bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of anti-peptide antibodies directed against an extracellular immunogenic epitope on the human α1-adrenergic receptor
Authors: FU M L X , WALLUKAT G , HJALMARSON A , HOEBEKE J
Published: July 2010

A synthetic peptide corresponding to amino acids 192–218 of the second extracellular loop of the human αIA-adrenergic receptor was used to raise antibodies in rabbits, Affinity-purified antibodies specifically recognized main bands with a molecular weight of about 68, 40 and 37 kD on the electrotransferred membrane proteins of rat ventricle membranes. The incubation of these antibodies with rat myocardial membranes resulted in a decrease in the number of binding sites for the specific radiolabelled α1-antagonist prazosin. These antibodies were also able to mimic the effects of agonist stimulation as demonstrated by a positive chronotropic effect on cultured cardiomyocytes. These results constitute the first immunochemical evidence of the presence of both the A and B subtypes of the α1-adrenergic receptor in the heart. They also confirm that the second extracellular loop of the α1-adrenergic receptors is an immunologically and functionally important domain.
Platelet surface IgG in patients receiving infusions of Fab fragments of a chimaeric monoclonal antibody to glycoprotein IIb-IIIa
Authors: CHRISTOPOULOS C
Published: July 2010

Platelet surface immunoglobulin G (PSIgG) was measured ex vivo in nine patients with stable angina pectoris receiving continuous (48–96 h) infusions of Fab fragments of a chimaeric MoAb (human IgG with murine variable regions) to platelet glycoprotein IIb-IIIa. PSIgG was measured using flow cytometry (FC) and an Fc-specific anti-IgG polyclonal antibody, which did not crossreact with the chimaeric Fab fragment (c7E3-Fab). A variable but statistically significant (P < 0·05) elevation of PSIgG was present within 24 h after the onset of the infusion, and was more marked (P < 0·01) several days after the end of the infusion despite an exponential fall in platelet surface c7E3-Fab post-infusion. PSIgG returned to normal within 2 weeks after the end of the infusion. The timing of IgG recruitment to the platelet surface suggested the pre-existence in the patients' plasma of IgG binding to c7E3-Fab-bearing platelets. None of the patients developed thrombocytopenia. In order to assess the incidence of IgG bindable to c7E3-Fab-bearing platelets in controls clinically comparable to the c7E3-Fab infusion patients, normal platelets coated with either chimaeric (c) or murine (m) 7E3-Fab were incubated with plasmas from 21 patients with ischaemic heart disease, and recruitment of IgG to the platelet surface was measured by FC. Fourteen of the 21 plasmas contained IgG bindable to c7E3-Fab-coated platelets, whereas only one of the 21 plasmas contained IgG bindable to m7E3-Fab-coated platelets (a highly significant difference, P < 0 0·01). These findings indicate that Infusions of Fab fragments of the chimaeric anti-platelet antibody 7E3 are often associated with elevations in PSIgG, which are probably due to pre-existing “naturally occurring” antibodies to the Fab fragments of chimaeric (but not murine) 7E3, and most probably other chimaeric MoAbs. The possible clinical significance of such ex vivo measured activities is at present a matter for speculation, and requires further study.
HLA associations with inclusion body myositis
Authors: GARLEPP M J , LAING B , ZILKO P J , OLLIER W , MASTAGLIA F L
Published: July 2010

Inclusion body myositis (IBM) is defined clinically by a characteristic pattern of progressive proximal and distal limb muscle weakness and resistance to steroid therapy, and histologically by the presence of distinctive rimmed vacuoles and filamentous inclusions as well as a mononuclear infiltrate in which CD8+ T cells are predominant. Muscle damage is believed to be mediated by autoimmune mechanisms, but very little information is available on the immunogenic features of IBM. MHC class I and DR antigens were typed on 13 caucasoid patients with IBM using standard serological techniques or by allele-specific oligonucleotide typing. Complement components C4 and properdin factor B (Bf) were typed by immunofixation after electrophoresis. Restriction fragment length polymorphisms (RFLP) in the class ITT region were analysed using cDNA probes for C4 and 21-hydroxylase (CYP21) after Taq 1 digestion. IBM was associated with DR3 (92%), DR52 (100%) and HLA B8 (75%). The phenotype data were examined for likely haplotypes by considering together the alleles at the class T, DR and complement loci along with the C4 and CYP21 RFLP. Ten of the DR3+ subjects had a 6 4-kb C4-hybridizing fragment characteristic of a deletion of C4A and CYP21-A. These patients probably carried all, or at least the class II and III regions, of the extended haplotype marked by B8/C4A*Q0/C4Bl/BfS/DR3/DR52, which has been associated with several autoimmune diseases and is present in 11% of the healthy Caucasoid population. Of the remaining subjects, two had evidence of the extended haplotype marked by B]8/C4A3/C4BQ*0/BfFl/DR3, which is present in less than 5% of the healthy population and has been associated with insulin-dependent diabetes mellitus. These data provide support for an autoimmune etiology for, and genetic predisposition to, IBM.
Serologic markers of lupus nephritis in patients: use of a tissue-based ELISA and evidence for immunopathogenic heterogeneity
Authors: BERNSTEIN K A , KAHL L E , BALOW J E , LEFKOWITH J B
Published: July 2010

