bluuebunny/crossref_metadata_2025 huggingface dataset:
Study of antibody and T cell responses in rabbits immunized with synthetic human B cell epitope analogues of La (SSB) autoantigen
Authors: Yiannaki E , Vlachoyiannopoulos P G , Manoussakis M N , Sakarellos C , Sakarellos-Daitsiotis M , Moutsopoulos H M , Tzioufas A G
Published: March 2003

The aim of this study was to investigate the immunogenicity of four synthetic peptides, representing linear B cell epitopes of the human La/SSB autoantigen: 145–164 aa (p1), 289–308 aa (p2), 301–318 aa (p3) and 349–364 aa (p4), in rabbits. New Zealand White rabbits were immunized with each of the above peptides attached in four copies on tetrameric sequential oligopeptide carriers (SOC) in duplicate. Control immunizations were also performed (one rabbit each, immunized with Freud's complete adjuvant alone or with the SOC carrier alone). Animals were bled at regular intervals and sera were analysed for anti-La/SSB activity by ELISA assays using as antigen the various synthetic peptides, as well as the whole La/SSB protein. Four months after the last immunization, the animals were killed and peripheral blood mononuclear and spleen cells were co-cultured with either the peptides, the SOC carrier, or 27 peptides, covering the entire length of the human La/SSB molecule (23 amino acids long, overlapping by eight residues to each other). A specific, IgG, anti-peptide antibody response was detected, initially directed against the priming peptide, and subsequently expanded to the other La/SSB synthetic peptides. The antibody titres remained high, even 4 months after the last immunization. Sera from rabbits immunized with either p2 or p3 reacted also with the whole La/SSB protein, as was demonstrated by ELISA and immunoblot assays. No reactivities against either Ro60 or Ro52 autoantigen were found. Rabbit spleen cell reacted not only with the epitope used for the immunization but also with other La/SSB peptides. Immunization of rabbits with the major human La/SSB B cell antigenic determinants, linked to SOC carrier, induces strong and sustained antibody and T cell responses against multiple epitopes of the human La/SSB protein. Thus, La/SSB B cell linear epitopes are probably capable also of functioning as T cell epitopes, in this experimental animal.
Cytokines, IgG subclasses and costimulation in a mouse model of thyroid autoimmunity induced by injection of fibroblasts co-expressing MHC class II and thyroid autoantigens
Authors: Yan X-M , Guo J , Pichurin P , Tanaka K , Jaume J C , Rapoport B , Mclachlan S M
Published: March 2003

AKR/N mice injected with fibroblasts expressing MHC class II (RT4.15HP cells) and the TSH receptor (TSHR) develop antibodies similar to those in Graves' disease. We were unable to analyse the subclass of these antibodies because of unexpectedly high non-specific binding by ELISA or flow cytometry. The non-specific binding reflected generalized immune activation which occurred even when the fibroblasts did not express the TSHR. However, the IgG subclasses were determined for thyroid peroxidase (TPO) antibodies induced using TPO-expressing RT4.14HP cells and found to be IgG2a > IgG1. This Th1 pattern is consistent with spontaneous secretion of interferon-gamma (but not IL-4 or IL-10) by splenocytes from injected mice. The Th1 bias was related to fibroblast injection because conventional immunization of the same mouse strain with purified TPO and adjuvant induced a Th2 response (IgG1 >> IgG2a). Further, untransfected fibroblasts themselves induced powerful, non-specific proliferative responses when used as antigen-presenting cells (APC) in vitro. Flow cytometry revealed that the RT4.15HP fibroblasts (and TSHR- and TPO-transfected derivatives) expressed B7-1. Unexpected constitutive expression of this key molecule may bypass the requirement for up-regulation of other costimulatory molecules involved in T cell stimulation. Our data support the concept that RT4.15HP fibroblasts present the TSHR (or TPO), at least for initiating the immune response. However, the accompanying generalized immune stimulation creates difficulties for analysis of TSHR-specific T and B lymphocytes. On the other hand, extension of the model to TPO, an easier antigen to study, will facilitate analysis of murine T cell responses likely to resemble those in human thyroid autoimmunity.
Increased interferon-gamma (IFN-γ), IL-10 and decreased IL-4 mRNA expression in peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE)
Authors: Csiszár A , Nagy G Y , Gergely P , Pozsonyi T , Pócsik É
Published: March 2003

Cytokines are important regulators of lymphocyte function in SLE. However, the profile of Th1 and Th2 cytokines produced by circulating lymphocytes in human SLE has not been clearly elucidated. The aim of the present study was to characterize the gene expressions of the Th1-type cytokine IFN-γ, and the Th2-type cytokines IL-10 and IL-4 in PBMC of 15 patients with SLE and 10 healthy individuals by a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). Our results showed that expression of IFN-γ (P = 0·0004) and IL-10 (P = 0·002) transcripts were significantly increased in PBMC of patients with SLE compared with healthy controls. By contrast, expression of IL-4 transcripts in PBMC of patients with SLE was significantly decreased compared with the healthy controls (P = 0·0008). Primary sources of IL-10 were B cells and monocytes, with variable contribution of T cells as detected in various fractions of PBMC of patients with SLE (P = 0·049). These findings support the hypothesis that the enhanced production of IFN-γ by mononuclear cells may trigger inflammatory responses, together with the enhanced production of IL-10 resulting in autoantibody production by B cells in human SLE.
Activation of signal transduction and apoptosis in healthy lymphomonocytes exposed to bystander HIV-1-infected cells
Authors: Abbate I , Dianzani F , Capobianchi M R
Published: March 2003

