bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and characterization of autoantibodies against catalase and α-enolase in patients with primary sclerosing cholangitis
Authors: Orth T , Kellner R , Diekmann O , Faust J , Meyer Zum Büschenfelde K-H , Mayet W-J
Published: March 2003

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease of unknown aetiology. Recent studies have shown that genetic factors and both cellular and humoral immunological abnormalities are important in the pathogenesis of PSC. The most prominent autoantibodies in PSC are anti-neutrophil cytoplasmic antibodies (ANCA). The autoepitopes of ANCA in PSC are not well defined. The aim of this study was to identify corresponding ANCA autoantigens in patients with PSC. A biochemical approach with enrichment and partial purification of soluble neutrophil proteins, detection of autoantibodies by Western blot and partial amino acid sequencing were used. Two new autoantigen/autoantibody systems in patients with PSC were detected: catalase and α-enolase. The presence of catalase autoantibodies in 9/15 (60%) and α-enolase autoantibodies in 4/15 (27%) was confirmed by ELISA and Western blot. Furthermore, we showed immunoreactions of PSC sera with human biliary epithelial cells, showed the reduction of fluorescence in anti-catalase absorption experiments and observed partial co-localization of anti-catalase antibodies and PSC sera in double-staining experiments on biliary epithelial cells. The anti-catalase antibody-positive PSC patients had a more severe course of disease with a significantly higher alkaline phosphatase compared with the anti-catalase-negative PSC patients (P < 0.06). All ulcerative colitis control sera were anti-catalase antibody-negative. The identified antigens catalase and α-enolase can partly explain the ANCA fluorescence on ethanol-fixed and formaldehyde-fixed granulocytes in patients with PSC. Catalase is an important anti-oxidant enzyme and prevents cell damage from highly reactive oxygen-derived free radicals. Catalase autoantibodies might play a pathogenic role in patients with PSC. Our findings support the hypothesis that oxidative stress is one of the pathogenic mechanisms in patients with PSC.
Anti-retroviral therapy reverses HIV-associated abnormalities in lymphocyte apoptosis
Authors: Johnson N , Parkin J M
Published: March 2003

The objective of this study was to assess the role of anti-retroviral therapy (ART) on the susceptibility of peripheral blood lymphocytes (PBL) from HIV-1-infected individuals to activation-induced apoptosis and in comparison with changes in CD4 lymphocyte counts. Eleven symptomatic HIV+ patients were studied. Ex vivo apoptosis was measured in phytohaemagglutinin (PHA)-stimulated PBL and CD4 subsets by flow cytometry, at baseline and after 1 month (4–6 weeks) and 2/3 months of ART. Six patients had extended studies of the effects of therapy to a maximum of 21 months. Lymphocyte apoptosis was significantly elevated in HIV+ patients at baseline (median 22% compared with 7.5% in HIV− risk-matched controls; P < 0.05). This decreased to control levels on ART (7.4% at 4–6 weeks, P < 0.01, and 6.2% at 8–12 weeks, P < 0.05, compared with baseline). Similar changes occurred in the CD4+ subpopulation. The decrease in apoptosis was maintained for several months, but the effect was rapidly lost if ART was discontinued. CD4 counts showed a reciprocal relationship to changes in apoptosis. The association of changes in apoptosis with those in CD4 counts suggests a link between programmed cell death and lymphocyte depletion. Apoptosis reduced in some individuals without any reduction in viral load, suggesting apoptosis may be influenced by factors in addition to the overall extent of HIV replication.
Tumour necrosis factor (TNF) gene polymorphism influences TNF-α production in lipopolysaccharide (LPS)-stimulated whole blood cell culture in healthy humans
Authors: Louis E , Franchimont D , Piron A , Gevaert Y , Schaaf-Lafontaine N , Roland S , Mahieu P , Malaise M , De Groote D , Louis R , Belaiche J
Published: March 2003

TNF-α is involved in infectious and immuno-inflammatory diseases. Different individuals may have different capacities for TNF-α production. This might determine a predisposition to develop some complications or phenotypes of these diseases. The aims of our study were to assess the inter-individual variability of TNF-α production and to correlate this variability to a single base pair polymorphism located at position −308 in TNF gene. We studied 62 healthy individuals. TNF-α production after LPS stimulation was evaluated using a whole blood cell culture model. The TNF gene polymorphism was studied by an allele-specific polymerase chain reaction. Other cytokines produced in the culture, soluble CD14 concentrations and expression of CD14 on blood cells were also measured. Among the 62 individuals, 57 were successfully genotyped. There were 41 TNF1 homozygotes and 16 TNF1/TNF2 heterozygotes. TNF-α production after LPS stimulation of whole blood cell culture was higher among TNF2 carriers than among TNF1 homozygotes (929 pg/ml (480–1473 pg/ml) versus 521 pg/ml (178–1307 pg/ml); P < 0.05). This difference was even more significant after correction of TNF-α production for CD14 expression on blood cells. In conclusion, the single base pair polymorphism at position −308 in the TNF gene may influence TNF-α production in healthy individuals.
Abnormal T cell receptor V gene usage in myasthenia gravis: prevalence and characterization of expanded T cell populations
Authors: XU B-Y , Giscombe R , Söderlund A , Troye-Blomberg M , Pirskanen R , Lefvert A K
Published: March 2003

