bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary pulmonary hypertension: immunogenetic response to high-mobility group (HMG) proteins and histone
Authors: Morse J H , Barst R J , Fotino M , Zhang Y , Flaster E , Fritzler M J
Published: November 2003

HLA class II alleles (DNA typing) and antibodies to HMG-1,2,14,17 proteins and H1 histone were determined in three predominantly Caucasian groups of patients with pulmonary hypertension (PHT). Forty-four adults had primary pulmonary hypertension (PPH), 42 children had PPH, and 41 children had PHT associated with anatomically large congenital pulmonary to systemic shunts (PHT+shunt). The HLA class II alleles in the Caucasian patients were compared with those of 51 healthy Caucasian controls. Eight (18%) of 44 sera from adults with PPH bound HMG-14 and 23 (52%) bound H1. None of 42 sera from children with PPH bound either HMG-14/17 or HMG-1/2, whereas four (10%) bound H1. In the PHT+shunt group of 41 children, two (5%) bound HMG-14, one (3%) bound HMG-17, four (10%) bound HMG-1 and/or HMG-2, and six (15%) bound H1. Among the 12 HMG antibody-positive patients, HLA-DQ6 was present in nine of 10 HLA typed patients (six PPH adults and three PHT+shunt children), seven of whom had antibodies to HMG-14 and one to HMG-17. The 100% frequency of HLA-DQ6 in seven Caucasian patients with antibodies to HMG-14/17 was statistically significant when compared with the 41% frequency of -DQ6 present in 51 healthy Caucasian controls (pc = 0.027, pc = Bonferroni correction, OR = 21.3). In contrast, when compared with controls, 25 patients with PPH and anti-H1 antibodies (21 adults and four children) had increased frequencies of HLA-DQ7 and -DR5 (60% versus 29%, P = 0.010, OR = 3.6 and 48% versus 22%, P = 0.018, OR = 3.4), which were not significant after correction. In essence, antibodies to HMG-14 and to H1 proteins were present predominantly in adults with PPH, suggesting that the pattern of response to HMG-14/17 was similar to that previously reported in systemic lupus erythematosus (SLE) and drug-induced autoimmunity. This is the first report of an association between autoantibodies directed against HMG and H1 with immunogenetic markers. These data suggest that a subset of patients with PPH may have an autoimmune disease.
Different Gm allotype amounts in human intravenous immunoglobulin (IVIG) preparations; survival of foreign Gm allotypes in immunodeficient patients
Authors: Oxelius V-A , Eibl M M
Published: November 2003

IVIG is used as standard replacement therapy in primary antibody deficiency. IVIG consists mainly of IgG. IVIG preparations were investigated with respect to Gm allotypes, which are characterized by various amino acid epitopes in the constant heavy chains of the IgG subclasses IgG1, IgG2 and IgG3. The alternative allelic Gm allotypes G1m(a) and G1m(f) of IgG1, G2m(n) and G2m(”) of IgG2 and G3m(g) and G3m(b) of IgG3 were measured by sensitive competitive ELISAs for G1m(a), G1m(f), G2m(n) and G3m(b). IgG subclass levels were quantified by radioimmunodiffusion (RID). Gm allotype quantities differed significantly in various IVIG products, with different products having half or double the amount of the different Gm allotypes. The results show the effect of the different manufacturing processes, but also indicate different physicochemical properties of Gm allotypes within the same IgG subclass. The different contents of Gm allotypes might be one reason for the variable levels of specific antibodies found in IVIG products. Immunodeficient patients with homozygous expression of Gm allotypes from IGHCG1, IGHCG2 and IGHCG3 were tested after infusion of foreign Gm allotypes. A prolonged survival was found for the G2m allotype, G2m(n), compared with G1m allotypes. Different half-lives were found for the alternative G1m(a) and G1m(f) allotypes, within the same IgG1 subclass.
Characterization of the antigenic determinants of the Leishmania infantum histone H3 recognized by antibodies elicited during canine visceral leishmaniasis
Authors: Soto M , Requena J M , Quijada L , Gomez L C , Guzman F , Patarroyo M E , Alonso C
Published: November 2003

In the present study we show that sera from dogs naturally infected with the protozoan parasite Leishmania infantum contain antibodies that specifically react with the parasite histone H3. Using synthetic peptides covering the complete sequence of the protein we located the linear antigenic determinants within the 40 amino-terminal amino acids of the molecule. In addition to the complete form of the protein (rLiH3), two regions of the Leishmania histone H3 were expressed as recombinant proteins: the rLiH3-Nt fragment containing the 39 amino-terminal amino acids and the rLiH3-Ct fragment containing the 90 carboxyl-terminal residues. Competition experiments using the protein fragment rLiH3-Nt as competitor confirmed that the antigenic determinants of histone H3 are confined to the amino-terminal domain. This domain, which is believed to be exposed on the nucleosome surface, is also the most evolutionarily divergent region of the L. infantum histone H3. Visceral leishmaniasis (VL) sera do not react with mammalian histones, an indication that the anti-histone response elicited during Leishmania infection is triggered by the parasite histone. The results of the prevalence of anti-histone H3 antibodies in canine VL sera together with the sequence-specific characteristics of the amino-terminal region of L. infantum histone H3 indicate that the recombinant protein rLiH3-Nt may be of use for diagnosis of canine VL.
The CD7− T cell subset represents the majority of IL-5-secreting cells within CD4+CD45RA− T cells
Authors: Reinhold U , Liu L , Sesterhenn J , Schnautz S , Abken H
Published: November 2003