Persistent activation of the immune system is one of the hallmarks of HIV-1 infection. In this study we analysed the induction of factors involved in cytokine signal transduction, such as STAT 1 proteins and IRF-1 mRNA, in normal peripheral blood mononuclear cells (PBMC) exposed to HIV-infected cells, and the induction of apoptosis. Western blot analyses and reverse transcriptase-polymerase chain reaction results indicate that both cells infected with a X4 strain and cells infected with a R5 strain are able to increase intracellular levels of STAT 1α and β proteins as well as IRF-1 mRNA. This effect was prevented by neutralizing antibodies against interferon-alpha (IFN-α). HIV-1-infected cells dose-dependently induced apoptotic commitment in normal PBMC, as revealed by DNA fragmentation analysis, but this was not accompanied by an increase of caspase-3 activity, even if a slight up-regulation of IL-1β-converting enzyme mRNA was detected. Apoptosis induction could be abrogated mainly by antibodies against tumour necrosis factor-alpha (TNF-α) and, to a lesser extent, by antibodies against IFN-γ. All these findings suggest that uninfected PBMC can undergo activation of signal transduction and apoptosis after exposure to bystander HIV-infected cells, subsequent to the induction of cytokines such as IFNs and TNF-α.
CD4+ and CD8+ clonal T cell expansions indicate a role of antigens inankylosing spondylitis; a study in HLA-B27+ monozygotic twins
Authors: Duchmann R , Lambert C , May E , Höhler T , Märker-Hermann E
Published: March 2003

Ankylosing spondylitis (AS) is a complex genetic disease in which both MHC and non-MHC genes determine disease susceptibility. To determine whether the T cell repertoires of individuals with AS show signs of increased stimulation by exogenous antigens, CD4+ and CD8+ T cell subsets of five monozygotic HLA-B27+ twins (two concordant and three discordant for AS) and CD8+ T cell repertoires of three healthy HLA-B27+ individuals were characterized by TCR β-chain (TCRB) CDR3 size spectratyping. Selected TCRB-CDR3 spectra were further analysed by BJ-segment analysis and TCRB-CDR3 from expanded T cell clones were sequenced. In an analysis of all data (519/598 possible TCRB-CDR3 spectra), AS was associated with increased T cell oligoclonality in both CD8+ (P = 0·0001) and CD4+ (P = 0·033) T cell subsets. This was also evident when data were compared between individual twins. Nucleotide sequence analysis of expanded CD8+ or CD4+ T cell clones did not show selection for particular TCRB-CDR3 amino acid sequence motifs but displayed sequence homologies with published sequences from intra-epithelial lymphocytes or synovial T cells from rheumatoid arthritis patients. Together, these results provide support for the hypothesis that responses to T cell-stimulating exogenous or endogenous antigens are involved in the induction and/or maintenance of AS.
Novel mutations and defective protein kinase C activation of T-lymphocytes in ataxia telangiectasia
Authors: García-Pérez M A , Allende L M , Corell A , Varela P , Moreno A A , Sotoca A , Moreno A , Paz-Artal E , Barreiro E , Arnaiz-Villena A
Published: March 2003

Three ataxia telangiectasia (AT) patients have been characterized immunologically and molecularly. Patient 1 presents two nondescribed splicing mutations which affect exons 15 and 21 of the ATM gene. The maternal defect consists of a G > A transition in the first nucleotide of the intron 21 donor splicing site which results in a complete deletion of exon 21. The paternal mutation consists of an A > C transversion in the intron 14 acceptor splicing site which produces a partial skipping of exon 15. Two abnormal alternative transcripts were found, respectively, 17 and 41 nucleotides shorter. Patient 2 presents a homozygous genomic deletion of 28 nucleotides in the last exon of the gene. This deletion changes the normal reading frame after residue 3003 of the protein and introduces a premature stop codon at residue 3008 that could originate a truncated ATM protein. Patient 3, a compound heterozygote, presents a defect which consists of a G > A transition in the first nucleotide of intron 62 donor splicing site which results in a complete deletion of exon 62. The results obtained during a three year period in the proliferation assays show an impaired PMA (phorbol myristate acetate) activation in specific T lymphocyte activation pathways (CD69, CD26, CD28, CD3, PHA, PWM and Con A mediated) but not in others (CD2, ionomycin, and Ig surface receptor). The possible link among specific ATM mutations and abnormal immune responses is unknown.
Expression of RCAS1 and FasL in human trophoblasts and uterine glands during pregnancy: the possible role in immune privilege
Authors: Ohshima K , Nakashima M , Sonoda K , Kikuchi M , Watanabe T
Published: March 2003

Pregnancy is an immunological balancing act. Trophoblasts do not express MHC class I or II, except HLA-C and G, but express Fas ligand (FasL), which confers immune privilege. RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) has recently been recognized to play a role in immune evasion of the tumour cells. We therefore studied the involvement of RCAS1 and FasL in the infiltration of NK cells by examining the curettaged uterine contents of 20 cases of early stage of pregnancy. The cases were clinically divided into two groups; curettage was performed (A) due to the absence of foetal heart beats, and (B) due to spontaneous uterine bleeding and abortion. In group A, RCAS1 was expressed in the uterine glands and extravillous cytotrophoblasts, as was FasL. Infiltration of NK cells around the uterine glands was scarcely detected. In contrast, in group B, expression of both RCAS1 and FasL was strikingly decreased in both the level of expression and the numbers of RCAS1/FasL-positive cells and massive infiltration of NK cells was frequently detected around the uterine glands. These findings suggest that a reduction in RCAS1 and FasL expression seems to be closely associated with activation and infiltration of maternal NK cells and destruction of uterine glands, resulting in rejection of the foetus. Thus, expression of RCAS1 and FasL in the uterine glands and cytotrophoblasts may play a role in the downregulation of the maternal immune response, thereby maintaining pregnancy at early stage.
Flow cytometric measurement of HLA-DR expression on circulating monocytes in healthy and sick neonates using monocyte negative selection
Authors: Kanakoudi-Tsakalidou F , Debonera F , Drossou-Agakidou V , Sarafidis K , Tzimouli V , Taparkou A , Kremenopoulos G
Published: March 2003

