bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoepitopes on autoantigen centromere protein-A (CENP-A) are restricted to the N-terminal region, which has no homology with histone H3
Authors: Muro Y , Azuma N , Onouchi H , Kunimatsu M , Tomita Y , Sasaki M , Sugimoto K
Published: March 2003

Anti-centromere autoantibodies (ACA) are commonly found in the serum of patients with a limited type of scleroderma and other systemic autoimmune diseases. CENP-A is one of the major antigens against ACA and a histone H3-like protein. To analyse the autoantigenic epitopes of CENP-A, a series of truncated peptides of human CENP-A were expressed in Escherichia coli and immunoblotting analysis was performed with 91 ACA+ sera. Eighty sera (88%) with the ACA reacted to the 52-amino acids N-terminal region which is not homologous to H3, while no sera reacted to the C-terminus which has a sequence similarity with H3. Moreover, ELISA was also employed in this study using two synthetic peptides corresponding to the amino acid sequences 3–17 (peptide A) and 25–38 (peptide B). Peptides A and B were reactive to 78 (86%) and 79 (87%) of ACA, respectively. Core antigens of hepatitis B virus (HBV) and hepatitis C virus (HCV) have similar sequences to peptide A and/or peptide B, but three sera containing HBV without ACA and five sera containing HCV without ACA were found to be reactive to neither peptide. Centromere localization of CENP-A is dependent on the H3-like C-terminal domain which is not autoantigenic, while the antigenic N-terminal domain, which might play unidentified functional roles, should be an important region for the induction of ACA.
Possible role of L-selectin in T lymphocyte alveolitis in patients with active pulmonary sarcoidosis
Authors: Kaseda M , Kadota J , Mukae H , Kawamoto S , Shukuwa T , Iwashita T , Matsubara Y , Ishimatsu Y , Yoshinaga M , Abe K , Kohno S
Published: March 2003

A number of adhesion molecules participate in the recruitment of inflammatory cells to the site of inflammation, and selectins together with their ligands are important in the early transient adhesion phase. In this study, we evaluated the role of L-selectin in T lymphocyte alveolitis in patients with active pulmonary sarcoidosis. We measured serum and bronchoalveolar lavage fluid (BALF) concentrations of soluble (s) L-selectin using an ELISA. Serum and BALF concentrations of s L-selectin were significantly elevated in patients with sarcoidosis compared with control healthy subjects and idiopathic pulmonary fibrosis (IPF) patients (P < 0.05 and P < 0.01, respectively). The lymphocyte surface marker was also examined in peripheral blood and BALF by flow cytometric analysis. The percentage of CD3+CD62L+ cells ( L-selectin-bearing T lymphocytes) was significantly lower in peripheral blood of sarcoidosis than in that of healthy subjects (P < 0.01). In contrast, the percentage of CD3+CD62L− cells ( L-selectin-negative T lymphocytes) in BALF of patients with sarcoidosis was significantly higher than in healthy subjects (P < 0.05) and IPF patients (P < 0.01). Furthermore, there was a significant correlation between serum concentrations of s L-selectin and the number of L-selectin-negative T lymphocytes in BALF (r = 0.535, P < 0.01). Our results suggest that L-selectin may be involved in T lymphocyte alveolitis in patients with active pulmonary sarcoidosis.
Clustering of distinct autoimmune diseases associated with functional abnormalities of T cell survival in children
Authors: Pignata C , Alessio M , Ramenghi U , Bonissoni S , Difranco D , Brusco A , Matrecano E , Franzese A , Dianzani I , Dianzani U
Published: March 2003

To ascertain whether alterations of lymphocyte switching off may be associated with clustering of autoimmune diseases in children, Fas- and C2-ceramide-induced cell death was evaluated on T cell lines derived from three patients affected by clustering of autoimmune disorders. Three patterns were found: patient 3 was resistant to Fas- and C2-ceramide, patient 1 was resistant to Fas, but sensitive to C2-ceramide, patient 2 was resistant to C2-ceramide, but sensitive to Fas. By contrast, Fas- and C2-ceramide-induced cell death was normal in five children with systemic juvenile rheumatoid arthritis, five children with insulin-dependent diabetes and 10 age-matched healthy controls. Surface expression of Fas was low in patient 1, but normal in patients 2 and 3. Together with normal Fas transcripts, patients 2 and 3 displayed a transcript 152 bp longer than the normal one retaining intron 5. Our data indicate that polyreactive autoimmune syndromes may be associated with heterogeneous alteration of the immune response switching-off system.
Evidence for recognition of novel islet T cell antigens by granule-specific T cell lines from new onset type 1 diabetic patients
Authors: Tree T I M , O'Byrne D , Tremble J M , Macfarlane W M , Haskins K , James R F L , Docherty K , Hutton J C , Banga J P
Published: March 2003

Type 1 diabetes is a T cell-mediated autoimmune disease where a number of islet β-cell target autoantigens have been characterized on the basis of reactivity with autoantibodies. Nevertheless, there remains uncertainty of the nature of another group of autoantigens associated with the secretory granule fraction of islet β-cells that appear to be targeted predominantly by autoreactive T cells. We have previously characterized CD4+, HLA-DR-restricted T cell lines from new onset type 1 diabetic patients that are specific for the secretory granule fraction of rat tumour insulinoma, RIN. The T cell line from the first patient, HS, proliferates in response to crude microsomal membranes prepared from a recently established, pure human islet β-cell line NES2Y. In addition, the HS line also responds to secretory granule fractions prepared from a murine tumour insulinoma grown in RIP-Tag mice, showing the recognition of species-conserved antigen(s) in β-cells. Using partially matched antigen-presenting cells, the HS T cells and another line derived from a second patient, MR, were shown to be restricted by disease-associated DRB1*0101 and DRB1*0404 alleles, respectively. Neither the HS or MR T cell lines proliferate in response to a large panel of candidate islet cell antigens, including insulin, proinsulin, glutamic acid decarboxylase, the protein tyrosine phosphatase IA-2/phogrin, imogen-38, ICA69 or hsp60. Our data provide compelling evidence of the presence of a group of antigens associated with the secretory granule fraction of islet β-cells recognized by the T cell lines, whose definition may contribute to our knowledge of disease induction as well as to diagnosis.
Enhancement of natural killer (NK) cell cytotoxicity and induction of NK cell-derived interferon-gamma (IFN-γ) display different kinetics during experimental infection withTrypanosoma cruzi
Authors: Une C , Andersson J , Eloranta M-L , Sunnemark D , Harris R A , Örn A
Published: March 2003