The usage of T cell receptor (TCR) Vα/Vβ chains on cells from 38 patients with myasthenia gravis (MG) was determined by flow cytometry. There was a decreased number of cells expressing Vβ2 in CD8+ and Vβ3 in CD4+ cells in patients compared with healthy individuals. Abnormal expansions of T cells using particular TCR Vα/Vβ gene products were found in 18/38 patients. A significantly higher usage of Vβ13 was observed but there was no restriction with regard to other TCR Vα/Vβ. Expanded cells belonging to both CD4+ and CD8+ were present in MG patients while restricted to the CD8+ population in healthy individuals. To elucidate the role of the expanded populations, we studied characteristics of the expanded and non-expanded T cells from MG patients who had persistent T cell expansions over more than 2 years. The cells were analysed with regard to phenotype, cytokine secretion, cytokine mRNA expression and reactivity with the autoantigen, the acetylcholine receptor. The characteristics of the expanded populations in MG clearly differed from those found in healthy individuals. More cells in the CD4+ expanded populations expressed HLA-DR and there was also a tendency for higher expression of CD25, CD28 and CD57. The number of cells spontaneously secreting cytokines was higher in the expanded populations. A dominant Th1-type cytokine secretion and mRNA expression was noted. Autoantigen-reactive CD4+ T cells were largely restricted to the expanded populations.
Increased number of T cells committed to IL-5 production after respiratory syncytial virus (RSV) infection of human mononuclear cellsin vitro
Authors: Thurau A M , Streckert H J , Rieger C H L , Schauer U
Published: March 2003

We examined changes in the cytokine profile of T cells induced by in vitro infection with RSV. Isolated mononuclear cells from 27 healthy adults and six infants were infected with RSV at a concentration of 3 MOI (multiplicity of infection). After 48 h cells were restimulated with phorbol ester and ionomycin in the presence of monensin for 5 h. The intracellular expression of viral antigen, the cytokines IL-2, IL-4, IL-5, interferon-gamma (IFN-γ), and the expression of surface markers were assessed by immunofluorescent staining and flow cytometry. There was a significant (P < 0.001) rise of IL-5 expression in RSV-infected cultures in comparison with uninfected cultures from the same individuals, whereas there were no changes in the expression of the other lymphokines. The increase in IL-5 generation depended on viable infectious RSV rather than inactivated virus. RSV infection as well as IL-5 production in T cells were confined to the CD8 subpopulation. However, there was no simultaneous expression of RSV antigen and IL-5. Purified T cells did not show any increase in IL-5 generation. However, when the rate of RSV infection was enhanced in monocytes by means of a specific monoclonal antibody, co-cultured T cells displayed an increase of IL-5 production compared with samples with ordinary low rate RSV infection. It is therefore likely that the increased commitment of lymphocytes to produce IL-5 after RSV infection in vitro is mediated by monocytes or other antigen-presenting cells.
Accumulation of immunoglobulin-containing cells in the gut mucosa and presence of faecal immunoglobulin in severe combined immunodeficient (scid) mice with T cell-induced inflammatory bowel disease (IBD)
Authors: Bregenholt S , Brimnes J , Reimann J , Claesson M H
Published: March 2003

Scid mice transplanted either with a gut wall graft or with low numbers of purified CD4+ T cells from immunocompetent syngeneic donor mice show clinical signs of IBD 3–4 months post-transplantation. The disease is mediated by mucosa-infiltrating CD4+ TCRαβ+ T cells. The pathology of 52 individual colon segments obtained from 20 gut wall- or CD4+ T cell-transplanted diseased scid mice was evaluated by histology and the numbers of infiltrating immunoglobulin-containing cells were determined. In particular, cells positive for IgM, IgA and non-inflammatory immunoglobulin isotypes such as IgG1 and IgG2b were found to accumulate in colon segments displaying the most severe histopathology, including inflammatory cellular infiltration, epithelial hyperplasia and ulcerative lesions. Compared with colon segments of normal C.B-17 mice, the lesional scid colon shows increased levels of cells positive for the IgG classes. Faecal extracts of the CD4+ T cell-transplanted scid mice revealed the presence of all six murine immunoglobulin isotypes. Disease progression was accompanied by an increased level of excreted IgM and IgG3 and decreased levels of IgA. It is concluded that locally secreted immunoglobulins may play an immunomodulating role in the pathological changes observed in the present model of T cell-induced inflammatory bowel disease.
The low molecular weight Dextran 40 inhibits the adhesion of T lymphocytes to endothelial cells
Authors: Termeer C C , Weiss J M , Schöpf E , Vanscheidt W , Simon J C
Published: March 2003

Dextrans are complex colloidal macromolecules widely used as haemorrheologic substances and anti-thrombotic agents. Here we describe a novel function of Dextran 40 by demonstrating an inhibition of T lymphocyte adhesion to endothelial cells (EC). We applied an established microassay in which constitutive and tumour necrosis factor-alpha (TNF-α)-induced binding of mouse T lymphoma cells (TK-1) to mouse endothelioma (eEND.2) cells is mediated by the interaction of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on EC with their counter-receptors the LFA-1 heterodimer (CD11a/CD18) and VLA-4 on T cells. Dextran 40 in therapeutically achievable levels (2–32 mg/ml) reduced both constitutive and TNF-α-stimulated TK-1 adhesion to eEND.2. Selective preincubation of eEND.2 or TK-1 revealed that Dextran 40 acted exclusively on the T cells. To explore further the mechanisms by which Dextran 40 interfered with TK-1 adhesion, their LFA-1 and VLA-4 expression was analysed by FACS. The surface expression levels of neither receptor were affected by Dextran 40. However, confocal microscopy revealed that Dextran 40 interfered with the activation-dependent capping and clustering of LFA-1 and VLA-4 on the surface of TK-1. We conclude that Dextran 40 inhibits the capacity of TK-1 T cells to adhere to eEND.2 endothelial cells and thus may be useful for therapeutic intervention in diseases associated with enhanced T lymphocyte binding to microvascular endothelium.
Anti-inflammatory effects of a new tumour necrosis factor-alpha (TNF-α) inhibitor (CNI-1493) in collagen-induced arthritis (CIA) in rats
Authors: ÅKERLUND K , ERLANDSSON HARRIS H , TRACEY K J , WANG H , FEHNIGER T , KLARESKOG L , ANDERSSON J , ANDERSSON U
Published: March 2003