The aim of this study was to investigate the effect of prematurity, neonatal sepsis, respiratory distress syndrome (RDS) and perinatal asphyxia on monocyte HLA-DR expression of neonates using a flow cytometric method based on monocyte negative selection. The subjects were one hundred and thirty-one neonates (59 healthy, 44 septicaemic, 20 with RDS and eight with perinatal asphyxia) and 20 healthy adults. Monocyte HLA-DR expression was measured using one-colour HLA-DR labelling in a gate for monocytes obtained using the combination of CD3-CD19–PE/CD15–FITC MoAbs. In addition, the common dual staining method using MoAbs against two CD14 epitopes (TUK4, MO2) was evaluated. With the one-colour HLA-DR labelling higher purity and recovery values of monocytes were achieved than with the dual labelling method. Healthy neonates had significantly lower percentages of HLA-DR+ monocytes than adults (69 ± 13% versus 91·5 ± 2·5%) and comparable mean fluorescence intensity (MFI) (119 ± 25 versus 131 ± 26). Values did not differ significantly between healthy term and preterm neonates. Preterm neonates with RDS had a significantly lower percentage of HLA-DR+ monocytes than the healthy preterm neonates. In neonates with asphyxia both parameters were comparable to those of the healthy ones. Septicaemic neonates presented significantly lower values of both parameters than the healthy, RDS and asphyxiated neonates. Monocyte negative selection provides a reliable estimation of HLA-DR expression on monocytes. Expression of monocyte HLA-DR is lower in healthy neonates in comparison with adults and is further decreased in neonates with sepsis and RDS, but it is not influenced by prematurity and perinatal asphyxia.
Involvement of the ERK mitogen-activated protein kinase in cell resistance to complement-mediated lysis
Authors: Kraus S , Seger R , Fishelson Z
Published: March 2003

Sublytic doses of complement desensitize cells and make them resistant to lytic complement doses. This process, named complement-induced protection, requires calcium ion influx, protein kinase C activation and protein synthesis. The involvement of the extracellular signal-regulated kinase, ERK, in cell desensitization by sublytic complement was examined in erythroleukaemia K562 cells and in COS-7 cells. As shown here, ERK is activated in K562 and COS-7 cells within 10 min of sublytic immune attack and then shows a decline and a second peak of activation at 20 min. C7- and C8-deficient human sera have a small effect on ERK activity. However, a significant increase in ERK activation is observed when C7 or C8, respectively, is added back to these sera. Complement-induced ERK activation was blocked in cells treated with GF109203X or Go6976, two selective PKC inhibitors, as well as by treatment with PD098059, an inhibitor of MEK1, the ERK kinase. PD098059 treatment also sensitized K562 cells to complement-mediated lysis and prevented complement-induced protection. COS-7 cells transfected with a dominant-negative MEK plasmid were incapable of undergoing the process of complement-induced protection. In conclusion, cell desensitization by sublytic doses of the complement membrane attack complex involves a signalling cascade that includes PKC-mediated ERK activation.
Differential induction of cellular responses by live and dead Leishmania promastigotes in healthy donors
Authors: Nylén S , Mörtberg U , Kovalenko D , Satti I , Engström K , Bakhiet M , Akuffo H
Published: March 2003

The most effective protection against human leishmaniasis has been achieved following vaccination with live promastigotes. Killed promastigotes + BCG can protect, albeit to a lower degree. To explore what mechanisms may be involved in these differences, the ability of live and dead promastigotes to induce immune responses were evaluated in vitro. The data showed that live and dead promastigotes differ in their ability to induce proliferation and cytokine production. Cytokine gene expression of Th1 related cytokines (IL-12, IFNγ and TNFα) in adult PBMC was more evident to live than to heat killed promastigotes. This was coupled with significantly higher number of IFNγ secreting cells induced by live than killed promastigotes. However, α-IL-12 antibodies did not block the IFNγ response induced by live promastigotes. Proliferative responses were variable. In contrast to adult PBMC no IFNγ secreting MNC could be detected in cord blood. However, in these cells the live promastigotes consistently induced higher proliferative response compared to dead. Implications of these findings are discussed.
Inter-individual differences in cytokine release in patients undergoing cardiac surgery with cardiopulmonary bypass
Authors: Roth-Isigkeit A , Hasselbach L , Ocklitz E , Brückner S , Ros A , Gehring H , Schmucker P , Rink L , Seyfarth M
Published: March 2003

