bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec complex recognized by autoantibodies from patients withtype-1 autoimmune hepatitis
Authors: Costa M , Rodríguez-Sánchez J L , Czaja A J , Gelpí C
Published: March 2003

We previously described autoantibodies against a UGA serine tRNA–protein complex (tRNP(Ser)Sec) in patients with type-1 autoimmune hepatitis [1] and now define the specificity and frequency of this autoantibody and the DNA sequence encoding the tRNA(Ser)Sec-associated antigenic protein. The presence of anti-tRNP(Ser)Sec antibodies was highly specific for type-1 autoimmune hepatitis, as 47·5% of patients were positive compared with none of the control subjects. To characterize the antigenic protein(s), we immunoscreened a human cDNA library with anti-tRNP(Ser)Sec-positive sera. Two clones (19 and 13) were isolated. Clone 19 encodes a protein with a predicted molecular mass of 48·8 kD. Clone 13 is a shorter cDNA, almost identical to clone 19, which encodes a 35·9-kD protein. Expression of both cDNAs was accomplished in Escherichia coli as His-tagged recombinant proteins. Antibodies eluted from both purified recombinant proteins were able to immunoprecipitate the tRNA(Ser)Sec from a HeLa S3 cell extract, demonstrating their cross-reactivity with the mammalian antigenic complex. Recent cloning data relating to the target antigen(s) of autoantibodies in autoimmune hepatitis patients that react with a soluble liver antigen (SLA) and a liver-pancreas antigen (LP) have revealed that these two autoantibodies are identical and that the cloned antigen shows 99% amino acid sequence homology with tRNP(Ser)Sec.
Resistance/susceptibility toEchinococcus multilocularisinfection and cytokine profile in humans. II. Influence of the HLA B8, DR3, DQ2 haplotype
Authors: Godot V , Harraga S , Beurton I , Tiberghien P , Sarciron E , Gottstein B , Vuitton D A
Published: March 2003

Differences have been shown between HLA characteristics of patients with different courses of alveolar echinococcosis (AE). Notably the HLA B8, DR3, DQ2 haplotype was associated with more severe forms of this granulomatous parasitic disease. We compared IL-10, IL-5, interferon-gamma (IFN-γ) and tumour necrosis factor (TNF) secretion by peripheral blood mononuclear cells (PBMC) isolated from eight HLA-DR3+, DQ2+, B8+ AE patients and from 10 HLA-DR3−, DQ2−, B8− patients after non-specific mitogenic and specific Echinococcus multilocularis antigenic in vitro stimulation. PBMC from seven HLA-DR3+, DQ2+, B8+ healthy subjects and nine HLA-DR3−, DQ2−, B8− subjects were also studied as controls. PBMC from AE patients with HLA DR3+, DQ2+ haplotype secreted higher levels of IL-10 without any stimulation and after specific antigenic stimulation than did patients without this haplotype. Higher levels of IL-5 and IFN-γ were also produced by these patients' PBMC after stimulation with non-purified parasitic antigenic preparations; however, the specific alkaline phosphatase antigen extracted from E. multilocularis induced only Th2-type cytokine secretion. A spontaneous secretion of TNF by HLA DR3+, DQ2+ B8+ AE patients was also found. These results suggest that HLA characteristics of the host can influence immune-mediated mechanisms, and thus the course of AE in humans; specific antigenic components of E. multilocularis could contribute to the preferential Th2-type cytokine production favoured by the genetic background of the host.
Triamcinolone acetonide modulates permeability and intercellular adhesion molecule-1 (ICAM-1) expression of the ECV304 cell line: implications for macular degeneration
Authors: Penfold P L , Wen L , Madigan M C , Gillies M C , King N J C , Provis J M
Published: March 2003

Whilst animal studies and a pilot clinical trial suggest that intravitreal triamcinolone acetonide (TA) may be useful in the treatment of age-related macular degeneration (AMD), its mode of action remains to be fully elucidated. The present study has investigated the capacity of TA to modulate the expression of adhesion molecules and permeability using a human epithelial cell line (ECV304) as a model of the outer blood–retinal barrier (BRB). The influence of TA on the expression of ICAM-1 and MHC-I was studied on resting and phorbol myristate acetate (PMA)- or interferon-gamma (IFN-γ)- and/or tumour necrosis factor-alpha (TNF-α)-activated cells using flow cytometry and immunocytochemistry. Additionally, ECV304 cells were grown to confluence in uncoated Transwell chambers; transepithelial resistance (TER) across resting and PMA-activated cells was monitored. TA significantly decreased the paracellular permeability of ECV304 cells and down-regulated ICAM-1 expression, consistent with immunocytochemical observations. PMA-induced permeability changes were dose-dependent and TA decreased permeability of both resting and PMA-activated monolayers. MHC-I expression by ECV304 cells however, was not significantly affected by TA treatment. The modulation of TER and ICAM-1 expression in vitro correlate with clinical observations, suggesting re-establishment of the BRB and down-regulation of inflammatory markers are the principal effects of intravitreal TA in vivo. The results further indicate that TA has the potential to influence cellular permeability, including the barrier function of the retinal pigment epithelium (RPE) in AMD-affected retinae.
CD14+CD16+ monocyte subpopulation in Kawasaki disease
Authors: Katayama K , Matsubara T , Fujiwara M , Koga M , Furukawa S
Published: March 2003