Early immunological activation involves an initial phase of cytokine activity and involvement of cell types such as NK cells. Such early immune responses are often decisive in resolution of microbial infection. NK cells reduce parasitaemia and enhance survival in experimental Trypanosoma cruzi infection, although the nature of these protective effects is not well understood. In this study, a detailed analysis of innate cytokine induction in the absence and presence of NK cells during the first 8 days of infection was performed. Following intraperitoneal infection with a high dose of parasites, reverse transcriptase-polymerase chain reaction showed that splenic mRNA for IFN-γ appeared as a peak 24 h after infection and then reappeared 2–3 days later. In NK-depleted animals the first peak of IFN-γ was absent and the second wave was slightly delayed. mRNA for IL-12 and tumour necrosis factor-alpha (TNF-α) as well as IFN-α protein in serum was only recorded 24 h after infection, at the same time as the IFN-γ peak. NK depletion resulted in a small decrease of IL-12 mRNA levels, whereas TNF-α and IFN-α were not affected. NK cytotoxicity remained elevated throughout the 8 days and thus did not parallel the expression of IFN-γ production by NK cells. We conclude that NK cell cytokine production and cytolytic activity play different roles in response to challenge with T. cruzi.
Influence of age and HLA type on interferon-gamma (IFN-γ) responses to a naturally occurring polymorphic epitope ofPlasmodium falciparumliver stage antigen-1 (LSA-1)
Authors: Bucci K , Kastens W , Hollingdale M R , Shankar A , Alpers M P , King C L , Kazura J W
Published: March 2003

Antigenic polymorphism and HLA restriction may limit the immunogenicity of a subunit vaccine against liver-stage Plasmodium falciparum. We examined 59 clinical isolates and five laboratory clones of P. falciparum for polymorphism in the N- and C-terminal regions of LSA-1, evaluated binding of the corresponding peptides to selected HLA class I alleles, and measured IFN-γ responses in residents of a malaria-endemic area of Papua New Guinea where HLA-A*1101, -24, -B13, and -B40 are the most common class I alleles. LSA-1 polymorphism was limited to a single non-synonymous mutation encoding serine (S), proline (P), or threonine (T) at amino acid 85. Nine-mer 84–92 peptides with S, T, or P at the primary anchor position bound differentially to HLA-A11, -A2, and -B7. IFN-γ ELISPOT responses increased with age in malaria-exposed subjects: 14–16% and 30–36% of 2–5- and 6–54-year-olds, respectively, had ≥ 10 IFN-γ-secreting cells/106 peripheral blood mononuclear cells when stimulated with at least one peptide variant (P < 0·05). IFN-γ responses to all three peptides were also greater for older than younger individuals. No children < 3 years old had lymphocytes that responded to all three 84–92 peptides, whereas 45% of adults (mean age 48 years) had aggregated IFN-γ responses. These data support the notion that age-related cumulative exposure to P. falciparum increases the frequency of IFN-γ responses to polymorphic epitopes of liver-stage antigens such as LSA-1.
A single measurement of CD38CD8 cells in HIV+, long-term surviving injecting drug users distinguishes those who will progress to AIDS from those who will remain stable
Authors: Froebel K S , Raab G M , D'alessandro C , Armitage M P , Mackenzie K M , Struthers M , Whitelaw J M , Yang S
Published: March 2003

This study compares the predictive power of a single measurement of CD8+CD38+, CD8+CD45RO+ or CD8+CD38+CD45RO+ subpopulations in predicting progression to AIDS in a cohort of HIV+ long-term surviving injecting drug users. The results showed that both the total CD8+ percentage, and the CD8+CD38+ and CD8+CD38+CD45RO+ subpopulations of cells all individually predicted progression to AIDS. In combination with CD4, only the CD8+CD38+ subpopulation enhanced the predictive power of the CD4 percentage alone. The CD8+ percentage correlated negatively with the CD4 percentage and the CD8+CD45RO+ subpopulation did not predict disease progression. The proportion of CD8+CD38+ cells identified which patients with a moderate CD4 level were more likely to progress to AIDS, and conversely, which patients with a low CD4 count were likely to remain clinically stable. The results were consistent irrespective of whether time was measured from the date of seroconversion, or from the date of the test. This study is the first to measure these markers in HIV-infected injecting drug users, and in long-term survivors. The results demonstrate the considerable added value of the CD8+CD38+ cell percentage over the CD4 count alone, in predicting HIV clinical progression.
MHC class I pathway is not required for the development of crescentic glomerulonephritis in mice
Authors: Li S , Holdsworth S R , Tipping P G
Published: March 2003