Absence of CD7 is a stable phenotype in a subset of normal human T cells. Most circulating CD7− T cells express the CD4+CD45RO+CD45RA− memory phenotype. We analysed CD4+CD45RA− peripheral blood lymphocytes that were separated into CD7+ and CD7− for their in vitro cytokine secretion in response to different stimuli. The CD4+CD7− subpopulation was found to secrete significantly higher levels of IL-5 compared with the CD4+CD7+ subset upon stimulation with ionomycin/phorbol myristate acetate (PMA) plus anti-CD28 MoAbs. In contrast to IL-5 secretion, IL-4 and interferon-gamma (IFN-γ) secretion was not significantly different in CD7+ and CD7− T cells upon stimulation in vitro. The data indicate that the CD4+CD7− T cell represents the majority of IL-5-secreting cells within the population of CD4+CD45RA− memory T cells. Since CD4+CD7− T cells were found to be enriched in various skin lesions associated with eosinophilic infiltration, the results of our study support the hypothesis that skin-infiltrating CD7− T cells are one of the major sources of IL-5 responsible for the development of eosinophilic inflammation in certain skin diseases.
A therapeutic human IgG4 monoclonal antibody that depletes target cells in humans
Authors: Isaacs J D , Wing M G , Greenwood J D , Hazleman B L , Hale G , Waldmann H
Published: November 2003

It is traditionally held that human IgG4 MoAbs should not deplete target cells in vivo, as this isotype is inactive in a number of in vitro assays that measure effector function. We have previously challenged this dogma, and the current study was designed to investigate the in vivo biological effects in humans of a MoAb of human IgG4 isotype. Nine patients with refractory rheumatoid arthritis (RA) fulfilling ARA criteria, and one with ankylosing spondylitis (AS) received a human IgG4 Campath-1 MoAb (with specificity against the pan-lymphocyte antigen CD52) as part of a two-stage therapeutic protocol. In stage 1, patients received a single dose of this MoAb. Stage 2, starting 48 h later, comprised a 5-day course of a human IgG1 Campath-1 MoAb with identical V-region (CAMPATH-1H), as previously used in the management of RA patients. The intervening 48 h provided a window of opportunity to monitor the biological effects of the IgG4 MoAb for comparison with the IgG1. The two MoAbs were also compared for in vitro biological activity. IgG4 depleted peripheral blood lymphocytes (PBL), albeit less efficiently than IgG1. It produced a first-dose reaction of similar intensity, although associated circulating tumour necrosis factor-alpha (TNF-α) levels were lower. TNF-α release from whole blood in vitro was also greater with the IgG1 MoAb. The study design did not permit conclusions to be drawn regarding therapeutic efficacy of the IgG4 MoAb. In summary, a human IgG4 Campath-1 MoAb depletes target cells in vivo. Importantly, this study demonstrates for the first time in humans that in vitro assays may not predict the in vivo effector function of therapeutic MoAbs.
Responsiveness of human polymorphonuclear cells (PMNL) to stimulation by a mannoprotein fraction (MP-F2) of Candida albicans; enhanced production of IL-6 and tumour necrosis factor-alpha (TNF-α) by MP-F2-stimulated PMNL from HIV-infected subjects
Authors: TOROSANTUCCI A , CHIANI P , QUINTI I , AUSIELLO C M , MEZZAROMA I , CASSONE A
Published: November 2003

IL-1β, IL-6, IL-8 and TNF-α production by PMNL from 21 HIV-infected (HIV+), including 11 full-blown AIDS, and 20 HIV-uninfected (HIV−) subjects (matched for age and sex to HIV+ ones) was studied by reverse transcriptase-polymerase chain reaction (RT-PCR) and ELISA. PMNL from both categories of subjects were strongly stimulated in their actual cytokine production by a mannoprotein fraction (MP-F2) of Candida albicans, as well as by the bacterial lipopolysaccharide (LPS). These stimulatory effects were apparently due to increased cytokine gene expression and were substantially reversed by the physiological inhibitor IL-10. However, PMNL from HIV+ subjects showed increased IL-6 and TNF-α gene expression and produced more IL-6 and TNF-α than PMNL from HIV− controls, under similar stimulation conditions. This difference could not be attributed to a given stage of HIV infection, any associated medication, or to a generalized increase of gene expression, as quantitatively similar β-actin and IL-1β transcripts were detected. Moreover, no significant difference in IL-8 production by the PMNL from HIV+ and HIV− subjects was observed. Our studies suggest that PMNL from HIV+ subjects might add to other cellular sources of IL-6 and TNF-α (e.g. monocytes-macrophages) in contributing to the cytokine-dysregulated pattern typical of the HIV+ patient.
T cell responses to natural human proteins in primary biliary cirrhosis
Authors: JONES D E J , PALMER J M , YEAMAN S J , BASSENDINE M F , DIAMOND A G
Published: November 2003

The study of T cell responses to autoantigens in human autoimmunity has been hampered by difficulties, firstly in identifying significant autoantigens, and secondly in the purification of authentic human proteins in sufficient quantities to allow characterization of antigen-specific T cell responses. In this study we have purified a human autoantigen, pyruvate dehydrogenase, retaining its enzymatic activity, and characterized autoreactive T cell responses to it in a human autoimmune disease, primary biliary cirrhosis. T cell responses to a mixture of the E2 and protein X subunits of human pyruvate dehydrogenase complex are seen in most affected patients, but in only a small minority of normal and chronic liver disease controls. By contrast, responses to whole pyruvate dehydrogenase complex occur with equal frequency in both groups. This suggests that responses to the E2 component/protein X of pyruvate dehydrogenase complex play a role in the pathogenesis of primary biliary cirrhosis. The availability of significant quantities of the human autoantigen in primary biliary cirrhosis makes this condition an interesting model in which to study true autoreactive human T cell responses.
Cytokine production in synovial tissue of mice with collagen-induced arthritis (CIA)
Authors: MÜSSENER Å , LITTON M J , LINDROOS E , KLARESKOG L
Published: November 2003