Kawasaki disease (KD) is an acute febrile illness caused by vasculitis, occurring in early childhood. We have demonstrated that the activation of monocytes/macrophages plays a central role during acute KD. Recently, it has been reported that the CD14+CD16+ monocyte subpopulation plays a more important role in inflammation. In this study, we investigated the peripheral blood CD14+CD16+ monocyte subpopulation by flow cytometry, and serum levels of IL-10 and IL-12 using a sandwich ELISA in 28 KD patients. We also investigated this subpopulation in patients with bacterial infections, mononucleosis and anaphylactoid purpura, since the cause of KD remains unknown. We observed an increase in the number of CD14+CD16+ monocytes with acute KD, which was a positive correlation with C-reactive protein levels, and we observed only the patients with severe bacterial infections had increased this subpopulation during the acute stage among control diseases. In addition, we found that the serum levels of IL-10, but not IL-12, were higher during acute KD. These data suggest that increased peripheral blood CD14+CD16+ monocytes are part of the regulatory system of monocyte function during acute KD.
Tumour necrosis factor-alpha (TNF-α) enhances lymphocyte migration into rheumatoid synovial tissue transplanted into severe combined immunodeficient (SCID) mice
Authors: Wahid S , Blades M C , De Lord D , Brown I , Blake G , Yanni G , Haskard D O , Panayi G S , Pitzalis C
Published: March 2003

Adhesion mechanisms play a major role in the recruitment of peripheral blood lymphocytes (PBL) which characteristically infiltrate rheumatoid arthritis (RA) synovium and other chronically inflamed tissues. Through a sequential series of complex integrated adhesion and signalling events, ‘multistep model of migration’, specific subsets of PBL are recruited into inflamed tissues. In this process both leucocyte receptors and microvascular endothelial (MVE) counter-receptors play a critical role. The MVE in particular, during an inflammatory state, is the target of various inflammatory mediators that cause the up-regulation of several cell adhesion molecules (CAM). One of the most important factors known to be a powerful inducer of MVE CAM is TNF-α. Conversely, blocking TNF-α causes a down-modulation of CAM expression. To test directly the capacity of TNF-α to induce cell migration into RA synovium we adapted a model in which synovial grafts were implanted into SCID mice subcutaneously. Using this model we demonstrate that: (i) transplants remain viable and become vascularized and fed by mouse subdermal vessels; (ii) the mouse vasculature connects to the transplant vasculature which maintains the ability to express human CAM; (iii) intragraft injections of TNF-α up-regulate the expression of human CAM, following the down-regulation which occurred 4 weeks post-transplantation; and (iv) the up-regulation of graft CAM is associated with increased human PBL migration into the transplants. This study provides direct evidence in vivo of the capacity of TNF-α to induce cell migration. In addition, it provides the experimental background for the optimal use of this model.
Glycosylphosphatidylinositol (GPI)-deficient Jurkat T cells as a model to study functions of GPI-anchored proteins
Authors: Bastisch I , Tiede A , Deckert M , Ziolek A , Schmidt R E , Schubert J
Published: March 2003

Many cell surface proteins attached to the membrane by GPI are involved in cell signalling. However, the role of the GPI membrane anchor itself remains poorly understood. GPI-defective cells from patients with paroxysmal nocturnal haemoglobinuria (PNH) are relatively resistant to apoptosis induction. We developed a Jurkat T cell model for GPI deficiency by isolating a GPI-negative mutant, which is defective in the GPI biosynthetic gene PIG-A. Using retroviral PIG-A gene transfer along with the transfer of a vector control, we obtained two genetically identical cell lines, distinguished only by expression of the PIG-A gene and, thus, their ability to produce GPI. Cell proliferation and survival were not affected by this difference. Apoptotic stimuli such as serum starvation and camptothecin exposure elicited similar responses. In contrast, GPI-defective Jurkat cells were more susceptible to Fas-mediated apoptosis than GPI-positive cells. These results indicate that a deficiency in GPI-anchored proteins, as is found in PNH, does not confer resistance to apoptosis.
Serum interferon-gamma-inducing factor/IL-18 levels in primary biliary cirrhosis
Authors: Yamano T , Higashi T , Nouso K , Nakatsukasa H , Kariyama K , Yumoto E , Kobayashi Y , Yamamoto K , Iwagaki H , Yagi T , Tanimoto T , Kurimoto M , Tanaka N , Tsuji T
Published: March 2003

Primary biliary cirrhosis is an autoimmune disease of the liver in which T helper 1 cytokines predominate over those of T helper 2 in the pathogenesis. Interleukin-18 (IL-18), for which the gene was recently cloned, is a novel T helper 1 cytokine, which augments interferon-gamma production. We designed this study to clarify the role of IL-18 in primary biliary cirrhosis and to examine whether serum IL-18 level can be a prognostic indicator for the disease. Serum IL-18 levels were measured using an enzyme linked immuno sorbent assay with mouse monoclonal antibodies. Twenty-two healthy volunteers, 31 patients with primary biliary cirrhosis (Scheuer's stage I, 13; II, 10; and IV, 8), 20 patients with autoimmune hepatitis, 11 patients with virus-related liver cirrhosis and six patients with obstructive jaundice were enrolled. Significant differences of serum IL-18 levels were observed between patients with Scheuer's stage IV and those with stage I, or II, virus-related liver cirrhosis and obstructive jaundice (P < 0·05). The IL-18 levels in primary biliary cirrhosis increased according to the disease progression, and fell promptly after living-related liver transplantation. Moreover, serum IL-18 levels in primary biliary cirrhosis were correlated with serum bilirubin concentrations and the Risk scores of the Mayo Clinic prognostic model for the disease. The IL-18 levels observed in patients with autoimmune hepatitis were also elevated, and correlated with the activity of the disease. These results indicate that serum interleukin-18 levels reflect the severity of primary biliary cirrhosis, the activity of autoimmune hepatitis, and may be an additive prognostic indicator in primary biliary cirrhosis.
Increased transforming growth factor-beta (TGF-β)-secreting T cells and IgA anti-cardiolipin antibody levels during acute stage of childhood Henoch–Schönlein purpura
Authors: Yang Y-H , Huang M-T , Lin S-C , Lin Y-T , Tsai M-J , Chiang B-L
Published: March 2003