MHC II and CD4+ T cells are required for anti-glomerular basement membrane (GBM) globulin-initiated crescentic glomerulonephritis (GN) in mice, but the role of MHC I and CD8+ T cells is unclear. The cytolytic function of CD8+ T cells requires recognition of peptide antigens presented on MHC I. CD8+ T cells can also perform helper functions via cytokine production. The contribution of MHC I to crescentic GN was investigated using TAP-1 gene knock out (TAP-1−/−) mice, which have deficient MHC I antigen presentation. Heterozygous TAP-1 mice have normal MHC I expression and developed GN with crescents in 42 ± 4% of glomeruli (normal 0%), proteinuria (9·1 ± 1·6 mg/20 h, normal 1·5 ± 0·3 mg/20 h) and impaired renal function (creatinine clearance 110 ± 8 μl/min, normal 193 ± 10 μl/min) following administration of sheep anti-mouse GBM globulin. TAP-1−/− mice, which have extremely low MHC I expression and reduced CD8+ T cells, developed similar GN with 39 ± 3% crescents, proteinuria (12·7 ± 4·3 mg/20 h) and impaired renal function (creatinine clearance 123 ± 20 μl/min). In vivo antibody-induced CD8 depletion did not attenuate crescent formation or protect renal function in C57Bl/6 mice developing GN, although significant reduction in proteinuria (5·3 ± 1·2 mg/20 h, P = 0·012) and glomerular recruitment of CD4+ T cells and macrophages were observed compared with control treated mice with GN. These data demonstrate that MHC I is not required for development of crescentic GN in mice. The MHC I-independent contribution of CD8+ T cells to proteinuria and inflammatory cell recruitment suggests that they may serve a ‘helper’ rather than cytolytic role in this disease.
Coxsackievirus B3 infection induces anti-flavoprotein antibodies in mice
Authors: Cicek G , Vuorinen T , Stähle I , Stepanek P , Freudenberg N , Brandsch R
Published: March 2003

Enteroviruses, the most common cause of acute myocarditis, are also supposed aetiological agents of dilated cardiomyopathy. Autoantibodies (anti-M7; Klein & Berg, Clin Exp Immunol 1990; 58:283–92) directed against flavoproteins with covalently bound flavin (αFp-Ab; Otto et al., Clin Exp Immunol 1998; 111:541–2) are detected in up to 30% of sera of patients with myocarditis and idiopathic dilated cardiomyopathy (IDCM). Mice inoculated with a myocarditic variant of coxsackievirus B3 (CVB3) were employed to study the occurrence of serum αFp-Ab following viral infection. The presence of αFp-Ab was analysed by Western blotting with the flavoprotein antigens 6-hydroxy-d-nicotine oxidase (6HDNO) and sarcosine oxidase (SaO). Of 10 sera from CVB3-infected mice, five showed a strong reaction with both antigens. The sera were reactive also to the mitochondrial covalently flavinylated proteins dimethylglycine dehydrogenase and sarcosine dehydrogenase. Sera of non-infected mice did not react with these antigens. A 6HDNO mutant protein with non-covalently bound FAD no longer reacted on Western blots with sera of CVB3-infected mice. Preincubation with FAD abolished or reduced the reaction of the sera with the 6HDNO antigen. At 2 weeks p.i. the αFp-Ab were of the IgM and IgG isotypes, at 7 and 9 weeks p.i. of the IgG isotype. The sera of CVB3-infected mice reproduced closely the antigenic specificity of the anti-M7 sera of patients, lending further support to the role of coxsackieviruses in the pathogenesis of IDCM.
Patients with inflammatory bowel disease (IBD) reveal increased induction capacity of intracellular interferon-gamma (IFN-γ) in peripheral CD8+ lymphocytes co-cultured with intestinal epithelial cells
Authors: Bisping G , Lügering N , Lütke-Brintrup S , Pauels H-G , Schürmann G , Domschke W , Kucharzik T
Published: March 2003

Intestinal epithelial cells seem to play a key role during IBD. The network of cellular interactions between epithelial cells and lamina propria mononuclear cells is still incompletely understood. In the following co-culture model we investigated the influence of intestinal epithelial cells on cytokine expression of T cytotoxic and T helper cells from patients with IBD and healthy controls. Peripheral blood mononuclear cells (PBMC) were purified by a Ficoll–Hypaque gradient followed by co-incubation with epithelial cells in multiwell cell culture insert plates in direct contact as well as separated by transwell filters. We used Caco-2 cells as well as freshly isolated colonic epithelia obtained from surgical specimens. Three-colour immunofluorescence flow cytometry was performed after collection, stimulation and staining of PBMC with anti-CD4, anti-CD8, anti-IFN-γ and anti-IL-4. Patients with IBD (Crohn's disease (CD), n = 12; ulcerative colitis (UC), n = 16) and healthy controls (n = 10) were included in the study. After 24 h of co-incubation with Caco-2 cells we found a significant increase of IFN-γ-producing CD8+ lymphocytes in patients with IBD. In contrast, healthy controls did not respond to the epithelial stimulus. No significant differences could be found between CD and UC or active and inactive disease. A significant increase of IFN-γ+/CD8+ lymphocytes in patients with UC was also seen after direct co-incubation with primary cultures of colonic crypt cells. The observed epithelial–lymphocyte interaction seems to be MHC I-restricted. No significant epithelial cell-mediated effects on cytokine expression were detected in the PBMC CD4+ subsets. Patients with IBD—even in an inactive state of disease—exert an increased capacity for IFN-γ induction in CD8+ lymphocytes mediated by intestinal epithelial cells. This mechanism may be important during chronic intestinal inflammation, as in the case of altered mucosal barrier function epithelial cells may become targets for IFN-γ-producing CD8+ lymphocytes.
Adrenalectomy enhances response of tumour necrosis factor-alpha (TNF-α) to haemorrhage in the conscious rat
Authors: Yamashita M , Yamashita M
Published: March 2003