The kinetics of cytokine production in arthritic limbs of mice with CIA was determined by using modified immunohistochemical techniques. Tissue cryostat sections of undecalcified whole paws were analysed for the presence of tumour necrosis factor-alpha (TNF-α), IL-6, IL-2, IL-4, IL-5 interferon-gamma (IFN-γ), transforming growth factor-beta 2 (TGF-β2) and TGF-β3. Locally produced TNF-α, IL-6 and TGF-β2 were observed within the lining layer, sublining and pannus at all stages of disease. The staining of TNF-α was particularly intense at the cartilage–pannus junction. In contrast to the monokines, IFN-γ and TGF-β3 were only expressed in scattered cells within the deeper layers of the synovia. Interestingly, IFN-γ was not present in the late phase of CIA, despite the continued presence of TNF-α and IL-6 in the pannus. Production of IL-2, IL-4 or IL-5 was not detected in any joint. The observed pattern of a relative paucity of T cell-derived cytokines and an abundance of monokines during the late phase of T cell-dependent CIA indicates that the synovial cytokine pattern previously described in rheumatoid arthritis (RA) is fully compatible with a pathogenic role of T cells. The temporal as well as spatial dissociation between expression of T cell-derived cytokines and monokines indicates that T cell-independent mechanisms may also be of importance in the triggering of monokine production during arthritis.
Effective treatment of experimental lupus nephritis by combined administration of anti-CD11a and anti-CD54 antibodies
Authors: KOOTSTRA C J , VAN DER GIEZEN D M , VAN KRIEKEN J H J M , DE HEER E , BRUIJN J A
Published: November 2003

Mice with chronic graft-versus-host disease (GVHD), induced by injection of DBA/2 lymphocytes in (C57BL10*DBA/2) F1 hybrids, develop a syndrome resembling systemic lupus erythematosus (SLE) with immune complex glomerulonephritis. In this model we evaluated the role of interactions between CD11a (LFA-1α) and CD54 (intercellular adhesion molecule-1 (ICAM-1)) molecules on leucocytes in the development of renal disease in systemic autoimmunity. Two weeks after induction of GVHD, when anti-nuclear autoantibodies were detected in the circulation and immune complexes had formed in the glomeruli, mice were injected twice per week with rat anti-CD11a and anti-CD54 MoAbs, or with their vehicle PBS, or with control rat IgG. MoAb treatment significantly lowered albuminuria and increased survival compared with control mice with GVHD. In the glomeruli of MoAb-treated mice there was markedly less binding of immunoglobulin and C3, while anti-renal tubular epithelium autoantibodies, but not anti-glomerular basement membrane autoantibodies, were significantly lowered in the circulation 4 weeks after disease induction. In addition, MoAb treatment inhibited the glomerular influx of CD11a+ cells and decreased development of histological abnormalities in the kidneys. Both rat IgG- and MoAb-treated mice developed anti-rat immunoglobulin antibodies. Furthermore, a marked splenomegaly with an increase of the T cell compartment was observed in MoAb-treated mice with GVHD. These results show that CD11a/CD54 interactions are crucial for the full-blown development of lupus nephritis in this model. Treatment aimed at blocking the activity of these molecules profoundly attenuated the development of renal disease in chronic GVHD even if started when first symptoms of SLE (i.e. anti-nuclear autoantibodies in sera and glomerular binding of immunoglobulins) were already detectable.
Up-regulation of the endothelial cell adhesion molecule intercellular adhesion molecule-1 (ICAM-1) by autoantibodies in autoimmune vasculitis
Authors: JOHNSON P A , ALEXANDER H D , McMILLAN S A , MAXWELL A P
Published: November 2003

Autoimmune vasculitis is characterized by the presence of autoantibodies, particularly anti-neutrophil cytoplasmic antibodies (ANCA) and anti-nuclear antibodies (ANA), in patient sera. These autoantibodies have an incompletely understood role in development of vascular injury. The expression or up-regulation of cell adhesion molecules is an early phase in the development of an inflammatory vascular lesion. Autoantibody-positive sera from patients with vasculitis were assessed for their ability to modulate adhesion molecule expression by human umbilical vein endothelial cells (HUVEC). Autoantibody-positive serum samples from 11 out of 21 patients with primary vasculitis produced substantial up-regulation of ICAM-1 on HUVEC. Autoantibody-negative samples did not produce adhesion molecule up-regulation. Up-regulation of adhesion molecules on HUVEC was observed with samples positive for ANA, a phenomenon not previously reported. Preincubation of the sera with purified antigens recognized by ANCA failed to block this activation. In addition, MoAbs to ANCA antigens were ineffective at inducing ICAM-1 up-regulation, suggesting that activation is independent of the molecular specificity of the antibody. This capacity of ANCA- and ANA-positive sera to up-regulate adhesion molecules on endothelial cells may be a factor in the vessel wall inflammation seen in ANCA-associated vasculitis.
IL-1 receptor antagonist inhibits recurrence of disease after syngeneic pancreatic islet transplantation to spontaneously diabetic non-obese diabetic (NOD) mice
Authors: SANDBERG J-O , EIZIRIK D L , SANDLER S
Published: November 2003

The effect of an IL-1 receptor antagonist on recurrence of hyperglycaemia after syngeneic pancreatic islet transplantation to spontaneously diabetic female NOD mice was investigated. The transplanted animals were treated with either the receptor antagonist (8.0 mg/kg body weight per day for 12–14 days) or PBS, delivered by subcutaneously implanted osmotic pumps. In the control animals, a transient normoglycaemia was achieved, but hyperglycaemia was generally observed 6 days after islet transplantation. Administration of IL-1 receptor antagonist had a clear protective effect against recurrence of hyperglycaemia until day 14, but after cessation of drug delivery hyperglycaemia re-appeared. The results indicate that continuous administration of the IL-1 receptor antagonist can prevent recurrence of the diabetogenic process in NOD mice. IL-1 receptor antagonist may therefore become a useful adjuvant immunomodulating therapy after human islet transplantation in insulin-dependent diabetes mellitus.
Oral administration of antibodies as prophylaxis and therapy in Campylobacter jejuni-infected chickens
Authors: TSUBOKURA K , BERNDTSON E , BOGSTEDT A , KAIJSER B , KIM M , OZEKI M , HAMMARSTRÖM L
Published: November 2003