Henoch–Schönlein purpura (HSP) is a small vessel vasculitis characterized by increased serum IgA and IgA-dominant immune complex deposition in lesions. The involvement of IgA implies a probable role for TGF-β, a major factor in IgA production, in the pathogenesis of HSP. Among IgA antibodies, serum IgA anti-cardiolipin antibodies (aCL) have been found in many diseases, including vasculitis. In addition to the clinical presentations and laboratory parameters, we further investigated the roles of IgA aCL and TGF-β in childhood HSP. Twenty-six Chinese children with the diagnosis of HSP were enrolled. Blood samples from these patients were collected at both acute and convalescent stages. Intracellular staining of lymphocytes was performed to enumerate type 1 (interferon-gamma-secreting), type 2 (IL-4-secreting), and type 3 (TGF-β-secreting) helper T cells. Serum levels of TGF-β were detected by ELISA. Serum IgA aCL of 21 of 26 patients at the acute stage, 11 of them at the convalescent stage, were measured by ELISA. The data showed that IgA aCL serum levels were significantly elevated in patients compared with healthy controls (P < 0·001), and those patients at the convalescent stage (P < 0·001). In addition, TGF-β-secreting T cells were significantly elevated during the acute stage, and decreased at the convalescent stage. Although more studies are needed, the high prevalence of IgA aCL and increased TGF-β-secreting T cells in children with acute HSP revealed some points which should permit a better understanding of the pathogenesis of HSP.
Role of IL-10 in invasive aspergillosis: increased resistance of IL-10 gene knockout mice to lethal systemic aspergillosis
Authors: Clemons K V , Grunig G , Sobel R A , Mirels L F , Rennick D M , Stevens D A
Published: March 2003

IL-10 is associated with a Th2 response, down-regulation of a Th1 response and macrophage activation. We assessed the role of IL-10 during systemic infection with Aspergillus fumigatus. Systemic aspergillosis was established in female C56Bl/6 IL-10−/– (KO) and wild-type (WT) C57Bl/6 mice by i.v. administration of 1 × 105−6 × 105 conidia of A. fumigatus. In two experiments, KO survived longer than did WT (P < 0·001). Determination of fungal burdens in the kidneys and brain showed that KO carried significantly lower burdens in both organs than did WT on day 3 (P < 0·001). Semiquantitative histological analyses showed fewer inflammatory foci/mm2 in brain and kidneys of KO than WT (P < 0·03 and < 0·001, respectively) and that extent of infection and associated tissue injury were greater in WT. Although beneficial in some bacterial infections, exogenous IL-10 has been shown deleterious in models of fungal infection. Our data indicate IL-10 is deleterious during systemic aspergillosis infection, increasing the host susceptibility to lethal infection. We speculate this might be related to greater Th2 or lesser Th1 responses, or down-regulation of macrophage responses, in WT compared with KO.
IL-10 stimulates production of platelet-activating factor by monocytes of patients with active systemic lupus erythematosus (SLE)
Authors: Bussolati B , Rollino C , Mariano F , Quarello F , Camussi G
Published: March 2003

IL-10 displays modulatory properties on the synthesis of platelet-activating factor (PAF), a potent inflammatory mediator of vascular injury. Despite the fact that IL-10 is considered to be an anti-inflammatory cytokine, IL-10 levels correlate with disease activity in SLE. Moreover, in SLE IL-10 is unable to exert its immunosuppressive and anti-inflammatory effects. We have investigated the ability of IL-10 to stimulate PAF production from monocytes of SLE patients. Spontaneous and IL-10-stimulated PAF production by peripheral blood monocytes was measured in active (n = 13) and inactive (n = 14) SLE patients and in 15 normal control subjects. We observed that monocytes derived from patients with active SLE, but not from controls or inactive SLE, spontaneously produced significant amounts of PAF. Moreover, IL-10 enhanced the synthesis of PAF from monocytes of active SLE patients only. IL-10-induced PAF production correlated with the severity of the disease and with the extent of proteinuria. These results indicate that IL-10 only stimulates the synthesis of PAF from monocytes of SLE patients when immunologically active, suggesting that IL-10 may possess a paradoxical proinflammatory effect in SLE by promoting the production of PAF, a secondary mediator of inflammation.
Hepatitis C virus (HCV) in lymphocyte subsets and in B lymphocytes expressing rheumatoid factor cross-reacting idiotype in type II mixed cryoglobulinaemia
Authors: Fornasieri A , Bernasconi P , Ribero M L , Sinico R A , Fasola M , Zhou J , Portera G , Tagger A , Gibelli A , D’Amico G
Published: March 2003

The IgMk rheumatoid factors (RF) of type II mixed cryoglobulinaemia (MC) react, in 95% of cases, with MoAbs against the cross-reactive idiotypes (CRI) Cc1 or Lc1 (corresponding to the products of the VH1 and VH4 genes). MC is closely associated with HCV infection, a virus which infects lymphocytes and may replicate in B cells. It has been suggested that HCV may induce clonal selection of B cells producing monoclonal IgMk RF in type II MC. To verify whether HCV is enriched in B cells, and in the subsets expressing Cc1 and Lc1 CRI, we studied peripheral blood lymphocytes from eight patients with MC and HCV RNA-positive sera. Seven patients had RF reacting with anti-Cc1, the other with anti-Lc1 CRI. Total lymphocytes, T cells, B cells, and Cc1+ or Lc1+, Cc1− or Lc1− B cells were purified using MoAb-coated magnetic beads. Lymphocyte subsets were then diluted to give a range of 1 × 106−1 × 103 cells and tested for HCV RNA by reverse transcriptase-polymerase chain reaction. HCV was found exclusively in B cells in seven out of eight patients. In three patients HCV was enriched in the Cc1+ cells. In one of these patients, HCV was found exclusively in Cc1+ cells, with Cc1− cells being HCV−. The data indicate that B cells from type II MC patients are almost constantly infected by HCV. In selected cases, B cell subsets expressing IgMk RF CRI are the prevalent cell type infected by HCV. Our data suggest HCV involvement in B cell dysregulation leading to cryoprecipitable IgMk RF production.
Neutrophil accumulation induced by bacterial lipopolysaccharide: effects of dexamethasone and annexin 1
Authors: Allcock G H , Allegra M , Flower R J , Perretti M
Published: March 2003