Cardiac surgery with cardiopulmonary bypass (CPB) leads to a systemic inflammatory response with secretion of cytokines (e.g. IL-6, TNF-α, IL-1β and sIL-2R). The objective of the following study was to investigate in vitro and in vivo cytokine responses and white blood cell counts (WBC) of patients with high versus low cytokine secretion after a coronary artery bypass grafting (CABG) procedure. Twenty male patients undergoing elective CABG surgery with CPB under general anaesthesia were enrolled in the study. On the day of surgery (postoperatively), serum levels of TNF-α and IL-1β were significantly higher in patients of the high IL-6 level group compared to the respective values in the patient group with low IL-6 levels. The inter-individual differences in IL-6 release in patients undergoing CABG surgery with CPB were accompanied by differences in the release of other cytokines, such as TNF-α, IL-1β and sIL-2R. To understand whether genetic background plays a role in influencing cytokine plasma levels under surgical stress, we examined the distribution of polymorphic elements within the promoter regions of the TNF-α and IL-6 genes, and determined their genotype regarding the BAT2 gene and TNF-β intron polymorphisms. Our preliminary data suggests that regulatory polymorphisms in or near the TNF locus, more precisely the allele set 140/150 of the BAT2 microsatellite marker combined with the G allele at −308 of the TNF-α gene, could be one of the genetic constructions providing for a less sensitive response to various stimuli. Our results suggest: (1) close relationships between cytokine release in the postoperative period, and (2) inter-individually varying patterns of cytokine release in patients undergoing CABG surgery with CPB.
Mechanism of NK cell activation induced by coculture with dendritic cells derived from peripheral blood monocytes
Authors: Amakata Y , Fujiyama Y , Andoh A , Hodohara K , Bamba T
Published: March 2003

Dendritic cells (DCs) have been regarded as one of the effective antigen-presenting cells, but the relationship between DCs and lymphocytes, in particular natural killer (NK) cells, remains unclear. In this study, we evaluated how DCs interact with both lymphocytes and NK cells using a coculture system. The number of lymphocytes increased significantly when cocultured with DCs (1.8-fold increase). In particular, the proliferation of NK cells was prominent. Furthermore, the coculture of DCs with lymphocytes induced a marked increase in IL-12 and IFN-γ secretion. When contact between the DCs and lymphocytes was prevented, the secretion of both IL-12 and IFN-γ was markedly reduced. IFN-γ production was completely blocked by an anti-IL-12 antibody, indicating that IFN-γ secretion was dependent on IL-12 secretion. The stimulating effect of the DCs on the proliferation of the lymphocytes was partially suppressed by anti-IL-12 antibodies, and was completely attenuated when cellular contact was prevented. Furthermore, the NK cell proliferation induced by coculture with DCs was significantly blocked by the inhibition of the interaction of either CD40–CD40L or CD28–B7 molecule. The coculture with DCs enhanced NK activity by 40%, and this was partially suppressed by anti-IL-12 antibodies and was completely blocked by the inhibition of cell-to-cell contact. These results indicate that the activation of NK cells by DCs is partially mediated by IL-12 secretion, and that direct contact between DCs and NK cells play a major role in this response.
Inhibition of p38 MAP kinase during cellular activation results in IFN-γ-dependent augmentation of IL-12 production by human monocytes/macrophages
Authors: Marriott J B , Clarke I A , Dalgleish A G
Published: March 2003

Interleukin-12 (IL-12) is a key immunomodulatory cytokine produced by antigen-presenting cells that promotes cellular immunity and enables the generation of protective immunity against intracellular pathogens and tumours. Therefore, modulation of IL-12 activity is a primary immunotherapeutic goal. However, little is known about its regulation. Signalling via p38 MAPK has been implicated in the control of inflammatory responses and is therefore a potential therapeutic target. We have used the highly selective p38 MAPK inhibitor (SB203580) to examine the effect of this pathway on the production of IL-12. Surprisingly, we found that SB203580 strongly up-regulated LPS induced IL-12p40 at the protein (intracellular and secreted) and mRNA levels in PBMC cultures. The effect on IL-12 was apparent using both T cell-independent and T cell-dependent stimuli but not in unstimulated cultures, indicating that activation signals are required. Furthermore, the production of IFN-γ by T cells is crucial as production was not increased in LPS-stimulated, purified adherent monocytes/macrophages without the addition of exogenous IFN-γ. These results provide evidence that p38 MAPK has an unexpected suppressive effect on IL-12p40 gene transcription, and suggests interplay between p38 MAPK- and IFN-γ -mediated signals in the regulation of IL-12 production by monocytes/macrophages. Furthermore, the importance of IL-12 as a key immunoregulatory cytokine suggests that the clinical application of pyrinidyl imidazole inhibitors, such as SB203580, may need to be reassessed.
Expression of membrane-type 1 matrix metalloproteinase in rheumatoid synovial cells
Authors: Honda S , Migita K , Hirai Y , Origuchi T , Yamasaki S , Kamachi M , Shibatomi K , Fukuda T , Kita M , Hida A , Ida H , Aoyagi T , Kawakami A , Kawabe Y , Oizumi K , Eguchi K
Published: March 2003

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is thought to be a putative regulator of pro-gelatinase A (MMP-2) in the rheumatoid synovium. In this study, we examined the effects of IL-1β, one of the inflammatory cytokines, on the expression of MT1-MMP and the activation of pro-MMP-2 using rheumatoid synovial cells. We also studied the effects of KE-298 (2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid), a new disease-modifying anti-rheumatic drug (DMARD), on MT1-MMP expression of rheumatoid synovial cells. Type B synovial cells (fibroblast-like synovial cells) were cultured with KE-298 (25–100 µg/ml) in the presence of IL-1β for 48 h. Activation of pro-MMP-2 secreted from synovial cells was analysed by gelatin zymography. Reverse transcription–polymerase chain reaction (RT–PCR) methods were used to detect MT1-MMP mRNA. MT1-MMP protein expression on synovial cells was examined by anti-MT1-MMP immunoblot. An active form of MMP-2 was demonstrated in the culture media conditioned by IL-1β-stimulated synovial cells. In addition, MT1-MMP mRNA and protein expression of rheumatoid synovial cells were increased by IL-1β treatment. KE-298 blocked this IL-1β-induced pro-MMP-2 activation and MT1-MMP expression, but did not affect IL-1β-induced tissue inhibitor of metalloproteinase-2 (TIMP-2) secretion from rheumatoid synovial cells. These findings indicate that activation of rheumatoid synovial cells by IL-1β results in the induction of MT1-MMP expression. Given that MT1-MMP promotes matrix degradation by activating pro-MMP-2, these results suggest a novel mechanism whereby cytokine may contribute to articular destruction in rheumatoid arthritis (RA). KE-298 may prevent this process by down-regulating MT1-MMP expression.
Differentiation of antineutrophil nuclear antibodies in inflammatory bowel and autoimmune liver diseases from antineutrophil cytoplasmic antibodies (p-ANCA) using immunofluorescence microscopy
Authors: Terjung B , Worman H J , Herzog V , Sauerbruch T , Spengler U
Published: March 2003