In order to assess the ability of various serologic assays to correlate with lupus nephritis, we analysed sera obtained from 60 patients with systemic lupus erythematosus (SLE), Patients were categorized as having active nephritis (group 1), active lupus without nephritis (group 2), inactive lupus with prior nephritis (group 3), or inactive lupus without prior nephritis (group 4), Three parameters were assessed including anti-dsDNA antibodies (Farr assay), immune complexes (C1q binding), and anti-C1q antibodies (salt-stable C1q binding). Additionally, glomerular binding activity (GBA) was measured using a new solid-phase immunoassay that detects immune elements by their ability to bind glomerular tissue. We found that patients with nephritis (group 1) exhibited higher mean values for each assay than patients in each of the other three groups (P= 0·001,0·009, 0·14, and 0·23 in the GBA, C1q, anti-dsDNA, and anti-C1q assays, respectively). The only assay which distinguished patients with nephritis (group 1) from patients having active disease without nephritis (group 2) was the GBA (mean 0·48 ± 0·09 versus 0·15 ± 0·04, (P 0·05), In terms of utility, all tests were specific for diagnosing nephritis among patients with lupus; however, only the GBA was reasonably sensitive. The information provided by the anti-dsDNA and C1q assays were not correlated with one another, nor additive to the GBA, Patients with false negative GBA tended to have received more intensive immunosuppression. The qualitative characteristics of GBA varied among patients with nephritis. These data suggest the pathogenesis of lupus nephritis is complex, and may be mediated by an array of immune elements. Moreover, the data indicate the potential utility for a broad tissue-based approach to detection of pathogenic immune elements over other, specific immunologic markers.
The development of transplant coronary artery disease after cardiac transplantation is correlated with a predominance of CD8+ T lymphocytes in endomyocardial biopsy derived T cell cultures
Authors: JUTTE N H P M , GROENEVELD K , BALK A H M M , OUWEHAND A J , LOONEN E H M , VAN DER LINDEN M , STRIKWERDA S , MOCHTAR B , CLAAS F H J , WEIMAR W
Published: July 2010

Long-term survival of heart transplant recipients is limited by the development of transplant coronary artery disease (TCAD). We analysed whether the development of TCAD is correlated with the incidence of acute rejection episodes, with the formation of anti-HLA antibodies or with the composition and function of T lymphocyte cultures derived from endomyocardial biopsies. TCAD was assessed by visual analysis of annually performed coronary angiograms and defined as the presence of all vascular changes, including minor wall irregularities. One year after transplantation, 31 of the 77 patients studied had TCAD (40%). The median age and mean number of HLA mismatches in patients with or without TCAD were highly comparable. The patient groups did not differ in incidence of acute rejection episodes, nor in percentage of endomyocardial biopsies yielding T cell cultures. At 1 year after transplantation, lymphocyte cultures from 18/31 TCAD+ patients (58%) and 27/46 TCAD- patients (57%) were analysed. The TCAD+ patients had, compared with the TCAD- patients, a higher median percentage of CD8+ T cells (71%versus 25%, P= 0·06) and a lower median percentage of CD4+ T cells (4%versus 40%, P= 0·04). Similar differences were found in a longitudinal analysis of the culture results of endomyocardial biopsies (EMBs) obtained during the first year. The cytotoxic reactivity of the cultures against donor HLA class I or class II antigens was comparable in the two groups, although a difference in recognition of heart specific antigens remains possible. The fact that EMB-derived cultures from TCAD+ and TCAD- patients differed in T cell phenotype populations gives some support to the hypothesis that cellular immunological processes are involved in the development of TCAD. However, while the median values differed, the overlap of the percentages of CD8+ cells in cultures from TCAD- and TCAD+ patients shows that other factors besides CD8+ T cells also play a role.
Progressive increase of CD7- T cells in human blood lymphocytes with ageing
Authors: KUKEL S , REINHOLD U , OLTERMANN I , KREYSEL H-W
Published: July 2010

CD7 is one of the major surface antigens expressed very early during T cell ontogeny. Lack of CD7 expression on mature T cells is regarded as a classical feature of malignant T cells in certain forms of cutaneous T cell lymphoma. Previously, we identified a CD7- subset of peripheral blood T lymphocytes in normal human individuals. In this study we determined the portion of CD7- T cells in the peripheral blood of healthy volunteers ranging in age from 8 months to 90 years (n= 85) and in cord blood of full-term infants (n= 14), Furthermore, this CD7- subset was characterized in detail by the use of MoAbs and three-colour flow cytometry. In cord blood no CD7- T cells could be detected. After birth, percentage and absolute number of CD7- T cells increased with age. Independently of age, most CD7- CD3+ cells belonged to the CD4+ subpopulation. Focusing on the latter we could demonstrate the predominance of the CD45RO+CD45RA- phenotype in the CD7- subset. Furthermore, CD7- T cells contained a higher number of cells expressing activation markers and the CD57 antigen, but a reduced number of CD62L+ cells in comparison with CD7+ T cells.
Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumour necrosis factor-alpha (TNF-α) activate human alveolar macrophages to inhibit growth ofMycobacterium aviumcomplex
Authors: SUZUKI K , LEE W J , HASHIMOTO T , TANAKA E , MURAYAMA T , AMITANI R , YAMAMOTO K , KUZE F
Published: July 2010

We investigated the effects of certain macrophage-active cytokines on the phagocytosis and growth inibition of Mycobacterium avium complex (MAC) by human alveolar macrophages (AM). We also evaluated the effects of pretreatment with each cytokine on the superoxide anion release (O2-) from AM. The cytokines that we used were recombinant GM-CSF, natural type TNF-α, recombinant interferon-gamma (IFN-γ). and recombinant IL-2, We found that phagocytosis by the various cytokine-stimulated AM was similar lo that of unstimulated AM. On the other hand, significant growth inhibition of MAC was observed in the macrophages treated with GM-CSF or TNF-α, while no growth inhibition of MAC was observed in the macrophages treated with IF'N-γ or IL-2. Pretreatment with alt cytokines tested enhanced the O2- release from AM. but there was no correlation between the enhancement of O2- release and the growth inhibition of MAC, Thus, we concluded that GM-CSF or TNF-α could activate AM to inhibit growth of MAC, probably not through the enhanced production of reactive oxygen intermediates.
Treatment of anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis with high-dose intravenous immunoglobulin
Authors: RICHTER C , SCHNABEL A , CSERNOK E , DE GROOT K , REINHOLD-KELLER E , GROSS W L
Published: July 2010