Annexin 1 (ANX-1) can reduce leucocyte migration in response to cytokines and chemokines in some rodent models of inflammation. However, its effectiveness against an inflammatory stimulus as strong as bacterial lipopolysaccharide (LPS) is unknown. Thus, we have examined whether ANX-1 can modulate LPS-induced neutrophil accumulation in the rat, as assessed by intravital microscopy and by myeloperoxidase (MPO) assay. The anti-inflammatory glucocorticoid, dexamethasone (DEX) was also studied for comparison. LPS superfusion induced adhesion of leucocytes to the endothelium and a subsequent increase in emigration from rat post-capillary venules over 2 h as assessed by intravital microscopy. Either ANX-1 or DEX was able to attenuate this adhesion and emigration of leucocytes. MPO activity in the lung, kidney and ileum was elevated after a 6-h exposure to LPS (intraperitoneal), indicating accumulation of neutrophils in these tissues. DEX attenuated the LPS-induced increase in MPO in the ileum but had no effect on MPO in the lungs or kidneys. This would suggest that the underlying mechanism by which neutrophils accumulate in the ileum, and more generally in the gastrointestinal compartment, is different from other vascular beds. ANX-1 had no effect on the LPS-induced increase in MPO activity in any of the tissues studied. Thus, from these data, ANX-1 appears to reduce leucocyte adhesion and emigration induced by a short-term (2 h), but not a longer (6 h) exposure to LPS.
Expression of IL-18 byMycobacterium avium-infected human monocytes; association withM. aviumvirulence
Authors: Shiratsuchi H , Ellner J J
Published: March 2003

Disseminated Mycobacterium avium infection is the most frequent bacterial infection in patients with advanced AIDS and also associated with interferon-gamma (IFN-γ) or IL-12 receptor deficiency. IFN-γ is a key cytokine in host defence against M. avium infection. Expression of IL-18, a potent IFN-γ inducer, and IFN-γ by human monocytes after infection with M. avium was examined. Monocytes were co-cultured with isogenic smooth-transparent (SmT: virulent) or smooth-domed (SmD: avirulent) M. avium strains (10 organisms per monocyte). Infection with the SmD strain induced significantly higher concentration of IL-18 and IFN-γ in culture supernatants than did the SmT strain. IFN-γ production in response to M. avium was partially inhibited by anti-human IL-18 MoAb. Both recombinant human IL-12 (77 ± 42 pg/ml, control versus 1492 ± 141 pg/ml, cultures with IL-12 1 ng/ml) and IL-18 (126 ± 37 pg/ml, control versus 2683 ± 864 pg/ml, cultures with IL-18 10 ng/ml) augmented M. avium-induced IFN-γ production. Freshly isolated uninfected monocytes expressed constitutive levels of IL-18. Following infection with M. avium, enhancement of IL-18 mRNA expression peaked at 3–6 h. IL-18 protein was detected in monocyte lysates as early as 1 h after infection with both SmT and SmD M. avium strains by Western blotting. Higher IL-18 expression by monocytes infected with the avirulent strain may result in more IFN-γ production, thus modulating its pathogenicity. Local induction of IL-18 may be important both for M. avium pathogenicity and host defence and become a potential candidate for immunotherapy.
Reduced systemic IgG levels against peptidoglycan in rheumatoid arthritis (RA) patients
Authors: Schrijver I A , De Man Y A , Melief M -J , Van Laar J M , Markusse H M , Klasen I S , Hazenberg M P , Laman J D
Published: March 2003

The gut flora is believed to play a role in the pathogenesis of RA. Peptidoglycan, a major cell wall component of Gram-positive bacteria, is a candidate antigen because of its capability to trigger production of proinflammatory cytokines, to induce arthritis in rodents, and because of its presence in antigen-presenting cells in RA joints. We investigated whether the systemic and local antibody levels against a peptidoglycan–polysaccharide (PG–PS) are related to the presence and disease activity of RA. Significantly lower levels of systemic IgG directed against PG–PS were found in healthy females compared with healthy males, and systemic IgA levels specific for PG–PS were negatively correlated with age. Levels of systemic IgG directed against PG–PS were significantly reduced in RA patients compared with sex- and age-matched healthy controls. Local (synovial fluid) levels of IgG did not correlate with disease activity whereas synovial fluid levels of IgA correlated positively with disease activity. These data suggest that IgG in healthy people mediates protection against spreading of PG to non-mucosal sites.
Elevated IL-5 levels in pleural fluid of patients with paragonimiasis westermani
Authors: Taniguchi H , Mukae H , Matsumoto N , Tokojima M , Katoh S , Matsukura S , Ogawa K , Kohno S , Nawa Y
Published: March 2003