Perinuclear antineutrophil cytoplasmic antibodies (p-ANCA) directed against cytoplasmic proteins of neutrophils have been studied extensively in patients with systemic vasculitides. Recent data indicate that antineutrophil antibodies in sera from patients with chronic inflammatory bowel diseases (IBD) or autoimmune liver disorders, currently called ‘atypical p-ANCA’, recognize a nuclear target antigen, rendering the term ‘ANCA’ inaccurate. Specific microscopic criteria to distinguish atypical p-ANCA from p-ANCA are lacking. We used planar and confocal laser scanning indirect immunofluorescence microscopy to examine the labelling characteristics of ethanol-, methanol- and formaldehyde-fixed neutrophils by antineutrophil antibodies in 153 serum samples from patients with IBD, autoimmune liver disorders, systemic vasculitides or healthy blood donors. On ethanol- or methanol-fixed neutrophils, multiple intranuclear fluorescent foci together with either a rim-like peripheral nuclear staining (‘type A’) or a combined cytoplasmic and peripheral nuclear staining (‘type B’) was noted exclusively with atypical p-ANCA in sera from patients with IBD or autoimmune liver disorders. Intranuclear foci, which probably corresponded to invaginations of the nuclear envelope, were not labelled by p-ANCA from patients with microscopic polyangiitis or cytoplasmic ANCA (c-ANCA) from patients with Wegener's granulomatosis. On formaldehyde-fixed neutrophils, atypical p-ANCA gave a fine rim-like staining of the nuclear periphery, whereas ANCA diffusely labelled the cytoplasm. To distinguish reliably between the patterns produced by atypical p-ANCA or p-ANCA, particularly p-ANCA, careful indirect immunofluorescence microscopy on ethanol- as well as on formaldehyde-fixed neutrophils is necessary, with particular emphasis on the presence of multiple intranuclear fluorescent foci.
The effect of IL-18 on IL-12-induced CD30 expression and IL-4 and IFN-γproduction by allergen and PPD specific T cells
Authors: Tarkowski M , Chrul S , Bodalski J
Published: March 2003

CD30 is expressed on activated T cells that, as has been suggested, preferentially produce IFN-γ. Interleukin 12 increases antigen-induced CD30 expression on T cells and IFN-γ production. Synthesis of IFN-γ can be augmented further by IL-18. The aim of our study was to investigate whether IL-18 affects the IL-12 induced CD30 expression and cytokine production by allergen or PPD specific T cells. Mononuclear cells of healthy or atopic volunteers were stimulated with PPD or allergen, respectively, to obtain specific T cell lines. T cells were restimulated with appropriate antigen and antigen-presenting cells in the presence of IL-12, IL-18 or a combination of these cytokines. After 3 days, expression of CD30 was investigated on CD4 and CD8 T cells and IFN-γ and IL-4 cytokine production was estimated in the culture supernatants. Flow cytometric analyses showed no effect of IL-18 on CD30 expression during IL-12 co-stimulation. At the same time after the optimal stimulation for CD30 expression, the levels of IFN-γ were high in PPD-stimulated cell lines but have not been up-regulated by IL-18. IFN-γ levels were much lower in allergen-stimulated T cells and although they were up-regulated by IL-12 there was no additional or synergistic effect from IL-18. IL-18, however, increased production of IL-4 in allergen-stimulated cell lines. Our studies provide new information about IL-18 activity on human cells and question its exclusive role in Th1 mediated responses.
The prevalence of autoantibodies in an elderly sub-Saharan African population
Authors: Njemini R , Meyers I , Demanet C , Smitz J , Sosso M , Mets T
Published: March 2003

In the present prospective, census-based study we have investigated the prevalence of organ-specific and non-organ-specific autoantibodies (AAb) in 152 unselected Cameroonians aged 60 years and older living in the community. AAb were detected in 49% of the participants. Non-organ-specific AAb (47%) predominated over organ-specific AAb (7%). Anti-TPO, anti-Tm, anti-Tg and anti-PC AAb were completely absent. RF was the most frequent AAb, being found in 57 (38%) cases. The prevalences of anti-SMA and RF were significantly higher in women than in men (respectively, P = 0·023 and P = 0·016). Higher serum concentrations of gammaglobulins were accompanied by a higher prevalence of RF (P < 0·0001) and a lower prevalence of ANA (P = 0·036). The overall prevalence of AAb was higher in the filaria-infected (60%) compared to the non-infected (42%) participants (P = 0·046). There was no significant influence of the vitamin D status, number of pregnancies, physical activity or medication use on the prevalence of AAb. In this study a heterogeneous pattern for the presence of the various AAb was found. Some AAb, which are commonly encountered in other studies on elderly subjects, were completely absent in this population. This diversified pattern of AAb prevalence therefore argues in favour of exogenous influences in the occurrence of AAb in elderly populations.
Myelin protein P0-specific IgM producing monoclonal B cell lines were established from polyneuropathy patients with monoclonal gammopathy of undetermined significance (MGUS)
Authors: KVARNSTRÖM M , SIDOROVA E , NILSSON J , EKERFELT C , VRETHEM M , SÖDERBERG O , JOHANSSON M , ROSÉN A , ERNERUDH J
Published: March 2003