Several factors have been implicated in TNF-α induction after haemorrhage, but the precise mechanism is unknown. To assess a possible role of adrenal glands on TNF-α induction after haemorrhage, adrenalectomized (ADX) and sham-adrenalectomized (Sham) rats were subjected to 20 ml/kg haemorrhage (approximately 30% of the total blood volume) over 5 min in the conscious state, and changes in serum TNF and hepatic TNF-α mRNA were analysed by cytotoxicity against L929 cells and by semiquantitative reverse transcription-polymerase chain reaction, respectively. Changes in plasma corticosterone (CS) and mean arterial blood pressure (MABP) were also detected. Plasma CS levels showed a rapid increase after haemorrhage in Sham rats, whereas there was no response in ADX rats. Responses of MABP were comparable in the two groups. Both serum TNF and hepatic TNF-α mRNA showed rapid increases after haemorrhage in the Sham group, and these increases were significantly augmented in the ADX group. Pretreatment with CS (100 μg/h) negated these augmentations in ADX rats. These results show that adrenalectomy enhances the response of TNF-α to 20 ml/kg haemorrhage in the conscious rat, and suggest that endogenous glucocorticoids may play an inhibitory role in the induction of TNF-α after haemorrhage.
Quantitative analysis of inflammatory cells infiltrating the cystic fibrosis airway mucosa
Authors: Hubeau C , Lorenzato M , Couetil J P , Hubert D , Dusser D , Puchelle E , Gaillard D
Published: March 2003

Airway inflammation represents a hallmark of the cystic fibrosis (CF) disease. However, the mucosal distribution of immune cells along the CF airways has not been clearly defined, particularly in intermediate bronchi and distal bronchioles. We analysed lung tissues collected at the time of transplantation from homozygous ΔF508+/+CF patients versus non-CF donors. Using immunohistochemistry, the distribution of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, polymorphonuclear neutrophils (PMN), mast cells, CD3+ T cells, including the CD4+ and CD8+ subsets, CD20+ B cells, CD38+ plasma cells and CD68+ macrophages, was analysed at lobar, segmental and distal levels of the bronchial tree. Using image cytometry, the number of cells per mm2 was assessed in the depth of the bronchial wall. In CF airways, alterations mainly consisted in lesions of the surface epithelium. Numerous immune cells were heterogeneously distributed all along the bronchial tree and mainly located in the mucosa, beneath the surface epithelium. Compared to non-CF donors, the lymphoid aggregates formed by B cells were significantly larger all along the CF airways (P = 0·001). The number of T lymphocytes was higher at the CF distal level (P = 0·035), where we observed an intense tissue damage. PMN preferentially accumulated (P = 0·033) in the CF surface epithelium, which overexpressed ICAM-1 but not VCAM-1 and E-selectin. These results highlight the nature of the inflammatory infiltrate in the CF airway mucosa and emphasize a prominent implication of PMN, B and T lymphocytes in the CF disease.
Non FcR-binding murine antihuman CD3 monoclonal antibody is capable of productive TCR signalling and induces proliferation in the presence of costimulation
Authors: Meijer R T , Yong S L , Ten Berge I J M , Van Lier R A W , Schellekens P T A
Published: March 2003

CLB T3/4.A is a non FcR-binding CD3 mAb of the murine IgA isotype, which may be used as an alternative for the mitogenic OKT3 mAb in the treatment of acute cellular rejection after organ transplantation. We studied TCR signalling and T cell activation in response to T3/4.A in normal human PBMC in vitro. T3/4.A induced a rapid rise in free cytoplasmic Ca2+, not different from the response to mitogenic CD3 mAb. However, protein tyrosine phosphorylation and, particularly, MAPK activation, were reduced as compared to mitogenic CD3 mAb. T3/4.A enhanced expression of both CD69 and CD25, but proliferation and detectable cytokine production did not occur. Addition of either CD28 mAb or IL-2 induced a strong proliferative response, which was accompanied by cytokine production. At higher mAb concentrations, T cell activation decreased, which correlated with TCR downmodulation. To exclude the possibility that activation by T3/4.A depends on interaction of murine IgA Fc with as yet unknown FcR, we showed that also with CD3 mAb F(ab′)2 fragments upregulation of activation molecules occurred, as well as proliferation in the presence of costimulation. We conclude that the non FcR-binding murine IgA mAb T3/4.A acts as a partial agonist and leads to proliferation and cytokine production only in the presence of appropriate costimuli. These findings may explain the mitigated cytokine release syndrome observed in vivo with some nonmitogenic CD3 mAbs.
The effect of interleukin-10 and of interleukin-12 on the in vitro production of anti-double-stranded DNA antibodies from patients with systemic lupus erythematosus
Authors: Tyrrell-Price J , Lydyard P M , Isenberg D A
Published: March 2003

IL-10 and IL-12 are cytokines which are important in regulating immune responses. Plasma levels of IL-10 and autoantibodies against double-stranded DNA (dsDNA) often mirror disease activity in patients with SLE. IL-12 secretion from SLE patients' blood mononuclear cells also correlates with disease activity, but has an inverse relationship. The aim of this study was to measure the effect of IL-10 and of IL-12 on the production of IgG autoantibodies from patients with SLE, both cross-sectionally and longitudinally.

Peripheral blood mononuclear cells (PBMC) were cultured with IL-10 (at 20 ng/ml or 2 ng/ml) or IL-12 (at 2 ng/ml or 0·2 ng/ml) or without cytokine and the supernatanants tested for the production of double-stranded DNA antibodies (dsDNA abs), single-stranded DNA antibodies (ssDNA abs) and total IgG antibodies (IgG abs) by ELISA. The BILAG disease activity index was recorded at each patient visit (a global score of six or more is regarded as active disease).