To investigate the pathogenic mechanisms of eosinophilic pleural effusion in patients with paragonimiasis, we measured the levels of IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma (IFN-γ) in pleural effusions. Samples were obtained from 11 patients with Paragonimus westermani infection. In addition, samples from 12 patients with pleural transudates, 16 with tuberculous pleurisy, seven with empyema and 20 with lung cancer were also examined. Eosinophilia was remarkable in peripheral blood (range 4–34%, median 23·4%) and pleural fluid (range 0–95%, median 71%) of paragonimiasis patients. IL-5 concentrations in pleural effusions of paragonimiasis were markedly higher than those in other groups. Although marked elevation of GM-CSF and IFN-γ levels was observed in pleural effusion of empyema and tuberculosis patients, it was marginal in the pleural effusion of paragonimiasis patients. In paragonimiasis patients, IL-5 levels in the pleural effusion correlated well with the percentage of eosinophils in peripheral blood and pleural fluid. Such a correlation was not observed between GM-CSF levels in pleural effusion and percentages of eosinophils in pleural fluid or peripheral blood. Our findings suggest that in paragonimiasis IL-5 in the local inflammatory site is particularly important in mediating eosinophilia in peripheral blood and pleural effusion.
Up-regulation of prostaglandin E receptor EP2 and EP4 subtypes in rat synovial tissues with adjuvant arthritis
Authors: Kurihara Y , Endo H , Akahoshi T , Kondo H
Published: March 2003

To evaluate the role of the prostaglandin E receptor (EP) subtypes in the development of inflammatory synovitis, we examined EP subtype mRNA distribution in the synovial tissue of rats with adjuvant arthritis and the effect of selective EP agonists on cytokine production by cultured rat synovial cells. We used reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization to measure the level of EP subtype (EP1, EP2, EP3, and EP4) mRNA expression in synovial tissues and cultured synovial cells from the arthritic joints of rats. RT-PCR and ELISA were used to analyse the effects of two selective EP agonists on IL-6 production by cultured rat synovial cells. EP2 and EP4 mRNA expression in inflamed synovial tissues was up-regulated. EP2 and EP4 mRNA were co-expressed in synovial macrophages and fibroblasts in inflamed tissues. EP4 and EP2 agonists both inhibited IL-1-induced IL-6 production. Our results suggest that prostaglandin E2 regulates the functions of synovial macrophages and fibroblasts through EP2 and EP4, which are induced by inflammatory stimuli in rats with adjuvant arthritis.
A defect in bone marrow derived dendritic cell maturation in the nonobesediabetic mouse
Authors: Strid J , Lopes L , Marcinkiewicz J , Petrovska L , Nowak B , Chain B M , Lund T
Published: March 2003

The pathogenesis of diabetes in the nonobese diabetic (NOD) mouse is characterized by a selective destruction of the insulin-producing β-cells in the islets of Langerhans mediated by autoreactive T cells. The function of T cells is controlled by dendritic cells (DC), which are not only the most potent activators of naïve T cells, but also contribute significantly to the establishment of central and peripheral tolerance. In this study, we demonstrate that the NOD mouse (H2: Kd, Ag7, E°, Db) shows selective phenotypic and functional abnormalities in DC derived from bone marrow progeny cells in response to GM-CSF (DCNOD). NOD DC, in contrast to CBA DC, have very low levels of intracellular I-A molecules and cell surface expression of MHC class II, CD80, CD86 and CD40 but normal β2-microglobulin expression. Incubation with the strong inflammatory stimulus of LPS and IFN-γ does not increase class II MHC, CD80 or CD86, but upregulates the level of CD40. The genetic defect observed in the DCNOD does not map to the MHC, because the DC from the MHC congenic NOD.H2h4 mouse (H2: Kk, Ak, Ek, Dk) shares the cell surface phenotype of the DCNOD. DC from these NOD.H2h4 also fail to present HEL or the appropriate HEL-peptide to an antigen-specific T cell hybridoma. However all the DC irrespective of origin were able to produce TNF-α, IL-6, low levels of IL-12(p70) and NO in response to LPS plus IFN-γ. A gene or genes specific to the NOD strain, but outside the MHC region, therefore must regulate the differentiation of DC in response to GM-CSF. This defect may contribute to the complex genetic aetiology of the multifactorial autoimmune phenotype of the NOD strain.
Fas/FasL mediated apoptosis of thyrocytes in Graves' disease
Authors: Sera N , Kawakami A , Nakashima T , Nakamura H , Imaizumi M , Koji T , Abe Y , Usa T , Tominaga T , Ejima E , Ashizawa K , Yokoyama N , Ishikawa N , Ito K , Eguchi K
Published: March 2003

We examined in the present study the possible involvement of Fas and its ligand (FasL) in the process of Graves' disease. Immunohistochemical analysis showed that few normal thyrocytes expressed Fas but many thyrocytes in Graves' disease expressed this molecule. The percentage of FasL-positive thyrocytes in Graves' thyroids was, however, less than in normal thyroids. Several apoptotic thyrocytes and infiltrating mononuclear cells (MNCs) were detected scattered throughout Graves' thyroid tissues and abundant proliferating cell nuclear antigen (PCNA)-positive thyrocytes were present. Apoptotic cells, as well as PCNA-positive cells, were scarcely detectable in normal thyroid glands, however. In vitro treatment of thyrocytes by IL-1β a cytokine found to be expressed in Graves' thyroid glands, increased Fas but reduced FasL expression. IL-1β-stimulated thyrocytes became sensitive to apoptosis by anti-Fas IgM monoclonal antibody (mAb). Activated T cells, which strongly expressed FasL, showed cytotoxic activity toward IL-1β-stimulated thyrocytes but not toward unstimulated thyrocytes. This cytotoxic activity involved the Fas/FasL pathway. Importantly, unstimulated thyrocytes could kill activated, but not resting, T cells. IL-1β-stimulated thyrocytes, with down-regulated FasL expression, could not efficiently kill activated T cells. The cytotoxic activity of unstimulated thyrocytes toward activated T cells was inhibited by anti-FasL mAb. Interestingly, unstimulated thyrocytes induced apoptosis in IL-1β-stimulated thyrocytes but not in unstimulated thyrocytes. These interactions were also blocked by anti-FasL mAb. Our results suggest that the apoptotic cell death of both thyrocytes and infiltrating MNCs found in Graves' thyroid glands is regulated by IL-1β through Fas/FasL interactions.
Phenytoin promotes Th2 type immune response in mice
Authors: Okada K , Sugiura T , Kuroda E , Tsuji S , Yamashita U
Published: March 2003

