order to assess the direct effects of cyclosporine A (CsA) and rapamycin on B cells, we utilized a two-segment culture system of highly purified B lymphocytes consisting of induction (activation) in the presence of the formalinized Staphylococcus aureus bacteria and IL-2. and differentiation, respectively, in the presence of various combinations of cytokines. Results show that rapamycin strongly inhibited production of both IgM and IgG measured at the end of the secondary culture supported by IL-2/IL-6, whereas CsA up-regulated the immunoglobulin production. The stimulatory effect of CsA was also observed when preactivated B cells were recultured in absence of any cytokines. These results show that rapamycin and CsA have dearly distinct effects on human B lymphocyte responses in vitro. Rapamycin is a more potent in vitro immunosuppressant of B lymphocytes than CsA. It is effective at significantly lower concentrations, and it does not stimulate either the proliferation or antibody production by preactivated B cells.
Histones interact with anionic phospholipids with high avidity; its relevance for the binding of histone–antihistone immune complexes
Authors: PEREIRA L F , MARCO F M , BOIMORTO R , CATURLA A , BUSTOS A , CONCHA E G D E L A , SUBIZA J L
Published: July 2010

Antibodies recognizing anionic phospholipids have been described in systemic lupus erythematosus (SLE) and other autoimmune diseases. Recent studies have shown that some of these antibodies may recognize a cardiolipin-binding protein (apolipoprotein H) rather than phospholipids. A similar possibility is conceivable for other cardiolipin-binding proteins that are targets of autoantibodies. In this study we have addressed whether this might be the case for histones, a set of highly cationic and widely distributed proteins that react in a well known autoantibody system. Our results indicate that: (i) histones bind to anionic phospholipids (cardiolipin and phosphatidylserine) with high avidity, but not to zwitterionic phospholipids (phosphatidylcholine); (ii) monoclonal and polyclonal antihistone antibodies recognize histones bound to cardiolipin; (iii) the addition of histones to serum samples containing antihistone antibodies often enhances their anticardiolipin reactivity. In addition, we have found that antihistone-producing hybridomas derived from MKL-lpr mice may show anticardiolipin activity due to the presence of histones in the cell culture supernatants with the resultant formation of immune complexes. Taken together, the results suggest a potential role for histones in the anticardiolipin activity detected in sera containing antihistone antibodies. These histone-phospholipid interactions should be taken into account when evaluating the pathogenic effects of antihistone antibodies or other autoantibodies reacting with nuclear components (e.g. nucleosomes) containing histones.
The cytotoxic analysis of T cell receptor Vδ+ T cell lines derived from the synovial fluid of rheumatoid arthritis patients
Authors: OHTA M , SATO N
Published: July 2010

We established six human T cell lines derived from rheumatoid arthritis synovial fluid (RASF). Phenotypically, T cell receptor (TCR) αδ T cells occupied the majority of these lines and most of them expressed the TCR Vi5l molecule. In contrast, Vδ2+ T cells, the majority population of peripheral blood 76 T cells, were rarely detected in these lines. To study the immunobiological roles of RASF Vδ1+ T ceils in RA development, their cytotoxic profile was studied. The results showed that these T cells selectively lysed Daudi, but not K562 cells. The cytotoxic response was MHC-unrestricted, and was inhibited by anti-CD3 MoAb. Moreover, the cold target inhibition assay showed that the cytotoxicity was competitively inhibited by autologous and allogeneic primarily cultured RA synovial cells as well as synovial sarconui and chondrosarcoma lines. However, PBL did not inhibit this cytotoxicity- These data suggest that Vδ1+ T cells in RASF may recognize the antigen which is commonly expressed on the surface of Daudi and the cells derived from RA synovium. We can assume that the cytotoxic Vδ1+ T cells are selectively expanded in RASF, playing a significant role for the pathogenesis of certain RA cases.
Analysis of the uveitogenic determinant in repeat structure of retinal interphotoreceptor retinoid-binding protein (IRBP)
Authors: INOUE H , TAKEUCHI M , TANAKA T , USUI M , ANDO S , TAGUCHI O
Published: July 2010

IRBP is a glycolipoprotein with a four-fold partially homologous repeat structure approximately 300 residues in length, and is one of the retinal antigens capable of inducing experimental autoimmune uveoretinitis (EAU) in susceptible animals by their active immunization. The most immunopathogenic peptide of bovine IRBP for FAU in Lewis rats is reported to be the sequence 1169–1191 (PTARSVGAADGSSWEGVGVVPDV) with two immunogenic motifs common to T cell epitopes (underlined). The uveitogenic site of peptide 1169–1191 was localized at the carboxyl terminus (peptide 1182 1191) and not al the amino acid terminus (peptide 1169–1182). Repeat peptides of sequenee 11 79–1191 containing the four homologous residues (1182W. 1186G. 1187V and 1189P). that is the peptides 271–283, 579 591 and 880892. all elicited EAU. Peptide 579–591 could not stimulate proliferation of lymphocytes from rats immunized with IRBP, but had the capacity to adoptively transfer EAU. The roie of the homologous residues was examined using analogues of the uveitogenic peptide 1182–1194. in which each homologous residue was substituted by giycine (G) or leucine (L) (1182W→G, 1186G→L, 1187V→G, and 1189 P→G). One analogue (1186G →L) strongly diminished the ability to induce FAU. while the other three analogues completely abolished the ability, indicating that these homologous residues were essential for the induction of FAU. In addition, the uveitogenic peptides tested in this study were found not to contain the major epitope for antibody production.
Evaluation of serum levels of soluble CD4, CD8 and β2-microglobulin in visceral human leishmaniasis
Authors: VITALE G , MOCCIARO C , MALTA R , GAMBINO G , SPINELLI A , GIORDANO C , STASSI G , ARCOLEO F , MILANO S , CILLARI E
Published: July 2010