A recently developed compound, a multivalent guanylhydrazone (CNI-1493) that inhibits TNF-α production by suppressing TNF-α translational efficiency, was administered in an experimental model of collagen type II-induced arthritis in DA rats. CNI-1493 was injected daily intraperitoneally either before the onset of arthritis or after the establishment of clinical disease. Prophylactic treatment with CNI-1493 significantly prevented or delayed the onset and suppressed the severity of arthritis in a dose-dependent manner. Therapeutic intervention with CNI-1493 in established joint disease also resulted in a significant reduction of clinical signs of arthritis in treated animals. No severe side-effects were noted when animals were treated with daily CNI-1493 doses up to 5 mg/kg. An immunohistochemical study was performed which demonstrated that CNI-1493 led to a reduced expression of TNF-α at the site of disease activity. Thus, CNI-1493 with documented inhibitory effects on TNF-α synthesis, has proven successful in ameliorating the course of arthritis in CIA. We believe that the use of a compound such as CNI-1493 with a defined mode of action provides a useful tool for dissecting and understanding important pathogenic mechanisms operating in the development of chronic arthritis.
Linomide suppresses experimental autoimmune neuritis in Lewis rats by inhibiting myelin antigen-reactive T and B cell responses
Authors: ZHU J , BAI X F , HEDLUND G , BJÖRK J , BAKHIET M , VAN DER MEIDE P H , LINK H
Published: March 2003

Linomide (quinoline-3-carboxamide) is a synthetic immunomodulator that suppresses several experimental autoimmune diseases. Here we report the effects of Linomide on experimental autoimmune neuritis (EAN), a CD4+ T cell-mediated animal model of acute Guillain–Barré syndrome (GBS) in humans. EAN induced in Lewis rats by inoculation with bovine peripheral nervous system (PNS) myelin and Freund's complete adjuvant was strongly suppressed by Linomide administered daily subcutaneously from the day of inoculation. Linomide dose-dependently delayed the interval between immunization and onset of clinical EAN, as well as the severity of EAN symptoms. These clinical effects were associated with dose-dependent down-modulation of PNS antigen-induced T and B cell responses and with suppression of the proinflammatory cytokines IL-12, interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) mRNA. In PNS sections, Linomide suppressed IL-12 and TNF-α, and up-regulated IL-10 mRNA expression. These findings suggest that Linomide could be useful in certain T cell-dependent autoimmune diseases.
Complement depletion aggravatesStaphylococcus aureussepticaemia and septic arthritis
Authors: SAKINIENE E , BREMELL T , TARKOWSKI A
Published: March 2003

The aim of the study was to assess the role of the complement system in Staphylococcus aureus arthritis and septicaemia. The murine model of haematogenously acquired septic arthritis was used, injecting intravenously toxic shock syndrome toxin-1 (TSST-1), producing S. aureus LS-1. Complement was depleted using cobra venom factor (CVF). Evaluation of arthritis was performed clinically and histopathologically. In addition, the effect of complement depletion on the phagocytic activity of leucocytes was assessed in vivo and in vitro. Six days after inoculation of S. aureus the prevalence of arthritis in decomplemented mice was three-fold higher than that in controls (91% versus 25%). The clinical severity of arthritis at the end of the experiment, expressed as arthritic index, was 7.3 and 1.9, respectively. These findings were confirmed by histological index of synovitis as well as of cartilage and/or bone destruction being significantly higher in decomplemented mice than in controls (9.8 ± 1.7 versus 4.9 ± 1.2, P < 0.05; and 7.9 ± 1.7 versus 3.0 ± 0.9, P < 0.05, respectively). Also, the septicaemia-induced mortality was clearly higher in decomplemented mice compared with the controls. CVF treatment significantly reduced in vivo polymorphonuclear cell-dependent inflammation induced by subcutaneous injection of olive oil and mirroring the capacity of polymorphonuclear cells (PMNC) to migrate and/or extravasate. Besides, the decomplementation procedure significantly impaired phagocytic activity of peripheral blood leucocytes in vitro, since the number of phagocytes being able to ingest bacteria decreased by 50% when the cells were maintained in decomplemented serum compared with those in intact serum. The conclusion is that complement depletion aggravates the clinical course of S. aureus arthritis and septicaemia, possibly by a combination of decreased migration/extravasation of PMNC and an impairment of phagocytosis.
Cyclooxygenase-1 and -2 are expressed by human T cells
Authors: PABLOS J L , SANTIAGO B , CARREIRA P E , GALINDO M , GOMEZ-REINO J J
Published: March 2003