The effects of chronic administration of phenytoin, a common anticonvulsive drug, on immune responses were studied in mice. Anti-keyhole limpet haemocyanin (KLH) IgE antibody response after KLH-immunization was enhanced in phenytoin-treated mice. Proliferative responses of spleen cells induced with KLH, concanavalin A (ConA), lipopolysaccharide and anti-CD3 antibody were reduced in phenytoin-treated mice. Accessory function of spleen adherent cells on ConA-induced T cell proliferative response was reduced in phenytoin-treated mice. KLH-induced IL-4 production of spleen cells was enhanced, while IFN-γ production was reduced in phenytoin-treated mice. In addition, production of IL-1α, but not IL-6 and IL-12 by spleen adherent cells from phenytoin-treated mice was reduced. Natural killer cell activity was reduced in phenytoin-treated mice. These results suggest that phenytoin treatment preferentially induces a Th2 type response. We also observed that plasma ACTH and corticosterone levels were increased in phenytoin-treated mice, and speculated that phenytoin might act directly and indirectly, through HPA axis activation, on the immune system to modulate Th1/Th2 balance.
Cytolytic complement activity in otitis media with effusion
Authors: Närkiö-Mäkelä M , Meri S
Published: March 2003

Otitis media with effusion (OME) is a chronic inflammation persisting in the middle ear cavity of at least 8 weeks duration. Middle ear effusion (MEE; n = 38), samples from children suffering from OME were investigated for their direct cytolytic activity or an ability to enhance complement lysis of unsensitized bystander cells. Thirteen of the 38 MEEs had direct endogenous haemolytic activity and 27 samples had an ability to enhance serum-initiated lysis. Using an enzyme immunoassay, high levels of terminal complement complexes (TCC) were detected in the MEE samples (mean 34·1 µg/ml, range 5–89 µg/ml). This indicated strong local complement activation that had progressed to the terminal stage. As one potential factor promoting complement activation we identified both monomeric and trimeric properdin in MEE by Western blotting. By stabilizing C3 and C5 convertases properdin accelerates the alternative and terminal pathways of complement. On the other hand, the membrane attack complex (MAC) inhibitor CD59, which was found to be extensively shed into the MEE in a functionally active form, may control excessive cytotoxicity of the MEE. In conclusion, intense complement activation, up to the terminal level, maintains ongoing inflammation in the middle ear cavity and can pose a threat to the local epithelium.
Pentoxifylline inhibits the synthesis and IFN-γ-inducing activity of IL-18
Authors: Samardzic T , Jankovic V , Stosic-Grujicic S , Popadic D , Trajkovic V
Published: March 2003

The effect of phosphodiesterase-inhibiting anti-inflammatory drug pentoxifylline (PTX) on LPS-induced IL-18 synthesis and IL-18-mediated IFN-γ-induction were investigated. In a dose-dependent manner PTX inhibited production of IL-18 in LPS-treated cultures of murine spleen cells and bone marrow-derived macrophages. Similarly, PTX treatment significantly reduced blood IL-18 levels and expression of spleen IL-18 mRNA in LPS-challenged mice. The inhibitory effect of PTX was specific for IL-18, since LPS-induced IL-12 p40 release was not suppressed either in splenocyte cultures or blood of LPS-injected animals. Synergistic induction of IFN-γ by combined IL-12/IL-18 treatment was also inhibited by PTX in vitro and in vivo. Experiments with IL-12 pretreatment of splenocytes, followed by IL-18 stimulation, revealed that PTX suppressed both IL-12 and IL-18 signals responsible for IFN-γ induction. These results suggest that interference with IL-18 synthesis and IFN-γ-inducing activity might contribute to anti-inflammatory actions of PTX.
Enhancement of cALL immunogenicity by co-culture with a CD154 expressing 293 cell line
Authors: Lee A J , Haworth C , Hutchinson R M , Patel R , Carter R , James R F L
Published: March 2003

Pre-B cell acute lymphoblastic leukaemia (cALL) commonly occurs in young patients and although successful conventional therapies are available (such as cytotoxic drugs and bone marrow transplantation) for a proportion of patients (approximately 30%) these are ultimately unsuccessful. Recurrence of disease is a result of the failure of the immune system to recognize these abnormal cells and down-regulation of crucial molecules required for cognate CD4+ T cell recognition has been postulated as a means of immune escape. In this study we show that an embryonic kidney cell line (293 cells) transfected with CD154 (40 L.1) are capable of not only maintaining the viability of primary ALL cells in culture but can also up-regulate the expression of a number of crucial molecules involved in antigen recognition. We show that 40 L.1 cell stimulation of primary ALL cell cultures can not only enhance the allogeneic and autologous MLR response to such cells but will also induce CTL effectors which are capable of lysing wild-type autologous ALL cells. It is therefore conceivable that such an approach could be used to generate an active anti-tumour response in patients, following conventional therapy, reducing the incidence of recurrence.
Anti-cytokeratin antibodies in sera of the patients with autoimmune hepatitis
Authors: Murota M , Nishioka M , Fujita J , Dobashi N , Wu F , Ohtsuki Y , Hojo S , Takahara J , Kuriyama S
Published: March 2003