In general, treatment with IL-10 caused PBMCs from patients with inactive disease to increase their antissDNA and dsDNA ab production (by upto 354% and 186%, respectively) while patients with active disease decreased their antibody production (by upto 91% and 97%, respectively). Overall there was a correlation between disease activity and change in antissDNA and dsDNA ab production (r = − 0·51; P = 0·03 and r = − 0·48; P = 0·042, respectively). Treatment with IL-12 at 0·2 ng/ml inhibited antissDNA and dsDNA antibody production, having the greatest effect on patients with active disease (decreasing antissDNA and dsDNA antibody production by upto 75% and 73%, respectively). This resulted in a significant correlation between disease activity and change in antissDNA antibody production (r = − 0·76; P = 0·03), but significance was not reached with antidsDNA antibody production (P = 0·06). Together these data suggest that the effect of these cytokines on antibody production by SLE PBMCs involves several factors; one of which is disease activity.
T cell responses to highly active antiretroviral therapy defined by chemokine receptors expression, cytokine production, T cell receptor repertoire and anti-HIV T-lymphocyte activity
Authors: Giovannetti A , Pierdominici M , Mazzetta F , Salemi S , Marziali M , Kuonen D , Iebba F , Lusi E A , Cossarizza A , Aiuti F
Published: March 2003

The immunological correlates of highly active antiretroviral therapy (HAART)-induced suppression of human immunodeficiency virus type 1 (HIV-1) replication have been investigated.

20 HIV-1-infected patients with mean CD4+ T cell count of 298/µl, plasma viral load of 4·7 log10 copies/ml and naive for protease inhibitors (PI) were studied during12 months of HAART. An increased number of both CD4+ and CD8+ naive T cells and a normalization of the frequency of CCR5- and CXCR4-expressing CD4+ T cells were readily observed after starting therapy. Single cell analysis of cytokine production after 12 months of HAART showed an increased number of interleukin (IL)-2-, but not IL-4- and (IFN)-γ-, producing T cells and a decreased percentage of CD8+ IFN-γ + cells. A correlation between the frequency of IFN-γ-producing T cells and that of memory, CCR5+ and CD95+ T cells was demonstrated in both CD4+ and CD8+ subsets. The diversity of T cell receptor (TCR) variable beta (BV) chain repertoire significantly increased after 12 months of HAART within the CD4+ but not the CD8+ T cell subset. However, the level of perturbation of the third complementarity-determining region (CDR3), was not significantly modified by effective therapy. The number of anti-HIV Gag and Pol cytotoxic T lymphocytes precursors (CTLp) decreased during HAART and highly correlated with the CD8 IFN-γ response. Ameliorated clinical conditions were observed in all patients in absence of any opportunistic infections during all the study period. These observations indicate that a better restoration of immunity may be obtained in patients starting HAART at less advanced stages of the disease.
Inosine monophosphate dehydrogenase (IMPDH) inhibitionin vitrosuppresses lymphocyte proliferation and the production of immunoglobulins, autoantibodies and cytokines in splenocytes from MRLlpr/lprmice
Authors: Jonsson C A , Carlsten H
Published: March 2003

The aim of this study was to evaluate the effects of the immunomodulating drug mycophenolic acid (MPA) on splenocytes in an animal model of systemic lupus erythematosus (SLE), using MRLlpr/lpr mice. MPA reversibly inhibits inosine 5′-monophosphate dehydrogenase, an enzyme involved in the de novo guanosine synthesis. Splenocytes were treated with MPA (at 1 or 10 µm), and stimulated with either lipopolysaccharide (LPS; 10 µg/ml) or concanavalin A (ConA; 1.25 µg/ml). In blocking experiments, guanosine (100 µm) was added to the cultures to inhibit the effects of MPA. Lymphocyte proliferation, enumeration of immunoglobulin producing cells (using ELISPOT) and quantification of anti-double-stranded (ds) DNA antibodies, IFN-γ and IL-10 (by ELISA) in supernatants were performed. In addition, cell viability was evaluated using propidium iodide and flow cytometry. We found that MPA-treated splenocytes had dramatically decreased mitogen-induced proliferation and number of immunoglobulin producing cells, down-regulated production of IFN-γ, IL-10 and IgM anti-dsDNA antibodies. The viability of MPA-treated cells was also decreased. All of the effect modulated by MPA could be neutralized by the addition of guanosine. We conclude that MPA has potent immunomodulating effects on both B and T lymphocytes, modulating not only proliferation, but also the production of cytokines, immunoglobulins and autoantibodies.
Different proteolytic mechanisms involved in FcγRIIIb shedding from human neutrophils
Authors: Middelhoven P J , Van Buul J D , Hordijk P L , Roos D
Published: March 2003

The Fcγ receptor type IIIb (CD16) is highly expressed on human neutrophils and is found in a soluble form in plasma and in other body fluids. Upon activation of neutrophils in vitro, FcγRIIIb is shed from the cell surface by proteolytic cleavage. We have now investigated the effect of metalloproteinase inhibitors and a serine proteinase inhibitor on the shedding of FcγRIIIb induced by phorbol 12-myristate 13-acetate (PMA) or cytochalasin B (cyto B) + N-formyl-methionyl-leucyl-phenylalanine (fMLP). Metalloproteinase inhibitors blocked to a large extent PMA-induced, but not cyto B + fMLP-induced shedding of FcγRIIIb. Inhibition of members of the ADAM (a disintegrin and metalloproteinase) family appeared most efficient. In contrast, the serine protease inhibitor N-methoxysuccinyl-alanine-alanine-proline-valine-chloromethylketone (MeOsuc-AAPV-CMK) largely blocked cyto B + fMLP-induced, but not PMA-induced shedding of FcγRIIIb. Metalloproteinase inhibitors in combination with the serine proteinase inhibitor resulted in full inhibition of FcγRIIIb shedding induced by either PMA or cyto B + fMLP. The shedding of FcγRIIIb that accompanied apoptosis was inhibited by 60% in the presence of inhibitors of metalloproteinases but was insensitive to inhibition of serine proteinases. These results show that distinct types of proteolytic enzyme are involved in the stimulus-induced shedding of FcγRIIIb from human neutrophils and suggest that these proteinases may become differentially activated under various physiological or pathological conditions.
Glioma-expressed antigen 2 (GLEA2): a novel protein that can elicit immune responses in glioblastoma patients and some controls
Authors: Fischer U , Struss A-K , Hemmer D , Pallasch C P , Steudel W-I , Meese E
Published: March 2003