In this uncontrolled study 15 patients with ANCA-associated systemic vasculitis, who were poor responders to conventional therapy, were treated with single or multiple courses of intravenous immunoglobulin (IVIG), 30g/day over 5 days. Clinical and serological evaluation was performed before and 4 weeks after IVIG. Six of the 15 patients experienced clinically significant benefit from IVIG. Improvement was confined to single organ manifestations (skin, ENT findings), no improvement was seen with conjunctivitis and scleritis, pericarditis or nephritis. No patient experienced complete remission after IVIG. Repeated courses of IVIG at 4-week intervals were no more effective than single courses. In six anti-proteinase 3 (PR3)-positive patients pretreatment sera were incubated with F(ab′)2 fragments of the IVIG preparation in vitro to measure the inhibitory effect of IVIG on anti-PR3 activity. An inhibition of anti-PR3 activity by 25–70% was observed; this did not correlate with clinical effects. Approximately 40% of patients benefited from IVIG treatment, though complete remission of disease activity did not occur. Neither clinical characteristics nor the inhibitory effect of the IVIG preparation on serum anti-PR3 activity in vitro predicted clinical response to this treatment modality.
Human autoantibodies directed against the RNA recognition motif of La (SS-B) bind to a conformational epitope present on the intact La (SS-B)/Ro (SS-A) ribonucleoprotein particle
Authors: RISCHMUELLER M , McNEILAGE L J , McCLUSKEY J , GORDON T
Published: July 2010

In systemic autoimmunity, the human B cell response to the La (SS-B) autoantigen is polyclonal and directed to both conserved and human-specific epitopes. This study has further characterized the B cell epitope(s) present within the conserved central region of the La protein, LaC (amino acids 111–242) containing the RNA recognition motif (RRM, aa 111–187). Ten overlapping and non-overlapping protein fragments spanning LaC were expressed in bacteria as NH2-terminal fusions with glutathione-S-transferase. The fusion proteins were tested by ELISA for reactivity with a panel of human anti-La sera in order to define the nature of the epitopes. Ninety-two percent of patient sera containing anti-La antibodies reacted with the region of La containing the RRM. Fine mapping of this reactivity using deletion mutants indicated that the deletion of 19 amino acids from either the NH2-terminal or COOH-terminal region of the RRM was associated with loss of antibody reactivity, suggesting that the immunodominant epitope expressed in this region is discontinuous. Autoantibodies affinity-purified from the La RRM fragment to remove other specificities immunoprecipitated newly synthesized native La (SS-B)/Ro (SS-A) complexes, providing additional evidence that autoantibodies were recognizing a conformational epitope. The findings indicate that the human autoantibody response to La involves recognition of a conformational determinant involving the conserved RRM region without necessarily interfering with the RNA-dependent association of the La/Ro ribonucleoprotein.
Characterization of murine monoclonal antibodies against 60-kD Ro/SS-A and La/SS-B autoantigens
Authors: VELDHOVEN C H A , PRUIJN G J M , MEILOF J F , THIJSSEN J P H , VAN DER KEMP A W C M , VAN VENROOIJ W J , SMEENK R J T
Published: July 2010

Small cytoplasmic ribonucleoproteins (scRNPs) are important autoantigens in patients with systemic lupus erythematosus and Sjögren's syndrome. MoAbs against these proteins were made by immunization of BALB/c mice with purified human recombinant 60-kD Ro/SS-A or 50-kD La/SS-B proteins. Five stable hybridoma cell lines were obtained, of which four secreted anti-Ro/SS-A antibodies (clones 1D8, 1D11, 2G10 and 6G8) and one produced anti-La/SS-B antibodies (clone 7F6). The MoAbs were further characterized using four different immunoassays: immunofluorescence, immunoblotting, RNA precipitation combined with Northern blotting, and recombinant protein precipitation. All lour MoAbs against Ro/SS-A recognized the native protein and one of them (2G10) recognized also intact scRNP particles. Interestingly, hY3-RNA was reproducibly not efficiently precipitated by MoAb 2G10. Epitope mapping using deletion mutants of the 60-kD Ro/SS-A antigen showed that MoAb ID8 recognized the C-terminal part of this protein, while 1D11 and 2G10 recognized distinct epitopes in the region between the RNP motif and the putative zinc finger domain. The epitopes recognized by these MoAbs lire highly conserved among species, and the epitope recognized by MoAb 2G10 may be identical to an autoepitope recognized by sera of patients. This is the first report describing the isolation and characterization of MoAbs of the IgG class against the 60-kD Ro/SS-A and La/SS-B autoantigens obtained by immunization with purified human recombinant proteins. These MoAbs can be of great use in studying the cellular processes in which scRNPs are involved, and may help to determine why these scRNPs become autoantigenic in autoimmune diseases.
Identification of HIV protein-derived cytotoxic T lymphocyte (CTL) epitopes for their possible use as synthetic vaccine
Authors: BRANDER C , PICHLER W J , CORRADIN G
Published: July 2010