In vitro, prostaglandins (PG) have strong inhibitory effects on T cell activation and proliferation and inhibitors of PG synthesis (NSAID) increase proliferation and activation of T cells. Although most studies have failed to demonstrate cyclooxygenase (COX) activity in lymphocytes, there is contradictory evidence on the synthesis of different PG. We have studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot the expression of COX-1 and -2 mRNA and protein in resting and activated peripheral blood or Jurkat T cells. Cells were activated by T cell receptor triggering with OKT3 antibodies and activation confirmed by flow cytometric analysis of surface CD69. COX enzymatic activity was measured by determination of arachidonic acid (AA)-induced PG synthesis. Both peripheral blood and Jurkat T cells expressed COX-1 and -2 mRNA and protein. COX-1 was constitutively expressed and did not change after OKT3 stimulation. COX-2 was inducible upon OKT3-induced activation. In spite of the presence of COX mRNA and immunoreactive protein, AA-induced PG synthesis was not detected at the EIA detection (p m) level. The potential role of cyclooxygenases in T cells deserves further study, since no PG of the studied series seem to be synthesized by T cells.
Antibodies to adult human endothelial cells cross-react with oxidized low-density lipoprotein and β2-glycoprotein I (β2-GPI) in systemic lupus erythematosus
Authors: WU R , SVENUNGSSON E , GUNNARSSON I , HAEGERSTRAND-GILLIS C , ANDERSSON B , LUNDBERG I , ELINDER L S , FROSTEGÅRD J
Published: March 2003

Cardiovascular manifestations are common in systemic lupus erythematosus (SLE). Oxidized low-density lipoprotein (oxLDL) is implicated in cardiovascular disease, especially atherosclerosis, and cross-reacts with antibodies to cardiolipin (aCL). β2-GPI is a plasma protein participating in the coagulating cascade, and is also cofactor for aCL, and some aCL have been shown to be directed against β2-GPI and/or complexes between β2-GPI and phospholipids. Lysophosphatidylcholine (LPC) is a phospholipid present both in oxLDL and in damaged endothelium, and we recently showed that LPC is involved in the antigenicity of oxLDL. Antibodies to endothelial cells (aEC) correlate with disease activity in SLE and vasculitis, and we recently showed that aEC are enhanced in cardiovascular diseases such as borderline hypertension and early atherosclerosis. aEC were determined using EC from adult V. Saphena Magna. Antibody levels were determined by ELISA. aEC of IgG type were enhanced in 184 patients with SLE compared with 85 healthy controls. There was a close correlation between aoxLDL, aCL, aLPC, aβ2-GPI and aEC. Binding of sera to EC was competitively inhibited by β2-GPI, LPC and oxLDL. Taken together, the data indicate that EC share antigenic epitopes with β2-GPI and with oxLDL, especially LPC. Phospholipids in EC membranes may thus be antigenic epitopes. β2-GPI may bind to these phospholipids, and become an autoantigen. LPC is formed by oxidation of phospholipids and/or proinflammatory factors leading to activation of phospholipase A2, and the findings indicate the potential role of both lipid oxidation and phospholipase A2 in SLE.
Alzheimer's β-amyloid peptides can activate the early components of complement classical pathway in a C1q-independent manner
Authors: BERGAMASCHINI L , CANZIANI S , BOTTASSO B , CUGNO M , BRAIDOTTI P , AGOSTONI A
Published: March 2003

β-Amyloid (β-A) accumulates in the brain of patients with Alzheimer's disease (AD) and is presumably involved in the pathogenesis of this disease, on account of its neurotoxicity and complement-activating ability. Although assembly of β-A in particular aggregates seems to be crucial, soluble non-fibrillar β-A may also be involved. Non-fibrillar β-A does not bind C1q, so we investigated alternative mechanisms of β-A-dependent complement activation in vitro. On incubation with normal human plasma, non-fibrillar β-A 1-42, and truncated peptide 1–28, induced dose-dependent activation of C1s and C4, sparing C3, as assessed by densitometric analysis of immunostained membrane after SDS–PAGE and Western blotting. The mechanism of C4 activation was not dependent on C1q, because non-fibrillar β-A can still activate C1s and C4 in plasma genetically deficient in C1q (C1qd). In Factor XII-deficient plasma (F.XIId) the amount of cleaved C4 was about 5–10% less that in C1qd and in normal EDTA plasma; the reconstitution of F.XIId plasma with physiologic concentrations of F.XII resulted in an increased (8–15%) β-A-dependent cleavage of C4. Thus our results indicate that the C1q-independent activation of C1 and C4 can be partially mediated by the activation products of contact system. Since the activation of contact system and of C4 leads to generation of several humoral inflammatory peptides, non-fibrillar β-A might play a role in initiating the early inflammatory reactions leading to a multistep cascade contributing to neuronal and clinical dysfunction of AD brain.
Are anti-neutrophil cytoplasmic antibodies (ANCA) clinically useful in inflammatory bowel disease (IBD)?
Authors: ROOZENDAAL C , KALLENBERG C G M
Published: March 2003

Since the first detection of ANCA in IBD, numerous studies have dealt with their prevalence, antigenic specificities, clinical significance, pathophysiological role, and their induction. This review summarizes the information obtained from those studies and shows that ANCA are not directly useful as diagnostic and prognostic factors in IBD. ANCA were detected in 50–85% of patients with ulcerative colitis (UC) and 10–20% of patients with Crohn's disease (CD). Multiple target antigens are recognized by these autoantibodies, including both cytoplasmic and nuclear proteins. A pathophysiological role for ANCA in IBD is far from clear. On the one hand, it is suggested that ANCA are genetic markers of susceptibility for IBD, and on the other hand, the induction of ANCA in those diseases may just be an epiphenomenon of chronic inflammation. We discuss recent evidence that ANCA may be induced by a break-through of tolerance towards bacterial antigens.
Presence of circulating abnormal CD34+ progenitors in adult Langerhans cell histiocytosis
Authors: MISERY L , ROUGIER N , CRESTANI B , FAURE M , CLAUDY A , SCHMITT D , VINCENT C
Published: March 2003