Glioma constitutes the most frequent brain tumour in man with glioblastoma as the most prevalent and malignant type. The average survival time of less than 16 months underlines the need for improvements in diagnosis and therapy. Here, we report the identification of a novel antigen termed glioma-expressed antigen 2 (GLEA2) causing a frequent immune response in glioma patients. Screening of 450 000 clones from a glioblastoma lambda zap expression library with autologous patient serum revealed a group of five serum-positive clones sharing a high sequence homology. Further sequence analysis showed a sequence homology to a hepatocellular carcinoma associated antigen 58 (HCA58). We localized the novel HCA homologous gene termed glioma-expressed antigen 2 (GLEA2) on chromosome 20 by somatic cell hybrid panel mapping. Using allogenic sera from 39 glioblastoma patients, we found an immune response against GLEA2 in 17 patients (43%). In addition, screening with allogenic sera from other glioma patients revealed GLEA2 directed antibodies in two out of five pilocytic astrocytomas and in one out of two astrocytomas. Unrelated tumour sera revealed no immune response and sera from healthy persons showed an immune response in two out of 14 cases (14%). Northern blot hybridization and RT-PCR showed ubiquitous GLEA2 gene expression in glioma and normal tissues. The novel HCA homologous gene, GLEA2, appears to induce a frequent immune response in glioma. In the light of the lack of useful glioma markers, it appears reasonable to consider GLEA2 as a potential future diagnostic marker.
MHC class I chain-like gene A (MICA) and its associations with inflammatory bowel disease and peripheral arthropathy
Authors: Orchard T R , Dhar A , Simmons J D , Vaughan R , Welsh K I , Jewell D P
Published: March 2003

MHC class I chain gene A (MICA) is a non-classical Class I gene which is expressed on the surface of epithelia without β2-microglobulin. The gene is found in the major histocompatibility complex (MHC) in tight linkage disequilibrium with human leucocyte antigen-B (HLA-B). Its precise function is unknown, but it interacts with γδ T cells of the intestinal immune system. This region of the MHC has been implicated in inflammatory bowel disease (IBD) pathogenesis by recent association mapping studies and this study was performed to examine the prevalence of MICA gene polymorphisms in IBD, in particular in type 2 peripheral arthropathy (PeA), which also has a strong HLA–B association. An assessment of the prevalence of MICA polymorphisms in IBD was made. Blood from 50 ulcerative colitis (UC) and 50 Crohn's disease controls was taken and MICA status determined using allele-specific PCR for 16 known alleles of MICA. A further 91 UC patients were recruited to confirm the results of this stage, and then the polymorphisms were studied in 52 type 1 and 45 type 2 PeA patients. The MICA status of these groups was compared with 118 blood and organ donor controls with appropriate correction for multiple comparisons. UC overall was associated with possession of MICA*007 in 32% compared to 11% of controls (Pc = 0·017). This association was confirmed in a second cohort of 91 patients (23% versus 11%, P= 0·02). These were independent of HLA class I status. Type 2 IBD PeA was associated with MICA*008 in 98% compared to 73% of controls (P = 0·0001). MICA*007 is associated with susceptibility to UC in our population and MICA*008 with type 2 IBD PeA. Further work is now required to assess the distribution and expression of MICA throughout the gut in health and disease.
T cell cytokines determine the severity of experimental IgA nephropathy by regulating IgA glycosylation
Authors: Chintalacharuvu S R , Nagy N U , Sigmund N , Nedrud J G , Amm M E L , Emancipator S N
Published: March 2003

Hyperfunction of Th2 cells and aberrant glycosylation of IgA have been proposed independently as factors in the pathogenesis of IgA nephropathy (IgAN), the most common form of glomerulonephritis. To investigate the relationship between Th2 cytokines and IgA glycosylation in the genesis of IgAN, we induced IgAN in C3HeB and BALB/c mice by oral immunization and intranasal challenge with Sendai virus. Although both strains of mice developed microhaematuria and glomerular IgA immune deposits to similar degrees, only BALB/c mice developed significant renal insufficiency. More profound reductions of terminal galactosylation and sialylation occurred in Sendai virus-specific IgA from BALB/c versus C3HeB mice, and splenocytes from immunized BALB/c mice produced more Th2 and less Th1 cytokines compared to C3HeB mice when stimulated with antigen in vitro. Furthermore, the decreased glycosylation of IgA elicited by Th2 cytokines in vitro was blunted by the addition of IFN-γ. We conclude that increased production of Th2 cytokines can lead to abnormalities in IgA glycosylation, which in turn promote heightened phlogistic responses to IgA immune complexes lodging in the glomerulus. We suggest that a relative or absolute increase in Th2 cytokine production in response to mucosal infection is a significant pathogenic factor in human IgAN.
B7-1 and B7-2 co-stimulatory molecules are required for mercury-induced autoimmunity
Authors: Bagenstose L M , Class R , Salgame P , Monestier M
Published: March 2003