Antibodies to cytokeratin (CK) are found in some patients with autoimmune hepatitis (AIH). We hypothesized that serum antibodies to CK8, CK18 and CK19 may be formed in patients with AIH. We established an enzyme-linked immunosorbent assay (ELISA) to quantify anti-CK8, anti-CK18 and anti-CK19 antibodies in sera of patients with AIH. In addition, we quantified circulating CK8:anti-CK8 antibody as well as CK18:anti-CK18 antibody immune complexes in patients' sera, by an enzyme-linked immunosorbent assay (ELISA). Furthermore, to evaluate the expression of CK8, CK18 and CK19 in liver tissue, immunohistochemical stainings were performed. Significantly high levels of anti-CK8, anti-CK18 and anti-CK19 antibodies were demonstrated in patients with AIH compared with normal volunteers and patients with chronic active hepatitis C (CH-C). In addition, these antibodies were significantly decreased after steroid treatment. Levels of CK8:anti-CK8 and CK18:anti-CK18 immune complexes in sera of patients with AIH were significantly high compared with those of patients with CH-C and normal volunteers. Immunohistochemically, CK8 or CK18 were absent from some hepatocytes of AIH. CK19 was aberrantly expressed in periportal hepatocytes in patients with AIH, but not CH-C. This is the first study to quantify anti-CK8, anti-CK18, anti-CK19 antibodies and immune complexes in patients with AIH. The clinical significance of anti-CK antibodies and their immune complexes of AIH is also discussed.
Occurrence of C-reactive protein in cryoglobulins
Authors: Weiner S M , Prasauskas V , Lebrecht D , Weber S , Peter H H , Vaith P
Published: March 2003

A previous case report described the formation of a complex between a monoclonal IgA with cryolabile properties and C-reactive protein (CRP). Our study provides the first evidence for the frequent occurrence of CRP in cryoglobulins (Cg) of all three types according to Brouet's classification. We performed a systematic immunochemical analysis of cryoglobulins from 18 patients by Western blotting and in 15 of 18 cryoprecipitates a single band (23 KD), immunoreactive with anti-CRP antibody, was demonstrable irrespective of the clonal composition of the cryoglobulins. This band was detectable in 4/5 of type I, in 6/8 of type II, and in 5/5 of type III cryoprecipitates, classified according to Brouet et al. In addition, the complement proteins C1q and C3 were present in nearly all CRP-containing cryoglobulins, presumably reflecting previous activation of the classical complement pathway at least. All three CRP-negative cryoprecipitates were derived from sera with low cryoglobulin content (1–2 g/l). Longitudinal investigation of 23 cryoprecipitates from seven patients confirmed that successful detection of CRP by Western blotting depends on the protein concentration of the cryoglobulins. Since complexed CRP was previously shown to be an effective activator of complement, via C1q binding, CRP may modulate pathophysiologic effects mediated by cryoglobulins in vivo.
The possible use of HLA-G1 and G3 in the inhibition of NK cell-mediated swine endothelial cell lysis
Authors: Matsunami K , Miyagawa S , Nakai R , Murase A , Shirakura R
Published: March 2003

The splicing isoform of HLA-G that is expressed in xenogeneic cells, and its effect on NK-mediated direct cytotoxicity was examined, using stable Chinese hamster ovary (CHO) cell or swine endothelial cell (SEC) transfectants. cDNAs of HLA-G (G1 and G3) and human β2-microglobulin were prepared and subcloned into the expression vector, pCXN. The transfected HLA-G1 was easily expressed on SEC, and co-transfection with human β2-microglobulin led to an enhanced level of HLA-G1 expression, as evidenced by flow cytometry. The expressed HLA-G1 significantly suppressed NK-mediated SEC cell lysis, which is an in vitro delayed-type rejection model of a xenograft. On the other hand, the swine leucocyte antigen (SLA) class I molecules could be up-regulated as the result of the transfection of human β2-microglobulin, but did not down-regulate human NK-mediated SEC lysis. The HLA-G3 was not expressed on CHO and SEC in contrast to HLA-G1, as the result of the transfection. The gene introduction of HLA-G3 in SEC showed no protective effect from human NK cells. However, indirect evidence demonstrated that HLA-G3 transfection resulted in HLA-E expression, but not itself, when transfected to the human cell line, 721.221, thus providing some insight into its natural function in human cells. The present findings suggest that the expression of HLA-G1 on the cell surface could serve as a new approach to overcoming NK-mediated immunity to xenografts.
In vitrocytokine production and growth inhibition of lymphoblastoid cell lines by CD4+ T cells from Epstein–Barr virus (EBV) seropositive donors
Authors: Wilson A D , Hopkins J C , Morgan A J
Published: March 2003

In vitro stimulation of peripheral blood lymphocytes (PBL) from healthy Epstein–Barr Virus (EBV) seropositive individuals with autologous lymphoblastoid cell lines (LCL) gives rise to CD4+ and CD8+ T cells both of which are cytotoxic for autologous lymphoblastoid cells. Activated EBV-specific CD4+ T cells are cytotoxic towards autologous LCL but, paradoxically, CD4+ T cells have also been shown to enhance tumour formation in SCID/Hu mice. Here, we show that despite being cytotoxic, CD4+ T-cell lines from different donors show considerable variation in their ability to inhibit the long-term growth of autologous LCLs in vitro. Following re-stimulation in vitro with PMA and ionomycin, CD4+ T cells produced IFNγ, TNFα, TNFβ, IL-2, IL-4, IL-10 and IL-13. TNFα, TNFβ and IL-10 production were also detected in LCL. IL-6 was only detected in trace amounts in either cell type. The ratio of IFNγ to IL-4 production varied between the CD4+ T-cell lines, indicating differences in the Th1/Th2 balance of the response. When CD4+ T cells were re-stimulated using autologous LCL as antigen-presenting cells, they produced more IL-4 and less IFNγ or IL-13 when compared with cells re-stimulated by phorbol myristate acetate (PMA) and ionomycin. Using two colour cytokine staining, we showed that many individual CD4+ T cells produced IFNγ along with either IL-4 or IL-13. Purified CD4+ T cells completely inhibited the outgrowth of autologous LCL in five out of nine cases, and partially inhibited outgrowth in the remaining four. There was no correlation between the pattern of CD4+ T-cell cytokine production and the capacity to inhibit outgrowth of autologous LCL. The killing of LCLs was contact-dependant and not mediated by soluble factors. We conclude that the ability of CD4+ T cells to inhibit autologous LCL growth is not directly related to T-helper cell cytokine production, but may depend on cytoxicity through surface ligands such as CD95L (FasL) and TNFα-related apoptosis-inducing ligand (TRAIL).
Distribution of lymphocytes of the αEβ7 phenotype and E-cadherin in normal human urothelium and bladder carcinomas
Authors: Cresswell J , Robertson H , Neal D E , Griffiths T R L , Kirby J A
Published: March 2003