CTL are by far the most important defence mechanisms against viral infections, and many attempts have been undertaken to induce protective CTL in vivo. In order to identify CTL epitopes for their possible use as peptide-vaccine candidates, HIV proteins were screened for peptide sequences which (i) fulfil the binding motif of the HLA-A2.1 molecule, and (ii) are involved in the natural immune response to HIV. From 73 nonameric peptides satisfying the binding motif, 20 peptides were synthesized and their binding to HLA-A2.1 was monitored by measuring the expression of HLA-A2.1 molecules on the cell surface of the mutant cell line T2. To evaluate the involvement in natural HIV infection, strongly binding peptides were used in cytotoxicity assays to assess their capacity to generate a peptide-specific CTL response in vitro. From 20 nonameric peptides synthesized, only five showed strong binding to HLA-A2.1. All five binding peptides had the secondary anchor residues, recently proposed by Ruppert et al. [1] to be required for binding to HLA-A2.1. The discrimination between bound and unbound peptides confirmed the importance of these secondary anchor residues which, beside the known binding motif, may dictate if a peptide can bind to HLA-A2.1 or not. In HIV− donors, no CTL activity against any of the HIV-derived peptides was detectable after a 12-day in vitro stimulation. In contrast, HIV-infected persons showed a cytotoxic response against peptide-labelled target cells, suggesting that they had developed upon HIV infection a cytotoxic immune response against the identified CTL epitopes.
Adhesion molecules intercellular adhesion molecule-1 (ICAM-1), ICAM-3 and B7 are not expressed by epithelium in normal or inflamed colon
Authors: BLOOM S , SIMMONS D , JEWELL D P
Published: July 2010

Adhesion molecules are involved in facilitating cell-mediated immune events. Because lymphocyte-epithelial cell interaction has been implicated in the pathogenesis of colonic inflammation, we analysed expression of a range of adhesion molecules on colonic epithelium in vitro and in vivo using flow cytometry, immunohistochemistry and in situ hybridization. Expression of ICAM-1 by cell lines HT29 and int407 was increased by proinflammatory cytokines interferon-gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α) and IL-1 but not by IL-6. Vascular cell adhesion molecule (VCAM) and E-selectin were not expressed. Immunohistochemistry using sections of inflamed colon from 16 patients with ulcerative colitis (UC), five patients with Crohn's disease (CD) and seven patients with normal colonoscopic biopsies, showed no expression of ICAM-1 on colonic epithelium. VCAM was seen in isolated lymphoid aggregates and E-selectin was expressed on endothelium. In situ hybridization showed no ICAM-1 or ICAM-3 mRNA in colonic epithelium. B7, the ligand for CD28, was not found on normal or inflamed colonic epithelium. The adhesion molecules ICAM-1, ICAM-3 and B7 are not involved in lymphocyte–epithelial cell interaction in the normal or inflamed colon. This may have implications for the development of T cell tolerance to intestinal luminal antigens.
Neutrophil adhesion molecules in term and premature infants: normal or enhanced leucocyte integrins but defective L-selectin expression and shedding
Authors: REBUCK N , GIBSON A , FINN A
Published: July 2010

The functional deficits of neonatal neutrophils are well documented and are thought to contribute to the increased susceptibility of newborn infants to infection. We measured the adhesion molecules L-selectin. CDlla/CD18 and CDltb/CDI8 on neutrophils from the cord blood of term (n= 22) and premature (n= 32) infants using a whole blood method with flow cytometry and quantitative bead standards to enumerate cell surface receptors. We also assayed plasma for the shed form of L-selectin (sL-selectin). Our results suggested that L-selectin expression on term infant neutrophils is lower than that on adult neutrophils (unstimulated and stimulated, both P < 0·001). but that stimulated premature infant cells express higher L-selectin than term infants (P < 0·05); it is possible that this deficiency is caused by physiological changes occurring around the normal time of parturition. We observed reduced sL-selectin in term infants (P < 0·001) compared with adults, and even lower eoncentrations in premature infants (P < O−001). The sL-selectin concentrations in plasma may be a refiection of granulopoiesis, which may be reduced in premature infants. Our results showed increased resting neonatal neutrophil expression of CD11b/ CD 18 compared with adults, and the absence of any neonatal deficit of the ability to up-regulate CD11b/CD18 expression on stimulation. These findings are contrary to previous reports. Further studies suggested that the isolation procedures used in previous reports reduces the capability of the cells to respond to a formyl methionine leucine phenylalanine (fMLP) stimulus. This effect is more marked in neonatal neutrophils, suggesting that the previously reported deficiency is in fact due to the isolation techniques used rather than the cells’ innate ability to up-regulale CD11b/ CDI8 expression. The results of our study lead us to propose that the adhesive function of neonatal neutrophils may be less defective than previously thought.
T cell receptor repertoire in the peripheral blood and intestinal mucosa of coeliac patients
Authors: Lahat N , Ben-Nun A , Cohen L , Kinarty A , Lerner A
Published: July 2010

The αβ and γδ T cell receptor (TCR) repertoire in the peripheral blood and intestinal mucosa of six coeliac children and six age-matched controls was analysed by reverse transcription and polymerase chain reaction (PCR). No TCR αβ and γδ restriction was observed in coeliacs and controls. However, Vγ3 was expressed only in coeliac peripheral and intestinal T cells. Vδ2 was strongly expressed in coeliacs and scarcely transcribed in control cells. The unique expression of these γδ TCR in coeliac patients suggests that Vγ3 and perhaps Vδ2 TCR-bearing lymphocytes may play a role in the pathogenesis of coeliac disease.
Altered Th1/Th2 cytokine profiles in the intestinal mucosa of patients with inflammatory bowel disease as assessed by quantitative reversed transcribed polymerase chain reaction (RT-PCR)
Authors: Niessner M , Volk B A
Published: July 2010