Passive immunity against gastrointestinal infections has recently been successfully applied as prophylaxis and therapy in patients in a variety of virally and bacterially induced infections. Campylobacter jejuni is frequently associated with acute diarrhoea in humans, and several species of animals have been shown to transmit the disease, although birds have been implicated as the main source of infection. We used bovine and chicken immunoglobulin preparations from the milk and eggs, respectively, of immunized animals for prophylactic and therapeutic treatment of chickens infected with C. jejuni. A marked prophylactic effect (a >99% decrease in the number of bacteria) was noted using either antibody preparation, whereas the therapeutic efficacy, i.e. when antibodies were given after the infection was established, was distinctly lower (80–95%) as judged by faecal bacterial counts. These observations may serve as a starting point for experiments aimed at elimination of the infection in an industrial or farm setting. It may also encourage future attempts to treat, prophylactically or therapeutically, patients with Campylobacter-induced diarrhoea.
A cross-reactive idiotype in scleroderma
Authors: VÁZQUEZ-ABAD D , TIAN L , ZANETTI M , ROTHFIELD N F
Published: November 2003

Autoantibodies to centromere proteins (anti-CENPs) and to topoisomerase-I are highly specific for scleroderma. Unlike most autoantibodies in other diseases, these autoantibodies are mutually exclusive. We have analysed the idiotypes (Ids) expressed by anti-CENP-B, antitopoisomerase-I, and IgGs from 20 scleroderma patients. Rabbit anti-Ids were prepared to antitopoisomerase-I from two scleroderma patients, and to anti-CENP-B from four patients. These six anti-Ids were used to study the purified autoantibodies from 20 scleroderma patients: four antitopoisomerase-I, 10 anti-CENP-B, and six purified IgG from scleroderma patients who were negative for both autoantibodies. In addition, we studied sera from 40 normal autoantibody-negative controls, and sera and purified immunoglobulins from 17 systemic lupus erythematosus (SLE) patients containing high titres of anti-double-stranded DNA, and/or autoantibodies to extractable nuclear antigens (ENA). Using direct binding, and competitive inhibition ELISAs and immunoblots, we identified an Id present in the heavy chains of all the affinity-purified antitopoisomerase-I, and anti-CENP-B. Interestingly, this Id was also present in the immunoglobulins of the scleroderma patients who had neither of the two autoantibodies. By contrast, cross-reactive Id-EM was not found in the sera or immunoglobulins from 17 SLE patients, or in the sera from 40 normal subjects. Several samples from two patients showed that this cross-reactive Id-EM was stable over time. The scleroderma disease-specific autoantibodies may be identified through a common structural feature at the variable region of the heavy chain: cross-reactive Id-EM.
The priming action of tumour necrosis factor-alpha (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on neutrophils activated by inflammatory microcrystals
Authors: BURT H M , JACKSON J K
Published: November 2003

We studied the effects of TNF-α or GM-CSF on the production of reactive oxygen species (as measured by chemiluminescence) and degranulation responses of neutrophils to opsonized inflammatory microcrystals. TNF-α in the 10–2000 pm or GM-CSF in the 2–200 pm concentration range caused the concentration-dependent amplification of neutrophil chemiluminescence responses to both calcium pyrophosphate dihydrate (CPPD) and monosodium urate monohydrate (MSUM) crystals. Degranulation responses, as measured by the extracellular release of the granule enzymes myeloperoxidase or lysozyme, were amplified by ≈ 50–100% for both MSUM or CPPD crystal-induced neutrophil activation when cells were pretreated with TNF-α at 2000 pm or GM-CSF at 75 pm.
Anti-Ro52 antibodies frequently co-occur with anti-Jo-1 antibodies in sera from patients with idiopathic inflammatory myopathy
Authors: RUTJES S A , VREE EGBERTS W T M , JONGEN P , VAN DEN HOOGEN F , PRUIJN G J M , VAN VENROOIJ W J
Published: November 2003

We analysed 112 idiopathic inflammatory myopathy (IIM) sera for the presence of anti-Ro, anti-La and anti-histidyl-tRNA synthetase (Jo-1) autoantibodies, and subsequently mapped B cell epitopes on the Ro52 protein recognized by anti-Ro52+ IIM sera. Sera were characterized by immunoblotting, ELISA and RNA precipitation. Both anti-Ro60 and anti-La activity was found in 4% of IIM sera. Anti-Ro52 antibodies were present in 20% of IIM sera. However, in anti-Jo-1+ IIM sera (21%), the frequency of the anti-Ro52 antibodies was found to be much higher (58%). No cross-reactivity between anti-Ro52 and anti-Jo-1 antibodies could be detected in these sera. To learn more about the nature of anti-Ro52 antibodies occurring in IIM sera, we analysed the major epitopes of the Ro52 protein targeted by anti-Ro52+ IIM sera by immunoprecipitation of in vitro translated Ro52 deletion mutants. The major epitope was mapped in the region bordered by amino acids 126 and 252. This part of the protein includes a long α-helical region which contains two potential coiled-coil domains as well as a leucine zipper motif. Although no difference in Ro52 epitope recognition between anti-Jo-1+ and anti-Jo-1+IIM sera could be observed, our results suggest that the autoimmune response against Ro52 and Jo-1 in IIM patients is coupled.
Complement coating of erythrocytes is reduced following their interaction with neutrophils in vitro without loss of complement receptor 1 (CR1)
Authors: IMRIE H J , JONES D R E
Published: November 2003