B7-1 (CD80) and B7-2 (CD86) molecules on antigen presenting cells play important roles in providing co-stimulatory signals required for activation and expansion of autoreactive T cells. Moreover, some reports have suggested that these molecules may have distinct functions in the differentiation of Th1 and Th2 cells. Mercury-induced autoimmunity in H-2s mice is characterized by lymphoproliferation of T and B cells, serum increases in IgG1 and IgE and production of antinucleolar antibodies (ANoA). The mechanisms responsible for the various manifestations of this syndrome have yet to be elucidated. To examine the contributions of B7 co-stimulatory molecules to this model, susceptible mice were treated with antibodies to B7-1, B7-2, or both during the development of mercury-induced autoimmunity. The combination of anti-B7-1 and anti-B7-2 antibodies prevented Hg-induced disease in H-2s mice. Additionally, single anti-B7-1 antibody treatment was sufficient to prevent Hg-induced ANoA production, but not IgG1 and IgE hypergammaglobulinaemia. Further, single antibody treatment with anti-B7-2 resulted in a partial reduction of ANoA titres but had no significant effect on total serum IgG1 and IgE levels. Taken together, these results indicate that B7-1 and B7-2 molecules are critical for the development of Hg-induced autoimmunity and suggest that the different manifestations of the syndrome are regulated by independent mechanisms.
Extra-articular cartilage affected in collagen-induced, but not pristane-induced, arthritis models
Authors: Hansson A-S , Lu S , Holmdahl R
Published: March 2003

Rheumatoid arthritis (RA) is a chronic inflammatory disease primarily affecting cartilaginous joints but also extra-articular tissues such as the nose and upper respiratory tract. We have investigated extra-articular cartilage involvement in two commonly used animal models for RA, collagen-induced and pristane-induced arthritis, by immunizing rats with different susceptibility to disease (LEW.1 A, LEW.1F and DA rats). We found that nasal and tracheolaryngeal cartilage is affected in LEW.1 A and DA rats to varying degrees in collagen-induced arthritis but not in any strain in the pristane-induced model. Antibodies to matrilin-1, a cartilage-specific protein expressed mainly in tracheolaryngeal and nasal cartilage but not in joints, were positively associated with the presence of inflammation in nasal cartilage. In contrast, no antibody response to matrilin-1 could be detected in pristane-induced arthritis. In addition, nasal vaccination with collagen type II prior to immunization in DA rats significantly decreased the antibody response to matrilin-1 at day 56, but not at earlier time points, indicating a late protective effect on extra-articular cartilage. We conclude that pristane-induced arthritis is a joint-specific model whereas collagen-induced arthritis affect joints as well as extra-articular cartilage. Furthermore, collagen immunization induces an antibody response to matrilin-1.
Placentally derived prostaglandin E2 acts via the EP4 receptor to inhibit IL-2-dependent proliferation of CTLL-2 T cells
Authors: KVIRKVELIA N , VOJNOVIC I , WARNER T D , ATHIE-MORALES V , FREE P , RAYMENT N , CHAIN B M , RADEMACHER T W , LUND T , ROITT I M , DELVES P J
Published: March 2003

A number of immunomodulatory molecules are present in the placenta, including cytokines, prostaglandins, progesterone and indoleamine 2,3-dioxygenase. An undefined factor capable of down-regulating T-cell activity has recently been reported [1] as being produced by short-term cultures of placental fragments. By careful repetition of these studies we have confirmed that chorionic villi isolated from term placenta produce a low molecular weight, heat stable factor capable of inhibiting the IL-2-dependent proliferation of mouse CTLL-2 cells. This activity was not due, however, to a previously unknown immunosuppressive molecule, but rather to prostaglandin E2 (PGE2). Expression of cyclooxygenase (COX)-2 was detected in the syncytiotrophoblast of chorionic villi explants using immunohistochemistry. Culture of the explants in the presence of the COX-1/COX–2 inhibitors indomethacin and diclofenac, or with the COX-2-selective inhibitor DFP, blocked the production of the immunosuppressive factor. The immunosuppressive activity was restored by adding PGE2 to the supernatants obtained from diclofenac-inhibited explants. A number of different receptors are involved in mediating the biological effects of prostaglandins. By utilizing selective antagonists of individual receptors, we have established that the immunosuppressive effect of PGE2 on CTLL-2 cells is exerted via the EP4 receptor. Thus, addition of an EP4-selective antagonist, but not of EP1 or EP3 antagonists, abolished the immunosuppressive effect of PGE2 on CTLL-2 cells. This may have implications for attempts to selectively manipulate T-cell responses.
Thein vivoeffects of tumour necrosis factor blockade on the early cell mediated immune events and syndrome expression in rat adjuvant arthritis
Authors: BUSH K A , KIRKHAM B W , WALKER J S
Published: March 2003

Anti-TNF therapy is effective in rheumatoid arthritis (RA); however, its mechanisms of action are incompletely understood. T cell-driven mechanisms are thought to play an important role in RA and the effects of TNF blockade on these mechanisms are unclear. Adjuvant arthritis (AA) is a T cell dependent model of inflammatory arthritis. The aims of this study were to investigate the effects of TNF blockade on in vivo T cell cytokine expression and to clarify the role of TNF in the inguinal lymph nodes (ILN) in early arthritis. AA was induced in male DA rats. Rats received either 3 mg/kg or 10 mg/kg PEG sTNF-RI at days 0, 2 and 4 postinduction or 10 mg/kg anti-TNF antibody on day of arthritis induction. Control rats received either saline or normal sheep serum. Paw volume was assessed every 3–4 days. Rats were sacrificed on days 0, 6, 13 and 21 postinduction. Ankles were removed for quantitative radiology and histology. Synovium and ILN were removed for cell culture and to determine mRNA expression of cytokines using semiquantitative RT-PCR. TNF and IFN-γ protein production was measured using a bioassay and an ELISA. TNF blockade did not suppress mRNA expression of T cell cytokines in the ILN of rats in the early phase of AA, suggesting ongoing T cell activity. TNF protein production by ILN cells in culture was reduced in PEG sTNF-RI treated rats, although mRNA expression was increased in the ILN prior to culture. Early administration of PEG sTNF-RI did not attenuate AA, in contrast to an anti-TNF antibody, which suppressed disease. A shorter half-life for the PEG sTNF-RI compared with the anti-TNF antibody or the development of anti-PEG sTNF-RI antibodies may account for these results.
Age-dependent preference in human antibody responses toStreptococcus pneumoniaepolypeptide antigens
Authors: LIFSHITZ S , DAGAN R , SHANI-SEKLER M , GROSSMAN N , FLEMINGER G , FRIGER M , NEBENZAHL Y MIZRACHI
Published: March 2003