Cytokines serve a central function as key factors in the regulation of the intestinal immune response and mediation of tissue damage in inflammatory bowel disease (IBD). Abnormalities in the expression of immunoregulatory cytokines such as IL-2, IL-4, IL-10 and interferon-gamma (IFN-γ) may indicate a dysregulation of intestinal immunity probably associated with pathogenic events. Therefore, cytokine mRNA concentrations were determined in the mucosa of patients with IBD at sites of active (n= 13) and inactive (n= 12) ulcerative colitis (UC), active (n= 11) and inactive (n= 11) Crohn's disease (CD) and in control patients (n= 14) using quantitative RT-PCR. IL-10 mRNA concentrations were significantly increased in patients with both active UC (P < 0.001) and active CD (P < 0.005) compared with control patients. IFN-γ mRNA concentrations were also significantly increased both in patients with active UC (P < 0.02) and active CD (P < 0.05) compared with control patients, whereas IL-2 mRNA levels were significantly (P < 0.02) increased only in active CD. IL-4 mRNA expression in the intestinal mucosa was frequently below the detection limit. Our results demonstrate that chronic intestinal inflammation in patients with CD is characterized by an increase of Th1-like cytokines. Furthermore, the increased IL-10 mRNA expression at sites of active IBD suggests that IL-10 is an important regulatory component involved in the control of the inflammatory response in inflammatory bowel disease.
Increased dimeric IgA-producing B cells in tonsils in IgA nephropathy determined byin situhybridization for J chain mRNA
Authors: Harper S J , Allen A C , BÉNÉ M-C , Pringle J H , Faure G , Lauder I , Feehally J
Published: July 2010

The origin of mesangial IgA deposits in IgA nephropathy (IgAN) remains obscure. A significant proportion of deposited immunoglobulin is dimeric (J chain-positive). Previous studies of J chain expression within lymphoid tissue in IgAN have utilized antibodies which other investigators have found to be non-specific. To address this problem, we have developed an in situ hybridization (ISH) method for the detection of J chain mRNA within IgA plasma cells. Tonsils from 12 patients with IgAN and 12 controls were studied using (i) non-isotopic ISH for J chain mRNA, and (ii) combined immunofluorescence (IF) and fluorescent ISH. J chain mRNA-positive cells were identified in germinal centres, and within the subepithelial and interfollicular zones. A greater number of J chain mRNA-positive cells were found in the germinal centres of patients (mean 57.7±4.6 cells/105μm2) compared with controls (mean 36.9±3.5 cells/105μm2) (P < 0.001). Combined IF and fluorescent ISH showed a greater proportion of J chain mRNA-positive interfollicular IgA cells in patient tonsils (3.2±3.4%) compared with controls (21±2.3%; P < 0.02). These results indicate a shift towards dimeric IgA production in the tonsils in IgAN. In addition, the finding of excess numbers of J chain-positive IgA-negative cells within germinal centres suggests that an abnormality may be present at the B cell differentiation stage before IgA switching. These results further highlight immune abnormalities within the tonsil as a central feature of abnormal polymeric IgA biology in this common form of glomerulonephritis.
Hydrocortisone enhances total IgE levels—but not the synthesis of allergen-specific IgE—in a monocyte-dependent manner
Authors: Bohle B , Willheim M , Baier K , Stadler B , Spitzauer S , Scheiner O , Kraft D , Ebner C
Published: July 2010

Recently, hydrocortisone (HC), when combined with human IL-4, has been reported to increase IgE levels in supernatants (SN) of in vitro cultured leucocytes. In this study we investigated the influence of HC on allergen-specific IgE synthesis. Moreover, we examined the relevance of different cell types in this respect. Peripheral blood mononuclear cells (PBMC), T cell-depleted PBMC, CD14-depleted PBMC and highly purified B cells from 10 allergic (birch pollen and/or grass pollen) patients and five non-allergic individuals were investigated. The cells were incubated with HC and/or recombinant human IL-4 (rIL-4) for 8 days. A considerable increase of total IgE was observed in HC/rIL-4-stimulated cultures compared with rIL-4 alone, HC alone or non-stimulated cultures. We demonstrate that this effect depends on the presence of monocytes in in vitro cultures. These results were seen in every experiment, irrespective of healthy or atopic state of the blood donor. The increase of IgE could not be attributed to a rise of birch pollen- and/or grass pollen-specific IgE in patients allergic to these allergens, as shown by IgE-immunoblot. Radioallergosorbent test (RAST) investigations of HC/rIL-4-stimulated cell cultures from allergic and non-allergic patients confirmed that HC/rIL-4-induced elevated IgE production was also not due to increased production of IgE, specific for important aero-allergens (pollens, house dust mite or animal dander). Therefore we conclude that newly synthesized IgE is not specific for allergens, but that sequential isotype switching in human B cells leads to increased polyclonal IgE production.
Depression in caregivers of demented patients is associated with altered immunity: impaired proliferative capacity, increased CD8+, and a decline in lymphocytes with surface signal transduction molecules (CD38+) and a cytotoxicity marker (CD56+ CD8+)
Authors: Castle S , Wilkins S , Heck E , Tanzy K , Fahey J
Published: July 2010