Monoclonal expansion of B cells and plasma cells, producing antibodies against ‘self’ molecules, can be found not only in different autoimmune diseases, such as peripheral neuropathy (PN), but also in malignancies, such as Waldenström’s macroglobulinaemia and B-type of chronic lymphocytic leukaemia (B-CLL), as well as in precancerous conditions including monoclonal gammopathy of undetermined significance (MGUS). About 50% of patients with PN-MGUS have serum antibodies against peripheral nerve myelin, but the specific role of these antibodies remains uncertain. The aims of the study were to establish, and characterize, myelin-specific B cell clones from peripheral blood of patients with PN-MGUS, by selection of cells bearing specific membrane Ig-receptors for myelin protein P0, using beads coated with P0. P0-coated magnetic beads were used for selection of cells, which subsequently were transformed by Epstein–Barr virus. The specificity of secreted antibodies was tested by ELISA. Two of the clones producing anti-P0 antibodies were selected and expanded. The magnetic selection procedure was repeated and new clones established. The cells were CD5+ positive, although the expression declined in vitro over time. The anti-P0 antibodies were of IgM-λ type. The antibodies belonged to the VH3 gene family with presence of somatic mutations. The IgM reacted with P0 and myelin-associated glycoprotein (MAG), and showed no evidence for polyreactivity, in contrast to other IgM CD5+ clones included in the study as controls. The expanded clones expressed CD80 and HLA-DR, which is compatible with properties of antigen-presenting cells. The immunomagnetic selection technique was successfully used for isolation of antimyelin protein P0-specific clones. The cell lines may provide useful tools in studies of monoclonal gammopathies, leukaemia, and autoimmune diseases, including aspects of antigen-presentation by these cells followed by T cell activation.
Immune response of the coeliac nasal mucosa to locally-instilled gliadin
Authors: TORRE P , FUSCO S , QUAGLIA F , ROTONDA M L L A , PAPARO F , MAGLIO M , TRONCONE R , GRECO L
Published: March 2003

We previously demonstrated a specific gluten-induced response in the rectal mucosa of coeliac patients. In the present study, we have evaluated the immune response to local gliadin challenge in the nasal mucosa of coeliac patients preliminary to exploring the feasibility of immune modulation by the nasal route. The local response to gliadin was evaluated on non-invasive scrapings of nasal mucosa. Cells harvested from the nasal scrapings of 21 coeliac patients and 12 healthy controls were counted after immunohistochemical staining. Six hours after gliadin challenge, the total number of cells was increased in coeliacs but not in controls. The increase was due principally to lymphoid cells and granulocytes. CD3+ cells doubled after gliadin challenge, but not after albumin control challenge. There was a similar rise in CD25+ cells, whereas the number of ICAM-expressing cells did not increase significantly. In control subjects, both gliadin and albumin induced a moderate but not significant increase in total cell number. In conclusion, the gliadin antigen provokes a mild inflammatory response in coeliac nasal mucosa.
Antigen presentation by macrophages is enhanced by the uptake of necrotic, but not apoptotic, cells
Authors: BARKER R N , ERWIG L-P , HILL K S K , DEVINE A , PEARCE W P , REES A J
Published: March 2003

The aim of this study was to determine whether phagocytosis of necrotic or apoptotic cells affects antigen presentation by murine bone marrow-derived macrophages. After uptake of necrotic neutrophils, macrophages were able to stimulate significantly higher T cell proliferation in vitro against both the recall antigen albumin and the mitogen concanavalin A. No such effect was seen following phagocytosis of apoptotic neutrophils. Flow cytometry revealed that, within 4h of ingestion, macrophages that had taken up the necrotic cells expressed higher levels of CD40 than those that had phagocytosed apoptotic cells. Macrophage cultures pulsed with apoptotic, but not necrotic, neutrophils contained higher levels of transforming growth factor β1, but lower concentrations of tumour necrosis factor α, compared to untreated controls. Our interpretation of these results is that macrophages that have taken up necrotic neutrophils co-stimulate T cells with greater efficiency due to rapid CD40 up-regulation, whereas those that have ingested apoptotic cells are not only ineffective in co-stimulation, but also secrete inhibitory cytokine.
Proteinase 3 and dihydrolipoamide dehydrogenase (E3) are major autoantigens in hepatitis C virus (HCV) infection
Authors: Wu Y-Y , Hsu T-C , Chen T-Y , Liu T-C , Liu G-Y , Lee Y-J , TSAY G J
Published: March 2003