Langerhans cell histiocytosis (LCH) is related to the proliferation of cells, which are similar to Langerhans cells (LC) but possess many abnormal characteristics. Lesions are widespread and this fact suggests that LCH cells or their precursors are present in the blood of patients. In five adult patients, we have isolated and cultured CD34+ blood progenitors of dendritic cells. We studied their phenotype by flow cytometry and their functional properties in mixed culture with heterologous lymphocytes and with autologous lymphocytes in the presence of tri-nitro-phenyl antigen (TNP). The amount of CD34+ precursors was dramatically higher than controls but a high mortality occurred during the in vitro differentiation. The phenotype of surviving cells was similar to LC phenotype (CD1a+, CD83+, Lag+) but some of them expressed CD2. These cells were able to induce T cell proliferation in mixed culture. They could not initiate primary response to TNP, except in a patient treated with thalidomide. In our hands, these CD34+ cells may be precursors of LCH cells.
Sodium butyrate blocks interferon-gamma (IFN-γ)-induced biosynthesis of MHC class III gene products (complement C4 and factor B) in human fetal intestinal epithelial cells
Authors: Kitamura K , Andoh A , Inoue T , Amakata Y , Hodohara K , Fujiyama Y , Bamba T
Published: March 2003

Human intestinal epithelial cells have been established as local sites for complement biosynthesis. In this study, we investigated the effects of IFN-γ and sodium butyrate on biosynthesis of MHC class III gene products (complement C4 and factor B) in the human fetal intestinal epithelial cell line INT-407. IFN-γ induced a dose- and time-dependent increase in C4 and factor B secretion. However, sodium butyrate dose-dependently inhibited IFN-γ-induced C4 and factor B secretion. These effects were also observed at the mRNA level. Immunoblotting indicated that IFN-γ induced a rapid activation of Stat1α, and fluorescence immunohistochemistry detected a translocation of Stat1α into the nucleus within 1 h. However, the translocation of Stat1α was not affected by the addition of sodium butyrate. Nuclear run-on assay indicated that IFN-γ induced a weak increase in the transcription rate of factor B gene, and sodium butyrate did not affect this response. IFN-γ and sodium butyrate induced a counter-regulatory effect on C4 and factor B secretion: IFN-γ acted as a potent inducer, but sodium butyrate potently abrogated these responses. These are mainly regulated through the post-transcriptional mechanism.
Onchocerciasis modulates the immune response to mycobacterial antigens
Authors: Stewart G R , Boussinesq M , Coulson T , Elson L , Nutman T , Bradley J E
Published: March 2003

Chronic helminth infection induces a type-2 cellular immune response. In contrast to this, mycobacterial infections commonly induce a type-1 immune response which is considered protective. Type-2 responses and diminished type-1 responses to mycobacteria have been previously correlated with active infection states such as pulmonary tuberculosis and lepromatous leprosy. The present study examines the immune responses of children exposed to both the helminth parasite Onchocerca volvulus and the mycobacterial infections, Mycobacterium tuberculosis and M. leprae. Proliferation of peripheral blood mononuclear cells (PBMC) and production of IL-4 in response to both helminth and mycobacterial antigen (PPD) decreased dramatically with increasing microfilarial (MF) density. Although interferon-gamma (IFN-γ) production strongly correlated with cellular proliferation, it was surprisingly not related to MF density for either antigen. IL-4 production in response to helminth antigen and PPD increased with ascending children's age. IFN-γ and cellular proliferation to PPD were not related to age, but in response to helminth antigen were significantly higher in children of age 9–12 years than children of either the younger age group (5–8 years) or the older group (13–16 years). Thus, there was a MF density-related down-regulation of cellular responsiveness and age-related skewing toward type 2 which was paralleled in response to both the helminth antigen and PPD. This parasite-induced immunomodulation of the response to mycobacteria correlates with a previous report of doubled incidence of lepromatous leprosy in onchocerciasis hyperendemic regions. Moreover, this demonstration that helminth infection in humans can modulate the immune response to a concurrent infection or immunological challenge is of critical importance to future vaccination strategies.
Analysis of the secretion pattern of monocyte chemotactic protein-1 (MCP-1) and transforming growth factor-beta 2 (TGF-β2) by human retinal pigment epithelial cells
Authors: Holtkamp G M , De Vos A F , Peek R , Kijlsta A
Published: March 2003

Retinal pigment epithelial (RPE) cells, situated between the neurosensory retina and the vascularized choroid, form part of the blood–eye barrier and are important for homeostasis of the outer retina. These cells are able to produce a variety of cytokines which may play a role in the maintenance of the immunosuppressive milieu inside the eye and in intraocular inflammatory responses. In the present study, we investigated whether RPE cells secreted the anti-inflammatory cytokine TGF-β2 and the proinflammatory cytokine MCP-1 in a polarized manner. Monolayers of human donor RPE cells were cultured on transwell filters. Secretion of TGF-β2 and MCP-1 at either the apical or basal side of the RPE cell monolayers, that were not treated or stimulated with IL-1β (200 U/ml), was analysed by ELISA. All three cell lines examined had a different TGF-β2 secretion pattern. In two of the three donor RPE cell lines tested, TGF-β2 secretion was polarized, but not in the same direction. TGF-β2 secretion was not up-regulated by stimulation with IL-1β. In contrast, IL-1β strongly induced MCP-1 secretion preferentially into the basal compartment of all RPE monolayers tested. These data indicate that human RPE cells are able to secrete TGF-β2 and MCP-1 in a polarized fashion. Our results suggest that MCP-1 can be secreted by RPE cells in the direction of choroidal vessels during inflammatory responses in the posterior part of the eye, which may limit damage to the neurosensory retina.
Externalization of tropomyosin isoform 5 in colon epithelial cells
Authors: Kesari K V , Yoshizaki N , Geng X , Lin J J-C , Das K M
Published: March 2003