Changes in relevant immune parameters, including function, were found to be associated with depression in elderly caregiver wives of demented patients. We studied the relationship between immune cell phenotype and T cell proliferative capacity of such caregivers to levels of stress and depression over the course of a support group intervention. The data indicate the strongest association between depression (of all stress parameters) and impaired T cell proliferative capacity. Depression was also most strongly (of stress parameters) associated with a shift in T cell populations with an increase in CD8+ T cells, and a reduced percentage of CD38+ cells in both CD8+ and CD4+ T cell populations. Since CD38 is a signal transduction factor, it was interesting that a decreased percentage of CD38+ cells correlated with impaired T cell function (proliferation). Another significant difference was the reduction in natural killer (NK) cells as well as the percentage of the CD56+ component of the CD8+ population. This latter subset is important in MHC-unrestricted cytotoxicity, and has been found expanded in healthy centenarians. This study shows that both chronic stress, and depression in particular, and age have deleterious effects on T cells, and together could significantly contribute to the higher risk of disease and mortality associated with being a caregiver of a demented individual.
The development of a novel immunotherapy model of human ovarian cancer in human PBL-severe combined immunodeficient (SCID) mice
Authors: Walker W , Gallagher G
Published: July 2010

The reported ability of SCID mice to accept xenografts of both human tumours and peripheral blood lymphocytes (PBL) provides the potential for the development of novel immunotherapy models in these animals. This study describes the development of a novel small animal model of human ovarian cancer. This was achieved by engrafting a human ovarian cancer cell line (Ovan-4) into the peritoneal cavity of immunodeficient SCID and immune reconstituted human PBL-SCID mice. When transplanted to SCID mice this cell line exhibited growth characteristics similar to the clinical disease observed in patients with Implantation of metastatic nodules onto the interior surface of the peritoneal wall. Reconstituted human PBL-SCID mice challenged with identical numbers of Ovan-4 cells exhibited a significant increase in survival time, suggesting a role for cells of the human immune system in preventing the development of this type of malignancy in vivo. Furthermore, vaccination of human PBL-SCID mice against Ovan-4 produced tumour-specific human antibodies in the serum of these animals. Animals reconstituted with CD8-depleted PBL exhibited increased serum immunoglobulin levels and produced enhanced anti-Ovan-4 activity after vaccination. Subsequent challenge of these animals with Ovan-4 revealed a further increase in survival time. These results suggest that human antibodies may have a role in immunity against ovarian cancer and could be of therapeutic value in this type of disease.
A systematic study of neutrophil degranulation and respiratory burstin vitroby defined immune complexes
Authors: Zhang W , Voice J , Lachmann P J
Published: July 2010

Defined immune complexes (IC) were used to compare the effect of antibodies of different classes and subclasses on neutrophil respiratory burst and degranulation. IC were made from 5-iodo-4-hydroxy-3-nitrophenacetyl (NIP) conjugated to bovine serum albumin (BSA) and chimaeric mouse-human anti-NIP monoclonal antibodies including lgA2, IgE and all four IgG subclasses. The activation of neutrophils by IC depended on antibody class and subclass, on antigen epitope density, on antigen : antibody ratio and on the medium used. The ability to generate the respiratory burst showed a different pattern to the ability to give rise to degranulation. Compared with other IC, IgA2 IC provided the strongest stimulus for neutrophil activation. IgG1 IC, IgG2 IC and IgG4 IC activated neutrophils moderately or weakly. IgG3 IC were unable to stimulate the respiratory burst, but could cause strong degranulation. IgE IC could hardly cause any neutrophil response. Neutrophil degranulation in response to IgG3 IC in serum-free medium or heat-inactivated serum was fast, and it quickly reached maximum. Degranulation caused by IgA IC was relatively slow. but gradually increased during incubation. The activity of IgG1 IC. IgG2 IC and IgG4 IC generated a respiratory burst increased with antibody excess and decreased with antigen excess. The activity of IgA2 IC, however, was not affected by change of antigen and antibody ratio. A specific role of serum, possibly due to complement, was found in enhancing degranulation. both temporally and quantitatively, by lgA2 IC.
Autoantibodies in human anti-Ro sera specifically recognize deproteinized hY5 Ro RNA
Authors: Boulanger C , Chabot B , MÉNard H A , BOIRE G
Published: July 2010

We report the existence of a Novel autoantibody specificity linked to anti-Ro antibodies. Sera from two patients with anti-Ro ribonucleoprotein (RNP) antibodies also contained antibodies that immunoprecipitated specifically either the deproteinized RNA component of the RohY5 RNP particle, or intact in vitro transcribed hY5 RNA. No serum recognized specifically the other hY RNAs. A mutant hY5 RNA with additional nucleotides (nt) at both extremities was not immunoprecipitated, possibly because of altered secondary structure. Following digestion of hY5 RNA with ribonuclease TI, the smallest immunoprecipitable RNA fragments were 27 and 31 nt long, and respectively mapped to the 5′and 3′ends of hY5 RNA, excluding the La-binding region. Base pairing between the 27 and 31 nt long fragments was required for recognition by antibodies. Our data indicate that the epitope bound by anti-hY5 RNA antibodies is conformational. We have previously reported that most anti-Ro sera contain a population of antibodies specific for the RohY5 RNP. Since antibodies to the deproteinized hY RNAs within anti-Ro sera are also restricted to anti-hY5 RNA, a direct role for the human-specific RohY5 particles in the immunization process leading to the production of anti-Ro antibodies is suggested.
Lysis of human macrophages by cytolytic CD4+ T cells fails to affect survival of intracellularMycobacterium bovis-bacille Calmette-Guérin (BCG)
Authors: Fazal N , Lammas D A , Rahelu M , Pithie A D , Gaston J S H , Kumararatne D S
Published: July 2010