Hepatitis C virus (HCV) infection has been found to be strikingly associated with autoimmune phenomena. The aim of the present study was to investigate the presence of various autoantibodies in patients with HCV infection. Anti-neutrophil cytoplamic antibody (ANCA), anti-dihydrolipoamide dehydrogenase (anti-E3), rheumatoid factor (RF), anti-dihydrolipoamide acetyltransferase (anti-E2), anti-SS-A/Ro (60 kD), anti-SS-A/Ro (52 kD), anti-SS-B/La, anti-topoisomerase II (anti-topo II), anti-cardiolipin (aCL), anti-dsDNA, anti-ssDNA, anti-nuclear antibodies (ANA), anti-proteinase 3 (anti-Pr3) and anti-myeloperoxidase (anti-MPO) were determined in sera from 516 patients with HCV infection, 11 with primary biliary cirrhosis (PBC) and 44 healthy controls. Assays employed were indirect immunofluoresence, the particle latex agglutination test, enzyme-linked immunosorbent assay (ELISA) and immunoblotting. ANCA, anti-E3 antibody and RF were positive in 278/516 (55·6%), 276/516 (53·3%) and 288/516 (56%) patients with HCV infection, respectively. Positivity for ANA was present in 15·8%, anti-ssDNA in 15·6%, anti-dsDNA in 8·5%, aCL in 5%, anti-SS-B/La in 4·1%, anti-SS-A/Ro (60 kD) in 3·9%, anti-E2 in 3·3% and anti-SSA/Ro (52 kD) in 1·2%, anti-MPO in 4·8%, anti-Topo II and anti-actinin in 0%. All sera with ANCA showed c-ANCA patterns and contained anti-PR3 specificity. HCV patients with ANCA showed a higher prevalence of skin involvement, anaemia, abnormal liver function and α-Fetoprotein (α-FP). HCV patients with anti-E3 antibodies showed a higher prevalence of liver cirrhosis, arthritis, abnormal liver function and elevated α-FP levels. The prevalence of autoantibodies was not affected by treatment with interferon-alpha (IFN-α). In conclusion, autoantibodies are commonly found in patients with HCV infection. There is a high prevalence of anti-E3, ANCA and RF in these patients. Proteinase 3 and E3 are the major target antigens in HCV infection. HCV may be regarded as a possible causative factor in ANCA-related vasculitis.
Predictors of HIV-specific lymphocyte proliferative immune responses induced by therapeutic vaccination
Authors: MOSS R B , WALLACE M R , STEIGBIGEL R T , MORRISON S A , GIERMAKOWSKA W K , NARDO C J , DIVELEY J P , CARLO D J
Published: March 2003

We treated a cohort of 38 HIV-infected individuals with a therapeutic vaccine (remune, HIV-1 Immunogen) in an open label study. We then determined whether baseline parameters, such as CD4 cell count, viral load and IgG levels, were predictive of the magnitude of the HIV-specific lymphocyte proliferative responses (LPRs). We demonstrate herein that there is a significant enhancement from baseline for both HIV and p24 antigen-stimulated LPRs after immunization. Using a responder definition of a stimulation index of >5 on at least two post-immunization time-points, 29/38 (76%) responded to HIV-1 antigen while 27/38 (71%) responded to native p24 antigen. Viral load and total IgG were negatively correlated, while CD4 cell counts were positively associated with the magnitude of the HIV antigen LPR. In a multivariable analysis, baseline CD4 was the best predictor of HIV antigen LPR post-immunization.
Transmural pressure induces IL-6 secretion by intestinal epithelial cells
Authors: KISHIKAWA H , MIURA S , YOSHIDA H , HIROKAWA M , NAKAMIZO H , HIGUCHI H , ADACHI M , NAKATSUMI R C , SUZUKI H , SAITO H , ISHII H
Published: March 2003

Although intestinal epithelial cells (IECs) are known as an important source for IL-6, it is not known whether mechanical forces affect IL-6 production. We investigated how transmural pressure modulates IL-6 synthesis and activation of transcription factors in IECs. Pressure was loaded onto IEC-18 cells by introducing compressed helium gas into the cell culture flask for 1–48 h. IL-6 release into the culture media was determined by cell proliferation bioassay using an IL-6-dependent mouse hybridoma cell line (7TD1). Exposure to pressure (80 mmHg) significantly enhanced IL-6 release into the culture media from IEC-18 cells at 12 h. Under control conditions, IL-6 secretion was directed to the basolateral side, but after exposure to pressure IL-6 secretion was increased in both the apical and basolateral sides. A nuclear factor kappa B (NF-κB) decoy reversed completely the pressure-induced increase of IL-6 secretion by IEC-18 cells. Pressure treatment enhanced IL-6 mRNA expression in IECs within 6 h. Pressure loading significantly enhanced the activation of both NF-κB and NF-IL-6 from 1h in the nuclear protein of IEC-18 cells as assessed by the electrophoretic mobility shift assay using FITC-conjugated specific primers. Increased phosphorylation of I-kappa B was also demonstrated in the cytosol of IEC cells within 1h by Western blot analysis. These results suggest a possible role for pressure loading in immune modulation of the intestinal mucosa by the stimulation of IL-6 release from intestinal epithelial cells.
T-cell specificity in murine autoimmune haemolytic anaemia induced by rat red blood cells
Authors: BARKER R N , SHEN C-R , ELSON C J
Published: March 2003

Autoimmune haemolytic anaemia (AIHA) can be induced in mice by repeated injections with rat red blood cells (RBC). Here we describe the identification of rat and murine RBC antigens recognized by T-cells from mice with this disease. Splenic T-cells from mice with AIHA proliferated in response to multiple murine RBC membrane components, each of which is recognized by rat RBC induced autoantibodies. Thus, there were responses to murine autoantigen fractions that correspond in apparent molecular mass with the anion channel Band 3, with spectrin from the membrane skeleton and with the high and low molecular mass glycophorins, and the equivalent fractions from rat RBC also stimulated proliferation by T-cells. It was confirmed that purified Band 3 from murine and rat RBC also elicited responses. In contrast with the results in AIHA, T-cells from healthy control mice failed to respond to the antigens from either species, with the exception of proliferation induced by murine spectrin in one experiment and weak responses elicited by rat Band 3. It is suggested that T-cells activated by multiple cross-reactions between rat and murine RBC proteins, and by epitope spreading, are necessary to drive autoantibody production in this model of AIHA.
Coupling complement regulators to immunoglobulin domains generates effective anti-complement reagents with extended half-lifein vivo
Authors: HARRIS C L , WILLIAMS A S , LINTON S M , MORGAN B P
Published: March 2003