Vulnerability to Streptococcus pneumoniae is most pronounced in children. The microbial virulence factors and the features of the host immune response contributing to this phenomenon are not completely understood. In the current study, the humoral immune response to separated Strep. pneumoniae surface proteins and the ability to interfere with Strep. pneumoniae adhesion to cultured epithelial cells were analysed in adults and in children. Sera collected from healthy adults recognized Strep. pneumoniae separated lectin and nonlectin surface proteins in Western blot analysis and inhibited on average 80% of Strep. pneumoniae adhesion to epithelial cells in a concentration-dependent manner. However, sera longitudinally collected from healthy children attending day care centres from 18 months of age and over the course of the following 2 years revealed: (a) development of antibodies to previously unrecognized Strep. pneumoniae surface proteins with age; (b) a quantitative increase in antibody responses, measured by densitometry, towards separated Strep. pneumoniae surface proteins with age; and (c) inhibition of Strep. pneumoniae adhesion to epithelial cells, which was 50% on average at 18 months of age, increased significantly to an average level of 80% inhibition at 42 months of age equalling adult sera inhibitory values. The results obtained in the current study, from the longitudinally collected sera from healthy children with documented repeated Strep. pneumoniae colonization, show that repeated exposures are insufficient to elicit an immune response to Strep. pneumoniae proteins at 18 months of age. This inability to recognize Strep. pneumoniae surface proteins may stem from the inefficiency of T-cell-dependent B-cell responses at this age and/or from the low immunogenicity of the proteins.
CD40 on salivary gland epithelial cells: high constitutive expression by cultured cells from Sjögren’s syndrome patients indicating their intrinsic activation
Authors: DIMITRIOU I D , KAPSOGEORGOU E K , MOUTSOPOULOS H M , MANOUSSAKIS M N
Published: March 2003

CD40 has been identified in an expanding list of haematopoietic and non-haematopoietic cells and has received an increased interest based on its role in a variety of cell-mediated responses and its potential to participate in the pathogenesis of chronic inflammatory disorders. Sjögren’s syndrome (SS) is an autoimmune exocrinopathy, which is characterized by chronic lymphocytic infiltration of exocrine glands and aberrant activation of epithelial tissues. We studied the expression of CD40 protein in cultured non-neoplastic salivary gland epithelial cell (SGEC) lines as well as in minor SG biopsies obtained from 17 SS patients and 12 controls. Immunocytochemical and flow cytometric analyses had revealed the occurrence of constitutively expressed CD40 molecules on the surface of long-term cultured SGEC lines, which could be further induced by interferon-gamma (IFN-γ) and IL-1β cytokines, but not tumour necrosis factor-alpha (TNF-α), IL-4, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IFN-α. Triggering of SGEC through CD40 enhanced the surface expression of the adhesion molecule intercellular adhesion molecule-1 (ICAM-1)/CD54, but not MHC class I and class II (HLA-DR) molecules. Spontaneous CD40 expression was significantly higher in SGEC lines derived from SS patients, compared with controls (P < 0·001), which is suggestive of their intrinsically activated status. In SG biopsies, CD40 was constitutively expressed by lymphocytes, ductal epithelial cells and endothelial cells but not by other glandular cell types, such as acinar cells, myoepithelial cells and fibroblasts. In addition, CD40L staining was also detected in 30–50% of the infiltrating lymphocytes in the biopsies of SS patients. Our findings indicate the immunoregulatory potential of SGEC and lend further support to a model of intrinsic activation in salivary epithelia in SS, whereby these cells actively participate in the induction and maintenance of lymphocytic infiltrates of patients.
Intravenous immunoglobulin (IVIG) preparations induce apoptosis in TNF-α-stimulated endothelial cells via a mitochondria-dependent pathway
Authors: NAKATANI K , TAKESHITA S , TSUJIMOTO H , SEKINE I
Published: March 2003

Endothelial cells (ECs) are a target in inflammation, and the death of EC is regulated by various factors. Although intravenous immunoglobulin (IVIG) preparations are known to be beneficial therapeutic agents for the treatment of autoimmune diseases and systemic inflammatory disorders, their mechanism of action have not yet been completely elucidated. The aim of the present study is to investigate the possible role of IVIG in EC apoptosis. We demonstrate herein that IVIG induced the apoptosis of human umbilical vein ECs (HUVECs) prestimulated by TNF-αin vitro, but not in unstimulated HUVECs, in a dose- and time-dependent manner, using a proportion of cells with hypodiploid DNA, DNA ladder formation and morphological changes. Anti-Fas MoAbs had no effect on the IVIG-induced apoptosis in the TNF-α-stimulated HUVECs. IVIG decreased the intracellular expression of anti-apoptotic proteins of the Bcl-2 family (A1 and Bcl-XL) while IVIG increased the intracellular expression of pro-apoptotic proteins (Bax and Bcl-XS) in the TNF-α-stimulated HUVECs. Furthermore, IVIG increased the intracellular production of reactive oxygen species and decreased the mitochondrial membrane potential (Δψm). Caspase-inhibitors inhibited the IVIG-induced apoptosis of the TNF-α-stimulated HUVECs. The present results show a novel action in which IVIG can induce the apoptosis of TNF-α-stimulated HUVECs through a mitochondrial apoptotic signalling pathway. These observations suggest that the clinical use of IVIG preparations may thereby regulate the cell death of activated ECs in inflammation.