Human CD4+, mycobacteria-specific, cytolytic T cell clones were used to lyse BCG-infected macrophages. and the effect on the subsequent growth and viability of the organisms was examined. The survival of released bacteria following cell lysis was assessed by both 3H-uridine labelling and colony-forming unit (CFU) estimation. The results indicate that even when effective antigen-specific or lectin-mediated cytolysis of the infected macrophages was achieved, there was no evidence for a direct mycobactericidal effect on the intracellular bacteria. This remained the case even if the period of co-culture of T cells and macrophages was extended up to 48 h. Pretreatment of the macrophages with interferon-gamma (IFN-γ) was not able to act together with T cell-mediated lysis to produce inhibition of mycobacterial growth.
A rare mediastinal tumour presenting with systemic effects due to IL-6 and tumour necrosis factor (TNF) production
Authors: Davies K A , Cope A P , Schofield J B , Chu C-Q , Mason J C , Krausz T , Smith P , Denman A M , Walport M J
Published: July 2010

Patients presenting with prolonged systemic illnesses with no specific clinical or serological defining features may be diagnosed as having atypical systemic vasculitides, but often turn out to have occult malignancies. Cytokines have been implicated in causing many of the systemic effects in such cases. In this study we describe a patient presenting after 2 years of a severe systemic illness with a marked acute phase response, due to an occult mediastinal angiomatoid malignant fibrous histiocytoma. Tumour resection was curative. We evaluated in detail the local and systemic production of cytokines induced by this tumour. Blood samples were taken pre- and postoperatively for cytokine studies. In vitro production of IL-2, IL-2R, IL-1β, IL-6 and TNF-α by cultured monocytes from the patient, as well as plasma cytokine levels, were measured by ELISA. Tumour cytokine production was also evaluated immunocytochemically, and by in situ hybridization with specific cDNA probes. Plasma IL-2R and IL-6, and IL-6 and TNF-α production by peripheral blood monocytes were markedly elevated before tumour resection, normalizing postoperatively. Most tumour cells and infiltrating lymphocytes stained with antibodies to IL-6, IL-6R and TNF-α, and expressed HLA class II. IL-6 and TNF- α mRNA production in the tumour was confirmed by in situ hybridization studies. We have described the first case of an occult angiomatoid malignant fibrous histiocytoma in the mediastinum. Studies of cytokine expression suggested that chronic TNF, IL-6, and IL-2 production by leucocytes and tumour cells in this patient was responsible for the severe systemic illness with which she presented.
IL-1 is expressed in human adrenal glandin vivo.Possible role in a local immune-adrenal axis
Authors: GonzÁLez-HernÁNdez J A , Bornstein S R , Ehrhart-Bornstein M , Gschwend J E , Gwosdow A , Jirikowski G , Scherbaum W A
Published: July 2010

IL-1 is an important mediator in the dialogue between the immune system and the hypothalamo-pituitary-adrenal axis. A direct influence of IL-1 upon adrenal steroidogenesis has been demonstrated in experimental animals. We therefore designed a study to see if IL-1 is expressed within the normal human adrenal gland. The combination of in situ hybridization and specific immunostaining to IL-1β was eminently suited to demonstrate both mRNA and protein production. The specific immunostaining of the different cells combined with in situ hybridization (IL-1) allowed us to identify the exact cellular source of IL-1. IL-1 mRNA occurred in the zona reticularis in 17α-hydroxyiase positive steroid cells surrounding the adrenomedullary cells. Some CD68+ macrophages in this zona showed a positive signal. A weak signal was seen to IL-1 mRNA in few chromaffin cells, while IL-1-like immunoreactivity was more frequent. We conclude that in the normal situation in man IL-1 is mainly expressed in specialized cortical cells. The occurrence of the major glucocorticoid inducing factor in the normal human adrenal gland itself provides evidence for an autocrine or paracrine reaction under physiological conditions.
Non-muscle myosin as target antigen for human autoantibodies in patients with hepatitis C virus-associated chronic liver diseases
Authors: VON MÜHLEN C A , CHAN E K L , PEEBLES C L , IMAI H , KIYOSAWA K , TAN E M
Published: July 2010

Three patients with hepatitis C virus (HCV)-related chronic liver disease were shown to have autoantibodies strongly reacting with cytoskeletal fibres of non-muscle cells. The heavy chain of non-muscle myosin microfilament was the main target for those autoantibodies, as determined by (i) cell and tissue immunofluorescence studies showing colocalization with an anti-myosin antibody prototype; (ii) primary reactivity in immunoblotting with a 200-kD protein, using either MOLT-4 cells, human platelets, or affinity-purified non-muscle myosin as antigen extract; and (iii) immunoblotting of similar immunoreactive fragments in papain-digested MOLT-4 cell extracts, by using those human sera and antibody prototype. Autoantibodies to non-muscle myosin heavy chain were not previously reported in patients with chronic liver diseases, especially in those associated with HCV infection.
Combined effects of FK506 (tacrolimus) and cyclophosphamide on atypical B220+ T cells, cytokine gene expression and disease activity in MRL/MpJ-lpr/lprmice
Authors: WOO J , WRIGHT T M , LEMSTER B , BOROCHOVITZ D , NALESNIK M A , THOMSON A W
Published: July 2010