Ulcerative colitis (UC) is associated with autoantibody response to a cytoskeletal protein, human tropomyosin (hTM) isoform-5 (hTM5). Because hTM5 is an intracellular protein, it may remain inaccessible to the autoantibodies. Therefore, we have investigated the possibility of externalization of hTM5 in colon epithelial cells. Freshly isolated colonic and small intestinal epithelial cells and LS-180 colon cancer cell line were examined for surface expression of hTM5 by flow cytometric analysis using hTM isoform-specific MoAbs. The extracellular release of hTM5 was determined by Western blot and radioimmunoprecipitation analyses. Physical association of hTM5 with a membrane-associated colon epithelial protein (CEP) was examined by co-immunoprecipitation of hTM5 with anti-CEP MoAb, and CEP with anti-hTM5 MoAb. Cell surface expression of hTM5 was observed in colonic epithelial and LS-180 cells but not in small intestinal epithelial cells. LS-180 cells spontaneously released hTM5 as well as CEP into the culture medium that was significantly stimulated by a calcium ionophore, A23187, but inhibited by phorbol-12-myristate-13-acetate, monensin and methylamine. Co-immunoprecipitation experiments revealed that hTM5 forms a complex with CEP. We conclude that hTM5 is externalized in colon but not in small intestinal epithelial cells. The physical association of hTM5 with CEP suggests a possible chaperone function of CEP in the transport of hTM5, a putative target autoantigen in UC.
Molecular determinants of polyreactive antibody binding: HCDR3 and cyclic peptides
Authors: Deng Y-J , Notkins A L
Published: March 2003

Human monoclonal antibody 63 (mAb63) is an IgM/λ polyreactive antibody that binds to multiple self and non-self antigens. The molecular basis of polyreactivity is still unclear. The present study was initiated to prepare a recombinant Fab of mAb63 and use it to study the determinants involved in polyreactivity. The baculovirus system was employed to express large amounts of mAb63 Fab in Sf9 cells. Our experiments showed that infected Sf9 cells secreted a soluble 50-kD Fab heterodimer that bound to multiple self and non-self antigens. The antigen-binding activity of mAb63 Fab was inhibited by both homologous and heterologous antigens. To study in more detail the molecular determinants involved in polyreactivity, the heavy chain complementarity-determining region 3 (HCDR3), which is known to play a key role in the binding of monoreactive antibodies to antigens, was subjected to site-directed mutagenesis. A single substitution, alanine for arginine, at position 100A resulted in complete loss of antigen-binding activity. The 19 amino acids comprising the HCDR3 of mAb63 were then synthesized and a cyclic peptide prepared. The cyclic peptide showed the same antigen-binding pattern as the parental mAb63 and the recombinant mAb63 Fab. A five amino acid motif (RFLEW), present in the HCDR3 of mAb63, was found by searching the GenBank in three of 50 other human polyreactive antibodies, but in none of nearly 2500 human antibodies thought to be monoreactive. It is concluded that HCDR3 plays a major role in polyreactivity and that in some cases cyclic peptides comprising the HCDR3, by themselves, may be polyreactive.
Interferon-alpha (IFN-α) stimulates anti-melanoma cytotoxic T lymphocyte (CTL) generation in mixed lymphocyte tumour cultures (MLTC)
Authors: Palmer K J , Harries M , Gore M E , Collins M K L
Published: March 2003

IFN-α administration after primary tumour resection improves the survival of melanoma patients at high risk of relapse. To investigate whether this response might be due to stimulation of anti-tumour immunity, the effect of IFN-α on anti-melanoma CTL generation in MLTC was measured. IFN-α increased both allogeneic and autologous anti-melanoma CTL generation from peripheral blood lymphocytes stimulated with irradiated primary melanoma cultures. IFN-α up-regulated MHC class I expression on primary melanoma cultures, whereas IFN-γ up-regulated both MHC class I and II expression. However, the effect of IFN-α on anti-melanoma CTL generation was often more potent than that of IFN-γ, equalling the effect of the optimal combination of IL-2 and IL-12. Pre-treatment of primary melanoma cultures with IFN-γ was sufficient for CTL generation in MLTC, whereas IFN-α needed to be present during the MLTC. While direct anti-proliferative effects of IFN-α on some tumour cells have been described, IFN-α did not inhibit proliferation of primary melanoma cultures. These results suggest that the clinical effects of IFN-α in melanoma patients may be immune-mediated.
Effect of IL-5, glucocorticoid, and Fas ligation on Bcl-2 homologue expression and caspase activation in circulating human eosinophils
Authors: Zangrilli J , Robertson N , Shetty A , Wu J , Hastie A , Fish J E , Litwack G , Peters S P
Published: March 2003

IL-5 is a potent eosinophil viability-enhancing factor that has been strongly implicated in the pathogenesis of IgE-mediated inflammation in vivo. Recently published data have suggested that IL-5 (and related cytokines) may act by altering the expression of the anti-apoptotic regulator Bcl-2 or its homologues, but this is controversial. The behaviour of the recently described pro-apoptotic cysteine proteases (caspases) in eosinophils after IL-5 treatment has not been explored. We examined the effect of IL-5 on the expression of four major Bcl-2 homologues, as well as on the expression/activation of key members of the caspase cell death cascade in cultured circulating human eosinophils. The effect of relevant inducers of eosinophil apoptosis (glucocorticoid and Fas ligation) on these regulatory proteins was also examined. We observed baseline expression of the anti-apoptotic Mcl-1 and pro-apoptotic Bax proteins in immunoblots of eosinophil lysates, but not Bcl-x, Bcl-2. IL-5 treatment had the effect of maintaining this basal level of expression over time without altering the balance of Bcl-2 homologues. The (upstream) caspase 8 and (downstream) caspase 3 proenzymes were detected in eosinophils at baseline, and were processed during spontaneous and stimulated eosinophil death. IL-5 completely blocked caspase processing in spontaneous and dexamethasone-induced cell death, and significantly slowed processing during Fas ligation. Our data do not support the theory that IL-5 acts by altering the balance of anti-apoptotic and pro-apoptotic Bcl-2 homologues, but suggest that it may act by regulating activation of the caspase cell death cascade.
Immunohistochemical study of lymphoid tissues in adjuvant arthritis (AA) by image analysis; relationship with synovial lesions
Authors: Carol M , Pelegrí C , Castellote C , Franch A , Castell M
Published: March 2003