We have investigated the interaction of complement-coated erythrocytes (E) with neutrophils, in vitro, to determine the effects of erythrocyte CR1. Complement-coating in vitro, to mimic immune complex uptake, caused a reduction in E-CR1, but subsequent interaction with neutrophils effected a removal of E-C3b and a return of E-CR1 to levels approximating that of uncoated erythrocytes. These data indicate that C3b, associated with immune complexes, may bind to E-CR1 in a reversible manner and that subsequent interaction with phagocytes need not necessarily result in cleavage (and loss of ) E-CR1. Further, we conclude that E-CR1 can apparently be reduced by epitope masking.
Up-regulated β1-integrin expression in autoimmune thyroid disorders
Authors: MARAZUELA M , DE LANDÁZURI M O , LARRAÑAGA E , SÁNCHEZ-MADRID F
Published: November 2003

Lymphocytic infiltration of the thyroid gland in autoimmune thyroid disorders requires, as a first step, their attachment to endothelial cells (EC) and, subsequently, interaction with thyrocytes and extracellular matrix proteins. Recent studies have focused on the pathophysiologic role of β1-integrins as adhesion receptors for extracellular matrix proteins and as cell-to-cell adhesion receptors. In this study, we examine by flow cytometry and immunohistochemical techniques the differences in expression of β1-integrins in thyrocytes and EC between normal thyroids and thyroid glands from patients with Graves’ disease (GD) and Hashimoto's thyroiditis (HT). Remarkably, we found an up-regulated de novo expression of very late antigen (VLA)-α6 subunit in thyrocytes in close proximity to lymphocyte infiltrates in GD and HT thyroid glands, with no reactivity in control thyroids. Moreover, interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α) and IL-1β produced a significant enhancement of VLA-α6 expression in vitro in thyrocytes in culture. In addition, an up-regulated expression of VLA-α5 and β1 subunits was found in thyrocytes from GD and HT glands, specifically in those areas more severely inflamed. VLA-α2 was basally expressed in middle size and large vessels in control glands, with an increased expression in vessels of all sizes in HT and GD glands. Dendritic cells in thyroid lymphoid follicles were also positive for VLA-β1, α2 and α6 subunits. These results indicate the existence of an up-regulatory process in the expression of β1-integrins, particularly the α6 subunit, in several cell types from inflamed GD and HT thyroid glands, suggesting that these integrins could play a relevant role in localizing and perpetuating the autoimmune response in the thyroid gland in autoimmune thyroid disorders.
The IL-1 system in HIV infection: peripheral concentrations of IL-1β, IL-1 receptor antagonist and soluble IL-1 receptor type II
Authors: KREUZER K-A , DAYER J-M , ROCKSTROH J K , SAUERBRUCH T , SPENGLER U
Published: November 2003

The proinflammatory cytokine IL-1β is thought to be involved in ongoing HIV disease. Furthermore, its naturally occurring inhibitors soluble IL-1 receptor type II (sIL-1RII) and IL-1 receptor antagonist (IL-1Ra) may play a pivotal role in regulating its biological action. To investigate the involvement of the IL-1 system we determined serum levels of IL-1β, IL-1Ra and sIL-1RII in 90 HIV+ patients. The obtained values were compared with markers of disease progression such as CD+ count, 5′-neopterin, β2-microglobulin and soluble tumour necrosis factor receptors (sTNF-R) p55 and p75 and then compared with C-reactive protein (CRP), granulocyte count, lL-6 and TNF-α. While IL-1Ra concentrations increased significantly with progressive CDC disease stages, sIL-1RII and IL-1β were not altered in our cohort. IL-1Ra showed statistical relation to decreasing CD4+ lymphocytes and increasing 5′-neopterin, β2-microglobulin, sTNF-R p55, sTNF-R p75. Furthermore, IL-1Ra correlated positively with serum IL-6, TNF-α, CRP and granulocytes. In contrast, sIL-1RII and IL-1β tended to show an inverse correlation or showed no significant relationship to all these parameters. Il-1β was measurable only in a limited number of samples. IL-1Ra showed a clear relationship to acute inflammatory events as well as to the different disease stages. Our data suggest a dissociation between IL-1Ra and sIL-1RII serum levels which may indicate that the two IL-1 binding proteins have different pathophysiological roles in HIV infection.
Role of tumour necrosis factor-alpha (TNF-α) in the induction of HIV-1 gp120-mediated CD4+ T cell anergy
Authors: KANEKO H , HISHIKAWA T , SEKIGAWA I , HASHIMOTO H , OKUMURA K , KANEKO Y
Published: November 2003

The HIV-1 envelope glycoprotein (gp120) is known to induce antigen-specific and non-specific CD4+ T cell anergy. We found that early T cell activation, as indicated by HLA-DP expression in the early G1 (G1A) phase of the cell cycle, and the inhibition of mitogen-mediated IL-2 production induced by gp120, required TNF-α produced by gp120-stimulated macrophages. In the presence of an antibody to TNF-α, these changes induced by gp120 were inhibited, while recombinant TNF-α induced similar abnormalities of CD4+ T cells, even in the absence of gp120. On the other hand, inhibition of the mixed lymphocyte reaction (MLR) in CD4+ T cells by gp120, which may be related to gp120-mediated down-regulation of CD4 expression on T cells and activation of protein tyrosine kinase p56lck in CD4+ T cells, was observed even in the absence of macrophage-derived TNF-α induced by gp120. These observations indicate that both TNF-α-dependent and independent events contribute to gp120-mediated CD4+ T cell anergy, and TNF-α appears to play an important role in inducing CD4+ T cell anergy in HIV-1 infection.
Streptozotocin-induced diabetes in mice lacking αβ T cells
Authors: ELLIOTT J I , DEWCHAND H , ALTMANN D M
Published: November 2003