Groups of female MRL/MpJ-lpr/lpr mice received either saline or FK506 (tacrolimus; 2mg/kg intraperitoneally) three times weekly, cyclophosphamide (CY; 20mg/kg) once monthly, or both drugs from 8 weeks of age. Median survival for untreated and CY-treated mice was 26 weeks, and for FK506- and FK5O6 + CY-treated groups was ± 44 weeks. Severity of skin lesions and lymph node hyperplasia was markedly reduced by the drug combination, whereas either drug alone was less effective. FK506 or CY alone delayed the onset of proteinuria, but by 24 weeks all of these animals were positive. In contrast, drug combination reduced the prevalence of proteinuria to ± 60% throughout the 44 weeks of study. Sequential monitoring of peripheral blood lymphocytes revealed that combination therapy but not monotherapy markedly reduced the proportion of atypical CD3+ B220+ and CD3+CD4-CD8- T cells. Neither FK506 nor CY affected the reduction in IL-2 and IL-4 mRNA levels observed in lymph nodes of diseased animals compared with normals. Although the drug combination also did not affect IL-2 mRNA levels, IL-4 mRNA transcripts were increased six-fold compared with saline-treated controls. IL-10 and interferon-gamma (IFN-γ) mRNAs were induced by FK506, CY and by the drug combination. Serum levels of anti-dsDNA antibodies were reduced in all treatment groups. These data demonstrate improved efficacy of combined T and B cell-directed immunosuppression in murine lupus, associated with marked inhibition of atypical T cells and selective augmentation of IL-4 within the affected lymphoid tissue.
Peritoneal lavage fluids stimulate NIH3T3 fibroblast proliferation and contain increased tumour necrosis factor and IL-6 in experimental silica-induced rat peritonitis
Authors: LEE H G , CHOI I , PYUN K H , PARK K W
Published: July 2010

Fibroblast proliferation is one of the earliest features of fibrosis, preceding collagen deposition in wound. The response to tissue injury is characterized by the infiltration of acute inflammatory cells, followed by persistence of macrophages. In vitro, macrophages are known to secrete monokines and growth factors which affect fibroblast proliferation and collagen synthesis. To investigate in vivo kinetics of fibroblast proliferating activity and monokine production in experimental peritonitis, silica was instilled intraperitoneally into rats, and peritoneal lavage fluids (PLF) and the culture supernatants of macrophages were analysed for NIH3T3 fibroblast proliferating activity and monokine production. NIH3T3 fibroblast proliferating activity in PLF was markedly elevated 3–5 days after intraperitoneal silica instillation, and peritoneal macrophages also spontaneously released these factors in vitro. Tumour necrosis factor (TNF) and IL-6 were also markedly increased after 3–5 days, and macrophages spontaneously released these monokines. PLF-induced fibroblast proliferation was blocked by anti-TNF antibody, and fibroblast proliferation was stimulated by the external addition of TNF or TNF and IL-6 in vitro. These results show high influx of fibroblast proliferating factors and monokines in peritoneal lavage fluid after 3–5 days of silica-induced experimental peritonitis, providing further evidence of involvement of macrophages, TNF, and possibly IL-6 in peritoneal fibrosis.
Do IgE-IgG complexes occur in the circulation?
Authors: BOLUDA L , BERRENS L
Published: July 2010

IgG anti-IgE autoantibodies in human allergic sera have been investigated using an enzyme immunoassay on microplates coated with anti-human IgE MoAb. After incubation with serum, the plates were developed with peroxidase-labelled anti-human IgE MoAb for the determination of total IgE levels, or with anti-IgG MoAb for evaluating IgG anti-IgE autoantibodies. Using this methodology, no correlation was found between total IgE and IgG anti-IgE levels in groups of sera of allergic individuals. Although the results obtained with enzyme-labelled anti-IgG are often interpreted as indicative of IgE-IgG complexes captured from the serum, molecular sieving on gel columns as well as direct ultrafiltration experiments through 300-kD membranes demonstrate that such complexes do not occur preformed in the circulation, but arise de novo on the anti-IgE-coated solid phase during in vitro incubation with human serum. It is suggested that IgG anti-IgE autoantibodies react with IgE only after the latter has undergone a conformational change, either by colloidal manipulation or after reaction with allergen.
Localization and identification of granzymes A and B-expressing cells in normal human lymphoid tissue and peripheral blood
Authors: KUMMER J A , KAMP A M , TADEMA T M , VOS W , MEIJER C J L M , HACK C E
Published: July 2010

Cytoplasmic granules from activated natural killer (NK) and cytotoxic T lymphocytes (CTL) contain a pore-forming protein, perforin, and several homologous serine proteinases called granzymes. Expression of these proteins correlates with the cytolytic potential of cytotoxic lymphocytes. Using a panel of MoAbs specific for human granzyme A and B, respectively, expression of these proteinases in non-pathological lymphoid tissue and peripheral blood lymphocyte (PBL) subpopulations was investigated. Using immunohistochemistry and double stainings, the phenotype of granzyme-expressing cells in lymphoid tissue was investigated. Granzyme-positive cells were detected in all lymphoid tissues tested. No large differences in the number and distribution between granzyme A- and granzyme B-positive cells were observed. The highest number of positive cells was located in the red pulp of the spleen. Significant numbers were detected in tonsil, lymph nodes, liver and thymus. Low numbers were present in the lamina propria of non-inflamed stomach, small intestine and colon. Phenotypic analysis and cell sorting showed that most of the granzyme-positive cells in lymphoid tissue and PBL consisted of CD3+ CD16+CD56+ lymphocytes. Hardly any granzyme-positive CD3+CD8+ CTL were present in peripheral blood. The synthesis of granzyme A as well as B by both CD3 CD16+ CD56+ and CD3+CD8+ cells in peripheral blood was increased upon IL-2 stimulation. These results indicate that in normal lymphoid tissue the predominant cytolytic cell population is formed by the NK cells, and activated CTL are rare.