The aim of this study was to examine leucocyte populations in lymphoid organs during AA and to ascertain the relationship with lesions in synovial joints. Popliteal lymph nodes, spleen and knee synovial membranes were removed from both healthy and AA rats at intervals of 3–4 days over a 3-week period. Cryostat sections were stained with MoAbs directed against lymphocyte and macrophage subpopulations, and studied by image analysis. Throughout the arthritic period, high numbers of ED1+ and ED3+ macrophages were seen in both lymphoid compartments and intercellular adhesion molecule-1 (ICAM-1) expression also increased in some zones of lymph nodes and spleen. The percentages of CD4+ and CD8+ cells rose in the splenic zones studied but fell in the lymph node cortex. Very few natural killer (NK) cells were found in lymphoid tissues, but the number rose after AA induction. In synovia from AA rats, ED2+ macrophages proliferated but α/β T cell infiltration was only occasionally observed, accompanied by ED1+ cells and ICAM-1 expression. In conclusion, synovitis developing after AA induction seems to be caused directly by macrophages and indirectly by lymphocytes placed both in popliteal lymph nodes and spleen.
Enhanced apoptosis of T cells in common variable immunodeficiency (CVID): role of defective CD28 co-stimulation
Authors: Di Renzo M , Zhou Z , George I , Becker K , Cunningham-Rundles C
Published: March 2003

CVID is a primary immune disorder in which hypogammaglobulinaemia may be associated with a number of T cell defects including lymphopenia, anergy, impaired lymphocyte proliferation and deficient cytokine secretion. In this study we show that T cells of CVID subjects, in comparison with control T cells, undergo spontaneous apoptosis in culture and markedly accelerated apoptosis after γ-irradiation. Although costimulation of the CD28 receptor following engagement of the TCR/CD3 receptor normally provides a second signal necessary for IL-2 secretion, CD28 costimulation in CVID does not significantly increase IL-2 production, nor does this combination of activators enhance the survival of irradiated CVID T cells, as it does for cultured normal T cells. Addition of IL-2 enhances CVID T cell survival, suggesting that the IL-2 signalling pathways are normal. CVID T cells have similar expression of Bcl-2 to control T cells. CD3 stimulation up-regulates T cell expression of bcl-xL mRNA for normal T cells, but anti-CD28 does not augment bcl-xL expression for CVID subjects with accelerated apoptosis. Defects of the CD28 receptor pathway, leading to cytokine deprivation and dysregulation of bcl-xL, could lead to poor T cell viability and some of the cellular defects observed in CVID.
Characterization of mercuric mercury (Hg2+)-induced lymphoblasts from patients with mercury allergy and from healthy subjects
Authors: Cederbrant K , Hultman P
Published: March 2003

Hg2+ induces lymphocyte proliferation when added to cell cultures from both healthy and mercury-allergic subjects. Consequently, when measuring DNA synthesis a possible Hg2+-specific response, resulting from proliferating memory cells, cannot be discriminated from a non-allergic response. The mechanism behind this non-allergic response is unknown but a superantigenic effect of Hg2+ has been suggested. In this study, five mercury-allergic patients, with oral lichen planus (OLP) lesions adjacent to dental amalgam and a positive patch test to Hg0, and five healthy subjects without amalgam were examined. The immunophenotype and the T cell receptor Vβ (TCR Vβ) repertoire of Hg2+-induced lymphoblasts as well as the expression of the lymphocyte activation markers CD23 and CD134 were analysed for possible differences between healthy and allergic subjects. The mechanism of Hg2+-induced proliferation was examined by comparing the TCR Vβ expression of Hg- and staphylococcal enterotoxin B (SEB)-activated lymphoblasts, the latter used as a positive superantigen control. It was not possible to discriminate between mercury-allergic and healthy subjects using the immunophenotype or the TCR Vβ profile of the Hg2+-induced lymphoblasts or the expression of CD23 and CD134. However, Hg2+-induced CD4+ lymphoblasts showed a skewing towards Vβ2. This relative increase in Vβ2 was only detected in the CD4+ but not in the CD8+ lymphoblast population. In conclusion, Hg2+ induced a proliferation-dependent skewing towards CD4+ but not CD8+ lymphocytes expressing Vβ2. In this respect Hg2+ differs from the classical bacterial superantigen SEB, which also stimulates unique TCR Vβ families among CD8+ cells.
Islet xenograft destruction in the hu-PBL-severe combined immunodeficient (SCID) mouse necessitates anti-CD3 preactivation of human immune cells
Authors: Gysemans C , Waer M , Laureys J , Depovere J , Pipeleers D , Bouillon R , Mathieu C
Published: March 2003