Multiple low-dose streptozotocin (MD-STZ) is widely used for the experimental induction of diabetes, but, as non-obese diabetic (NOD)-scid/scid mice have been found to display enhanced susceptibility to MD-STZ, whether or not the model is genuinely autoimmune and T cell-mediated has been unclear. Mice bearing a targeted mutation of the T cell receptor (TCR) α-chain were therefore used to assess whether TCR αβ+ cells are involved in the diabetogenic effects of MD-STZ injections. Young NOD mice lacking TCR αβ cells, when given five daily injections of 40 mg/kg STZ, developed diabetes at low frequency (2/12), despite the widespread destruction of pancreatic islet cells. By comparison, most normal control mice became hyperglycaemic (12/23). We conclude that whilst much of the tissue destruction observed in this model is due to the direct toxic effect of STZ, a significant amount is also due to the action of TCR αβ cells tipping the balance between tolerable and clinically damaging action on islet cells.
Antipolysaccharide antibodies in 450 children with otitis media
Authors: MISBAH S A , GRIFFITHS H , MITCHELL T , FREELAND A , HAENEY M R , CHAPEL H M
Published: November 2003

We have measured antibodies to pneumococcal and Haemophilus polysaccharides in a prospective study of 450 children aged 2–16 years with otitis media requiring grommets (ear tubes). Pneumococcal antibody levels were significantly higher in the 2–6 year (P < 0.004) and 7–10 year (P < 0.04) study groups in comparison with age-matched controls. There was no difference in Haemophilus antibody levels between the study and control group children for the age groups 2–6 years and 11–16 years. Haemophilus antibody levels were significantly lower in the 7–10 year (P < 0.003) group in comparison with age-matched controls. Eighty-eight out of 450 (19.6%) children had pneumococcal antibody levels below the 25th percentile. Nineteen out of 88 (21.6%) children with pneumococcal antibody levels below the 25th centile were test immunized with 23 valent Pneumococcal polysaccharide and unconjugated Haemophilus type b capsular polysaccharide. Of these 19 children (aged 4–11 years), five mounted suboptimal responses to both polysaccharide antigens, whilst one child failed to respond to Haemophilus polysaccharide alone. There was no significant difference in the prevalence of IgG subclass deficiency between the normal responders and poor responders to immunization (P= 0.12). We found no evidence of specific polysaccharide antibody deficiency in the vast majority of the 450 children studied. However, the significance of poor antibody responses to test immunization in a small minority of children with otitis media is unclear. Long-term follow up of these children is required to determine whether poor immunization responses herald the development of frank antibody deficiency.
Synovial fluid T cell clones from oligoarticular juvenile arthritis patients display a prevalent Th1/Th0-type pattern of cytokine secretion irrespective of immunophenotype
Authors: GATTORNO M , FACCHETTI P , GHIOTTO F , VIGNOLA S , BUONCOMPAGNI A , PRIGIONE I , PICCO P , PISTOIA V
Published: November 2003

The aim of the present study was to investigate the patterns of cytokine production by T cell clones raised from in vivo activated synovial fluid (SF) mononuclear cells (MNC) of five patients with oligoarticular juvenile arthritis (JA). Freshly isolated SF T cells were cultured in vitro with low dose recombinant IL-2 and subsequently cloned by limiting dilution. Sixty-four clones were obtained from the five patients studied. Fifty-nine clones were TCR α/β+, either CD4+ (n = 43) or CD8+ (n = 15). The remaining five clones were TCR γ/δ+, CD4−, CD8−. Clone immunophenotypes differed in the individual patients. Forty-four T cell clones were stimulated with phytohaemagglutinin (PHA) and phorbol myristate acetate (PMA) and supernatants tested for the presence of IL-2, IL-4, IL-5 and interferon-gamma (IFN-γ) by ELISA or bioassays. Cytokine mRNA accumulation was tested by reverse transcriptase-polymerase chain reaction (RT-PCR). Most of 44 clones tested released large amounts of IFN-γ irrespective of the immunophenotype. Of these, 27 were classified as Th1-type and 17 as Th0-type based upon the IFN-γ/IL-4 ratio in culture supernatants. Finally, when 10 representative T cell clones were tested for pro- and anti-inflammatory cytokines, gene expression by RT-PCR, all of them were found to express the granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-α), IL-10 and transforming growth factor-beta 1 (TGF-β1) genes, and half of them IL-6 and IL-8 mRNA. In conclusion, T cell clones, that represent the progeny of in vivo activated SF T cells from oligoarticular JA patients, display heterogeneous immunophenotypes, but all share the ability to produce large amounts of IFN-γ, with a predominant Th1/Th0 pattern. The expression of pro- and anti-inflammatory cytokine genes in these clones suggests that in vivo activated SF T cells modulate joint inflammation in a complex fashion.
The normally expressed κ immunoglobulin light chain gene repertoire and somatic mutations studied by single-sided specific polymerase chain reaction (PCR); frequent occurrence of features often assigned to autoimmunity
Authors: JUUL L , HOUGS L , ANDERSEN V , SVEJGAARD A , BARINGTON T
Published: November 2003

The expressed human κ light chain gene repertoire utilized by healthy individuals was studied by two different single-sided specific PCR techniques to avoid bias for certain V genes. A total of 103 rearranged κ sequences from peripheral blood mononuclear cells from healthy individuals were cloned from cDNA and assigned to the Vκ and Jκ germ-line genes with the closest overall homology. The use of cDNA rather than genomic DNA focused the analysis on activated B cells rich in mRNA. Accordingly, the sequences represented the applied repertoire and almost all were somatically mutated. V genes from the Jκ-proximal duplication unit of the κ locus were almost exclusively used. A total of 65% of the sequences could be assigned to four or five genes: A27 (humkv325), L6 (Vg), L2 (humkv328), and A3 and/or A19. N additions and P nucleotides were quite common and found in 32% and 21% of the sequences, respectively. Extended CDR3s more than nine residues in length were found in 18% of the sequences, and in 71% of cases this was due to insertion of an extra proline residue. This proline was usually explained from the germ-line sequences involved. These results are in good agreement with those of previous repertoire studies using potentially V-gene-biased techniques. Thus, it is clear that restricted V-gene usage, common N and P additions, and extended CDR3 regions are normal features and not, as has been claimed, characteristics of pathological autoantibodies.
Expression of lactoferrin on human granulocytes: analysis with polyclonal and monoclonal antibodies
Authors: AFELTRA A , CACCAVO D , FERRI G M , ADDESSI M A , DE ROSA F G , AMOROSO A , BONOMO L
Published: November 2003