Complement activation and subsequent generation of inflammatory molecules and membrane attack complex contributes to the pathology of a number of inflammatory and degenerative diseases, including arthritis, glomerulonephritis and demyelination. Agents that specifically inhibit complement activation might prove beneficial in the treatment of these diseases. Soluble recombinant forms of the naturally occurring membrane complement regulatory proteins (CRP) have been exploited for this purpose. We have undertaken to design better therapeutics based on CRP. Here we describe the generation of soluble, recombinant CRP comprising rat decay accelerating factor (DAF) or rat CD59 expressed as Fc fusion proteins, antibody-like molecules comprising two CRP moieties in place of the antibody Fab arms (CRP-Ig). Reagents bearing DAF on each arm (DAF-Ig), CD59 on each arm (CD59-Ig) and a hybrid reagent containing both DAF and CD59 were generated. All three reagents inhibited C activation in vitro. Compared with soluble CRP lacking Fc domains, activity was reduced, but was fully restored by enzymatic release of the regulator from the Ig moiety, implicating steric constraints in reducing functional activity. In vivo studies showed that DAF-Ig, when compared to soluble DAF, had a much extended half-life in the circulation in rats and concomitantly caused a sustained reduction in plasma complement activity. When given intra-articularly to rats in a model of arthritis, DAF-Ig significantly reduced severity of disease. The data demonstrate the potential of CRP-Ig as reagents for sustained therapy of inflammatory disorders, including arthritis, but emphasize the need for careful design of fusion proteins to retain function.
Increased expression of the secretory leukocyte proteinase inhibitor in Wegener's granulomatosis
Authors: OHLSSON S , FALK R , YANG J J , OHLSSON K , SEGELMARK M , WIESLANDER J
Published: March 2003

The secretory leucocyte proteinase inhibitor (SLPI) is a low molecular weight, tissue-specific inhibitor of proteases, such as elastase and cathepsin G. It is the major local protease inhibitor in the upper airways. Proteinase 3, the main autoantigen in Wegener's granulomatosis (WG), can degrade SLPI proteolytically. In addition, SLPI is sensitive to oxidative inactivation by myeloperoxidase-generated free oxygen radicals. SLPI also has an antimicrobial capacity that can be of interest, as infection is considered to play a role in the pathogenesis of WG. This study focuses on SLPI expression in patients suffering from WG, something that to our knowledge has not been explored hitherto. Serum samples and nasal biopsies were obtained from 12 Swedish WG patients, while buffy coats were obtained from 33 American WG patients. SLPI levels in serum were measured by means of ELISA and the protein was detected by means of immunohistochemistry in nasal biopsies. mRNA expression was studied by means of in situ hybridization on nasal biopsies and RT-PCR on leucocytes. IL-6 or ESR were measured as markers of inflammatory activity. Cystatin C or creatinine was measured as a marker of renal filtration. White blood cell counts were registered. In serum, we found close to normal SLPI levels, without any correlation to IL-6. Two patients had greatly elevated values, both of them suffering from severe renal engagement. Strong SLPI mRNA expression was found in nasal biopsies. RT-PCR on leucocyte mRNA showed normal or greatly elevated expression of SLPI mRNA, correlating with disease activity. Leukocyte SLPI expression seems to be up-regulated in active WG. Serum levels were measured in a small number of patients and were found to be close to normal. Lack of correlation to the acute phase response indicates a specific regulation. This might be linked to an altered protease/antiprotease balance. These findings could indicate that SLPI locally participates in the anti-inflammatory and perhaps antimicrobial response in WG.
Chlamydia trachomatis heat shock protein-60 induced interferon-γand interleukin-10 production in infertile women
Authors: KINNUNEN A , SURCEL H-M , HALTTUNEN M , TIITINEN A , MORRISON R P , MORRISON S G , KOSKELA P , LEHTINEN M , PAAVONEN J
Published: March 2003

Chlamydia trachomatis-associated tubal factor infertility (TFI) involves enhanced humoral and cell-mediated immune response to the chlamydial 60 kDa heat shock protein (CHSP60). We evaluated the role of CHSP60-induced immune response in TFI by studying lymphocyte proliferation and cytokine (interferon (IFN)-γ, interleukin (IL)-12 and IL-10) secretion in response to C. trachomatis elementary body (EB) and CHSP60 antigens in 57 women with TFI and in 76 women with other causes of infertility. Positive proliferative response of PBMC to CHSP60 was more common in the TFI group (20/57; 36%) than in the other groups (17/76; 22%) although the frequency or the median responses did not differ significantly (1·6, range 0·2–22·1 versus 1·4; 0·2–24·4). C. trachomatis EB induced significantly higher IFN-γ and lower IL-10 secretion in the TFI group compared to the other groups. The EB and CHSP60 induced IL-12 secretion was similar in all study groups and correlated with IFN-γ secretion in the other but not in the TFI group. The lack of correlation between EB-induced IL-12 and IFN-γ production and simultaneously found prominent IL-10 secretion in response to CHSP60 in the TFI group suggests that the CHSP60 may have a specific role in regulating the immune reactions during chlamydial infection and may consequently contribute to the immunopathogenesis of TFI.