Introduction of the hu-PBL-SCID mouse model has yielded a potentially useful tool for research in transplantation. The aim of this study was to define the conditions necessary for a reconstituted human immune system to destroy in a consistent manner rat islet xenografts in the alloxan-diabetic hu-PBL-SCID mouse. We examined different time points of hu-PBL reconstitution, different transplantation sites of the islets and several hu-PBL reconstitution protocols. Major differences in graft destruction were observed between the different hu-PBL reconstitution protocols, irrespective of timing of hu-PBL reconstitution or site of transplantation. Although preactivation of hu-PBL did not improve the level of hu-PBL chimerism, histological and immunohistochemical analysis of the grafts revealed a severe human lymphocytic infiltration and β cell destruction only in the grafts of mice receiving preactivated hu-PBL. This β cell injury resulted in impaired glucose tolerance, with in some animals recurrence of hyperglycaemia, and decreased insulin and C-peptide levels after glucose stimulation. Therefore, we conclude that activation of hu-PBL prior to transfer is essential in achieving xenograft infiltration and destruction in hu-PBL-SCID mice. The need for immune manipulation suggests that interactions between hu-PBL and xenografts in this model may be hampered by incompatibilities in cross-species adhesion and/or activation signals.
Expression of cyclooxygenase-1 (COX-1) in labial salivary glands of Sjögren's syndrome
Authors: Tominaga M , Migita K , Sano H , Fukui W , Kohno M , Tsubouchi Y , Honda S , Fukuda T , Nakamura H , Yamasaki S , Kawabe Y , Kawakami A , Eguchi K
Published: March 2003

COX plays an important role in inflammatory diseases such as rheumatoid arthritis. To determine the role of COX in Sjögren's syndrome (SS), we examined COX expression in the salivary glands of SS patients. We examined 15 patients with SS and two normal subjects. Labial salivary gland tissue samples were analysed immunohistochemically using anti-COX-1 and COX-2 antibodies. All biopsy samples from 15 patients with SS were stained for COX-1. In contrast, COX-1 immunostaining was not detected in normal salivary gland tissues. Co-expression of COX-1 and CD68 was confirmed by mirror section technique and double antibody immunostaining. This finding indicated that COX-1-expressing cells in SS salivary glands were infiltrating macrophages. In contrast to COX-1 staining, only a little COX-2 immunostaining was observed in salivary gland tissues from SS patients. These data suggest that COX-1 expression on infiltrating macrophages may contribute to the inflammatory process of salivary glands in SS.
Antigen-induced IL-17 response in the peripheral blood mononuclear cells (PBMC) of healthy controls
Authors: Lenarczyk A , Helsloot J , Farmer K , Peters L , Sturgess A , Kirkham B
Published: March 2003

IL-17 is a T cell cytokine with a complex and important role in the immune system. It has been detected in rheumatoid arthritis (RA) synovial membrane and found to stimulate the production of the proinflammatory cytokines IL-6, IL-8, tumour necrosis factor-alpha (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro. To date, there are few data available on the agents that stimulate IL-17 production. We therefore investigated the in vitro IL-17 response to a variety of mitogens and antigens, and compared the IL-17 response to interferon-gamma (IFN-γ), IL-4, IL-10 and TNF-α. In this study we used a type-0 antigen, tetanus toxoid (TT), a type-1 antigen, PPD from Mycobacterium tuberculosis, a potential type-2 rye grass (RG) antigen (Lol I) and an autoantigen SS.B (La), to stimulate PBMC from healthy controls. Cytokine mRNA was measured using semiquantitative reverse transcriptase-polymerase chain reaction and cytokine protein measured using specific ELISA techniques, while the frequency of IL-17-producing T cells was determined by flow cytometry. The mitogens concanavalin A, phytohaemagglutinin and phorbol myristate acetate/ionomycin induced a significant increase in IL-17, with the highest levels being produced by anti-CD3/anti-CD28 stimulation. The antigens TT and PPD significantly increased IL-17 mRNA expression over time, but failed to have such an effect at the protein level. IL-17 protein was also detectable in both antigen-specific (TT, SS.B) and non-specific T cell clones, but at levels lower than IFN-γ. IL-17 production did not correlate with either the type-1 cytokine IFN-γ or TNF-α or the type-2 cytokine IL-4 or IL-10 at either the mRNA or protein level.
Efficient generation of respiratory syncytial virus (RSV)-neutralizing human MoAbs via human peripheral blood lymphocyte (hu-PBL)-SCID mice and scFv phage display libraries
Authors: Nguyen H , Hay J , Mazzulli T , Gallinger S , Sandhu J , Teng Y-T A , Hozumi N
Published: March 2003

RSV is one of the major causes of pneumonia and bronchiolitis in infants and young children and is associated with high mortality. RSV neutralizing human antibody (hu-Ab) is known to mediate resistance to viral infection as well as to be an effective treatment for severe lower respiratory tract RSV infection. We have previously demonstrated that human primary and secondary immune responses can be established in severe combined immunodeficient mice engrafted with human peripheral blood lymphocytes (hu-PBL-SCID). By combining this animal model with the single-chain Fv antibody (scFv) phage display library technique, we were able to investigate further its clinical potential by generating a panel of human scFvs that exhibit both high F glycoprotein (RSV-F) binding affinities (∼108 M−1) and strong neutralizing activities against RSV infection in vitro. Sequencing analysis of the randomly isolated anti-RSV-F scFv clones revealed that they were derived from different VH families with mutations in the complementarity-determining region 1 (CDR1). The results suggest that: (i) RSV-F-specific human immune responses and affinity maturation can be induced in hu-PBL-SCID mice; and (ii) this approach can be applied to generate large numbers of human scFvs with therapeutic potential. Despite the fact that hu-PBL-SCID mouse and human scFv phage display library have individually been established, our approach contributes a simple and significant step toward the generalization of antigen-specific human monoclonal antibody (hu-MoAb) production and their clinical applications.