Lactoferrin (LF), an iron-binding protein present in specific granules of neutrophils, is expressed on membrane after granulocyte activation. It may represent a target for anti-neutrophil cytoplasmic antibodies (ANCA) in patients affected by some immunomediated diseases. We recently produced two MoAbs, AGM 2.29 and AGM 10.14, that recognize two spatially distant epitopes of human LF. In this study we perform a cytometric analysis in order to evaluate the expression of LF on the surface of granulocytes obtained from freshly drawn blood or after purification, in both the presence and absence of stimuli. Our results demonstrate that LF is not constitutively expressed on membrane of circulating neutrophils. After priming with phorbol myristate acetate (PMA) or tumour necrosis factor-alpha (TNF-α), an increased mean fluorescence intensity (MFI) was obtained on neutrophils stained with polyclonal anti-LF antibodies and with AGM 2.29. The kinetics of LF expression during activation demonstrated a progressive increase in MFI within 45 min. No increase in MFI was documented when primed granulocytes were stained with MoAb AGM 10.14, thus indicating that the epitope recognized by AGM 10.14 is not exposed at the cell surface. Following membrane permeabilization, performed in order to analyse the binding of anti-LF MoAbs to cytoplasmic LF, a marked increase in MFI was obtained by staining granulocytes with both anti-LF MoAbs. Indirect immunofluorescence (IIF) analysis confirmed that AGM 2.29 and AGM 10.14 reacted with human granulocytes, showing a cytoplasmic pattern on formalin–acetone-fixed neutrophils and a perinuclear one on ethanol-fixed cells.
Haemophilus influenzae and Streptococcus pneumoniae induce different intracerebral mRNA cytokine patterns during the course of experimental bacterial meningitis
Authors: DIAB A , ZHU J , LINDQUIST L , WRETLIND B , BAKHIET M , LINK H
Published: November 2003

Using in situ hybridization with radiolabelled oligonucleotide probes, we studied the mRNA expression of IL-1β, IL-4, IL-6, IL-10, IL-12, tumour necrosis factor-alpha (TNF-α), TNF-β, interferon-gamma (IFN-γ), and transforming growth factor-beta (TGF-β) in the brain during the lethal course of experimental meningitis in a rat model inoculated intracisternally with Haemophilus influenzae type b (Hib) or Streptococcus pneumoniae and in uninfected control rats inoculated with the same volume of PBS. The production of IL-1β, IL-4, IL-6 and IFN-γ was also evaluated by immunohistochemistry. In the brain of Hib-inoculated rats, there was marked mRNA expression of IL-1β, IL-6, TNF-α, IL-12 and IFN-γ. IL-1β, IL-6 and TNF-α were up-regulated throughout the observation period at 2, 8 and 18 h post-inoculation (p.i.), with similar patterns of induction. The Th1 cytokines IFN-γ and TNF-β were up-regulated within 8 h p.i. IL-10 and TGF-β were down-regulated at 18 h p.i., while IL-4 was not detected. In contrast, the brain of S. pneumoniae-inoculated rats showed lower levels of IL-1β, IL-6 and TNF-α, but higher levels of TNF-β and detectable mRNA expression of IL-4 when compared with Hib-inoculated rats. IL-12, IFN-γ, IL-10 and TGF-β exhibited similar patterns of induction in the brains of Hib- and S. pneumoniae-inoculated rats. At 18 h p.i., immunohistochemistry showed similar patterns of IL-1β, IL-4, IL-6 and IFN-γ as mRNA expression in the brains of Hib- and S. pneumoniae-inoculated rats. The differences of cytokine profiles induced by the two bacterial strains may imply that different immunomodulating approaches should be considered, depending on etiology.
Absence of streptococcal protein G (PG)-specific determinant in the Fab region of human IgG2
Authors: PEROSA F , LUCCARELLI G , DAMMACCO F
Published: November 2003

It has been previously reported that CH1 Fab protein G-contact site is responsible for the widespread recognition of mouse and human IgG Fab by PG. Here we present evidence that PG binding to F(ab′)2 is restricted, as indicated by the lack of reactivity with PG-Sepharose columns of a portion of F(ab′)2 fragments obtained by pepsin digestion of human IgG from a commercial immunoglobulin preparation for intravenous use or purified from sera of two healthy blood donors and two patients with polyclonal hypergammaglobulinaemia. Isoelectric focusing showed that F(ab′)2 fragments that did not bind PG focused in a lower pH range compared with those which did. Testing of the Fab fractions with MoAbs to κ and λ light chains or to γ1, γ2 and γ3-Fab subclass determinants showed that γ2-F(ab′)2 were mainly found in the PG non-reactive F(ab′)2 fraction, and that this distribution was not influenced by the L chain isotype. These results indicate that the PG-specific binding determinant(s) is not expressed in the F(ab′)2 region of most human IgG2.
Therapeutic effects of preferential induction of mite-specific T helper 0 clones
Authors: YAMASHITA N , TAKENO M , KANEKO S , MIZUSHIMA Y , SAKANE T
Published: November 2003