The levels of soluble CD4 (sCD4), sCD8 and β2-microglobulin (β2-M) were measured in sera from patients with visceral leishmaniasis during the course of infection. Levels of sCD4. sCD8 and β2-M were raised significantly above levels In normal sera and returned to the normal range after recovery. The decrease in the levels of CDS was related to a reduction of anaemia, leukopenia and thrombocytopenia. In contrast, sCD4 levels fluctuated during the period of infection. β2-M returned within normal range more rapidly than sCD8 secretion. Our results suggest that T cells are activated during infection, and that it is also possible that the raised levels of these soluble molecules play a role in the impairment of protective immunity.
Bacillus Calmette-Guérin (BCG) and immunoglobulins synergistically enhance mineral dust-induced production of reactive oxygen metabolites by human monocytes
Authors: NYBERG P , KLOCKARS M
Published: July 2010

The modulating effect of BCG and polyelonal immunoglobulin on mineral dust-induced production of reactive oxygen metabolites (ROM) by human monocytes was studied using luminoldependent chemiluminescence. BCG and immunoglobulin synergistically amplified the ROM production induced by chrysotile asbestos and quartz particles, and BCG caused a sharper dose response for poly-immunoglobulin added to the mineral dusts. Immunoglobulins did not affect zymosan yeast-induced ROM production, which was enhanced strongly by BCG. As there is evidence that phagocyte-derived ROM are of importance in mineral dust-induced lung injury, we suggest that the observed synergism between host response inflammatory mediators (polyimmunoglobulin) and exogenic Irritants (BCG) may contribute to the outcome of exposure of mineral dusts, and thus in part explain the individual variations in susceptibility to mineral dustinduced diseases.
Early impairment of gut mucosal immunity in HIV-1-infected children
Authors: QUESNEL A , MOJA P H , BLANCHE S , GRISCELLI C , GENIN C
Published: July 2010

This study was performed in 27 HIV-1+ children to characterize the IgA hyperglobulinaemia observed in the serum during the course of HIV-1 infection. By contrast with serum IgG, which increased very early, IgA elevation was related to the decrease of CD4+ cell percentage. It was demonstrated that IgA1 subclass increased selectively. Secretory IgA (SIgA) and IgA and IgG activity to gliadin, bovine serum albumin (BSA) and at a lower level to casein could be detected in the serum at the early stages of HIV infection, but SIgA levels and IgA activity to gliadin further increased during the course of immunodeficiency. By contrast, IgA and IgG activity to tetanus toxoid did not change. These data demonstrate that the hyper IgA, closely related to the degree of immunodeficiency, could be due in part to a disturbance of the gut mucosal immune system. Moreover, impaired intestinal immunity seems to appear very early, and to progress during the course of paediatric HIV-1 infection.
T cell differentiation and generation of the antigen-specific T cell repertoire in man: observations in MHC class II deficiency
Authors: MANNHALTER J W , WOLF H M , HAUBER I , MIRICKA M , GADNER H , EIBL M M
Published: July 2010

The circulating T cell pool of an MHC class II-deficient patient was shown to lack the MHC class II-specific T cell functions. This was demonstrated by the absence of MHC class Il-specific alloreactive T cells and a substantially decreased number of circulating CD4+ lymphocytes. The patient's T cells did respond to an allostimulus, although the restriction pattern of this reaction remains speculative. The function and distribution of peripheral T cell subsets from the patient resemble findings in MHC class II-deficient mice, which also lack interaction of T cell precursors with MHC class Il-bearing accessory cells during thymic differentiation. Our data support the concept that Tcell differentiation in humans is similar, and that the human MHC-restricted Tcell repertoire depends on prior interaction of T cell precursors with self MHC.
Restricted junctional diversity of T cell receptor δ gene rearrangements expressed in systemic lupus erythematosus (SLE) patients
Authors: OLIVE C , GATENBY P A , SERJEANTSON S W
Published: July 2010

SLE is an autoimmune connective tissue disorder affecting multiple organs, in which T cells may play a central role. This study investigated T cell receptor (TCR) γ/δ repertoire expression in peripheral blood mononuclear cells (PBMC) of SLE patients and healthy individuals using variable (V) gene family-specific polymerase chain reaction (PCR) amplification of TCR cDNA. The expressed Vγ repertoires were diverse in SLE and control PBMC, although VγIV gene rearrangements were barely detectable or not expressed in some patients. In contrast, δ chain expression was limited in all SLE patients, with Vδ transcripts rearranged primarily to the Vδ1 and Vδ2 genes, as opposed to control PBMC, in which all six Vδ genes were detected. To assess the clonality of TCR populations. cDNA clones containing rearranged Vδ1, Vδ2 and Vγ9 transcripts were sequenced from PBMC of both patients and controls. For controls, δ chain junctional region sequences showed extensive molecular heterogeneity, since virtually all 34 Vδ1 and 32 Vδ2 cDNA clones analysed were unique. A few Vδ9 cDNA clones (3/21) had the same junctional region sequence motif (EVQEL) encoded largely by the Vγ9 and joining (J) γP gene segments. Identical Vγ9 junctional sequences were found in SLE patients that did not contain the EVQEL motif present in normal peripheral blood γ/δ lymphocytes. Moreover, the predominant Vδ1-Jδ-consiant (C) δ and Vδ2-Jδ-Cδ gene rearrangements expressed in SLE PBMC showed restricted junctional diversity, but the oligoclonal δ transcripts were different in each patient. These findings suggest in vivo oligoclonal expansion of γ/δ T cells in the periphery of SLE patients in response to a limited number of nominal ligands. Whether γ/δ T cells contribute to the development of systemic autoimmunity remains to be investigated.