The primary aim of this work was to survey normal urothelium and transitional cell carcinoma (TCC) for the presence of T lymphocytes expressing the intraepithelial, CD103+ phenotype. This antigen defines the αEβ7-integrin. The adhesive counter-receptor for αEβ7 is E-cadherin, which is down-regulated during cancer progression. The secondary aim was to determine the pattern of distribution of CD103+ lymphocytes in relation to E-cadherin expression in bladder cancer. Cryostat sections of normal bladder and TCC were treated with antibodies specific for human CD103, CD3, CD8 and E-cadherin. Visualization was performed by immunoperoxidase or alkaline phosphatase development with light and confocal microscopy. Dual staining and serial sections were used to assess the relationship between these antigens. Four samples of normal bladder and 26 TCC samples were assessed. Occasional T lymphocytes (CD3+) were seen in normal urothelium and lamina propria. In the urothelium the majority of these T lymphocytes (71%) were also CD8+ and of these 68% expressed the CD103 marker. In the lamina propria 62% of the T lymphocytes were CD8+ and 56% of these expressed the CD103 marker. In carcinomas significantly greater numbers of CD103+ T lymphocytes were present in the surrounding stroma rather than infiltrating the carcinomas (P = 0·0006). Of those T lymphocytes infiltrating the tumours, 71% were CD8+ and of these 58% expressed CD103. In the surrounding stroma 52% of lymphocytes were CD8+ and 82% of this subset expressed CD103. Infiltration by CD103+ lymphocytes was not related to the intensity of E-cadherin expression. T lymphocytes of the CD103+ phenotype are present in normal urothelium where they may play a role in immunosurveillance. Rather than infiltrating into carcinomas, these cells predominate in the surrounding stroma which could suggest a failure of immune function.
Characterization of CD44 expressed on alveolar macrophages in patients with diffuse panbronchiolitis
Authors: Katoh S , Matsubara Y , Taniguchi H , Fukushima K , Mukae H , Kadota J , Matsukura S , Kohno S
Published: March 2003

Interleukin (IL)-8 may play an important role in neutrophil infiltration in the airways of patients with diffuse panbronchiolitis (DPB). Furthermore, alveolar macrophages could produce IL-8 subsequent to CD44-hyaluronic acid (HA) interaction. The purpose of this study was to evaluate the contribution of CD44 expressed on alveolar macrophages to the pathogenesis of DPB. We examined the concentration of soluble CD44 (sCD44) in bronchoalveolar lavage fluid (BALF) and CD44 expression on macrophages in BALF from patients with DPB before and after low-dose, long-term macrolide therapy. We also assessed the HA-binding ability of alveolar macrophages as a functional analysis of the CD44 molecule. The sCD44 concentration in BALF was significantly lower in patients with DPB than in healthy volunteers. Percentages of alveolar macrophages expressing low CD44 (CD44 low+) and HA-nonbinding alveolar macrophages were higher in patients with DPB compared with healthy volunteers. Furthermore, macrolide therapy normalized CD44 expression and HA-binding ability of macrophages in BALF from DPB patients. Our findings suggest that alveolar macrophage dysfunction could result from abnormalities of CD44 expression in patients with DPB and that these events could contribute to the pathogenesis of DPB.
Increased soluble p55 and p75 tumour necrosis factor-αreceptors in patients with hepatitis C-associated mixed cryoglobulinaemia
Authors: Kaplanski G , Marin V , Maisonobe T , Sbai A , Farnarier C , Ghillani P , Thirion X , Durand J M , Harlé J R , Bongrand P , Piette J C , Cacoub P
Published: March 2003

To investigate whether tumour necrosis factor α (TNFα) plays a role in the pathogenesis of hepatitis C virus-associated mixed cryoglobulinaemia (HCV-MC), we measured soluble TNFα and its soluble p55 (sTNFR1) and p75 (sTNFR2) receptors in the serum of patients with HCV-MC. TNFα, sTNFR1 and sTNFR2 were measured in the serum of 32 patients with HCV-MC, 18 patients with hepatitis C without MC (HCV) and 18 healthy volunteers, using specific immunoassays. Correlations between clinical and biological parameters and the concentrations of TNFα and sTNFRs were established by studying detailed clinical records of the 32 HCV-MC patients. Although higher, TNFα levels were not significantly different in HCV-MC patients compared with healthy or HCV controls. sTNFR1 and sTNFR2, however, were significantly higher in HCV-MC compared with controls or with HCV patients, and higher concentrations of sTNFR1 and sTNFR2 were observed in patients with severe visceral vasculitis, compared with patients with limited purpura. sTNFR1 concentrations positively correlated with fibrinogen levels but TNFα, sTNFR1 and sTNFR2 did not correlate with other biological parameters such as rheumatoid factor concentrations, CH50 or C4 values. These data suggest a role for TNFα in the pathogenesis of the immune complex-mediated vasculitis associated with HCV-MC.