The predominance of Th2 cytokine-secreting pattern in allergic asthma has been known as a cause and an accelerating factor, and Th1 suppresses these allergic phenomena, but the role of Th0 clones is obscure. Because Th1/Th2 differentiation has been determined by cytokine environment, we investigated how mite-specific helper T cells stimulated in different cytokine environments actually influenced IgE and IgG4 synthesis, which are known to be regulatory immunoglobulins for allergic response. Th0 clones, which were mainly established in the presence of IL-12, provided a great deal of help for IgG4 and IgG1 synthesis, but did not provide help for IgE synthesis, whereas Th2 clones helped IgE synthesis prominently, and IgG4 and IgG1 synthesis marginally. These characteristics of Th0 clones were also true for Th0 clones obtained from patients who were successfully treated with desensitization therapy. Furthermore, the differences in helper activity between Th0 and Th2 clones were not ascribed solely to soluble factors. These data indicate that IgE and IgG4 synthesis is differentially regulated by antigen-specific T cells, and that conversion or selection from Th2 to Th0 by the addition of IL-12 may exhibit therapeutic effects.
Human mast cells expressing recombinant proteinase 3 (PR3) as substrate for clinical testing for anti-neutrophil cytoplasmic antibodies (ANCA)
Authors: SPECKS U , WIEGERT E M , HOMBURGER H A
Published: November 2003

We have expressed conformationally intact, enzymatically active recombinant PR3 in HMC-1 cells (HMC-1/PR3 cells) that is recognized by C-ANCA. Here we directly compared the clinical utility of C-ANCA testing by indirect immunofluorescence (IIF) using HMC-1/PR3 cell cytospin versus polymorphonuclear neutrophil (PMN) cytospin preparations and commercially available anti-PR3 ELISA kits. Two hundred sera were tested independently by three investigators: 101 previously determined to be C-ANCA-positive by routine clinical laboratory testing using standard IIF on PMN cytospins, and 99 control samples chosen primarily because they contained antibodies against other cytoplasmic target antigens. Discrepant test results between the two cellular substrates were found in seven samples: 2/7 were PMN-positive and HMC-1/PR3 cell-negative (one Sjögren's syndrome, one hand injury); 5/7 were PMN-negative and HMC-1/PR3-positive (all Wegener's granulomatosis (WG)). All C-ANCA-positive WG patients were also positive on HMC-1/PR3 cells. IIF using HMC-1/PR3 cells was as sensitive as the most sensitive anti-PR3 ELISA (79.8% versus 80.7%, P = 0.739), and more sensitive than standard IIF C-ANCA testing using PMN cytospins (79.8% versus 75.2%, P = 0.025) or the anti-PR3 ELISA with the least false-positive test results (79.8% versus 63%, P < 0.01). These findings indicate that HMC-1/PR3 cells are a very sensitive antigen-specific substrate for clinical anti-PR3 ANCA testing which appears superior to standard C-ANCA testing using PMN cytospin substrates and anti-PR3 ELISA. Our results also suggest that in WG the C-ANCA fluorescence pattern is not caused by antibodies against target antigens other than PR3.
Impairment of natural killer (NK) cytotoxic activity in hepatitis C virus (HCV) infection
Authors: CORADO J , TORO F , RIVERA H , BIANCO N E , DEIBIS L , DE SANCTIS J B
Published: November 2003

In the present study, we evaluated the NK cell cytotoxic activity in a group of HCV-infected individuals. Although the number of NK cells present in the peripheral blood of the HCV-infected patients was comparable to non-infected individuals, spontaneous NK cytotoxicity was four-fold lower (P < 0.001) than in normal donors. This functional impairment was not overcome by depletion of adherent or B cells, and it was partially restored by short-term (18 h) stimulation with IL-2. However, long-term stimulation (72 h) with this lymphokine induced activated killer cell (LAK) activity comparable to normal controls. The reduction in NK cytotoxic response does not seem to be due to soluble suppressive factors, since incubation of normal peripheral blood mononuclear cells (PBMC) with infectious HCV serums for a 4-h period does not affect NK spontaneous cytotoxic activity. Successful in vitro infection of PBMC with HCV infectious serum also resulted in an impairment of NK cytotoxicity, suggesting that altered NK function is associated with HCV infection and may be responsible, at least in part, for the chronicity of the infection.
Differential effects of cytomegalovirus infection on complement synthesis by human mesangial cells
Authors: TIMMERMAN J J , BEERSMA M F C , VAN GIJLSWIJK-JANSSEN D J , VAN ES L A , VAN DER WOUDE F J , DAHA M R
Published: November 2003

Viruses may be eliminated by the host immune system via complement-mediated lysis of infected cells. Previously, we have demonstrated that the synthesis of complement factor B by renal mesangial cells (MC) is enhanced by interferon-alpha (IFN-α), -β and -γ. In the present study we investigate the effect of human cytomegalovirus (HCMV) infection on the production of complement factors by MC. The production of factor B, C2, C4 and factor H by mock-infected MC was 0.2 ± 0.4, 3.9 ± 6.8, 1.7 ± 0.8 and 149 ± 36 ng/106 cells per 72 h, respectively. In HCMV-infected MC cultures an induction of both factor B and C2 protein synthesis was observed up to 2.2 ± 1.1 and 156 ± 74 ng/106 cells per 72 h, respectively. The synthesis of C4 and factor H of 2.9 ± 2.0 and 146 ± 31 ng/106 cells, respectively, was not altered significantly. By Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis it was demonstrated that factor B and C2 mRNA expression were up-regulated in HCMV-infected cell cultures, whereas the levels of C4 and factor H mRNA were not changed. When MC cultures were inoculated with heat- or UV-inactivated HCMV no enhancement of factor B mRNA expression was observed. The enhanced expression was not blocked by phosphono acetic acid (PAA), suggesting that expression of the HCMV immediate early or early genes is sufficient to induce complement synthesis. We conclude that infection of MC cultures with HCMV selectively induces complement C2 and factor B production, probably mediated by interferons.