bluuebunny/crossref_metadata_2025 huggingface dataset:
PAI‐1 promoter 4G/5G genotype as an additional risk factor for venous thrombosis in subjects with genetic thrombophilic defects
Authors: Seguí Rafael , Estellés Amparo , Mira Yolanda , España Francisco , Villa Piedad , Falcó Cristina , Vayá Amparo , Grancha Salvador , Ferrando Fernando , Aznar Justo
Published: July 2010

Impaired fibrinolysis as a result of increased plasminogen activator inhibitor‐1 (PAI‐1) levels in plasma is a common finding in patients with deep vein thrombosis (DVT). A 4G/5G polymorphism in the promoter region of the PAI‐1 gene has been reported to influence the levels of PAI‐1. The 4G allele was found to be associated with higher plasma PAI‐1 activity (act), but contradictory results on the incidence of the 4G allele in DVT patients have been reported. The aim of this study was to analyse whether the PAI‐1 promoter 4G/5G genotype increases the risk of venous thrombosis in subjects with thrombophilic defects, and to determine the distribution of the PAI‐1 4G/5G genotype and its relation to plasma PAI‐1 levels in 190 unrelated patients with DVT in comparison with a control group of 152 healthy subjects. No differences between the 4G/5G allele distribution in the DVT group (0·43/0·57) and in the control group (0·42/0·58) were observed. However, the presence of the 4G allele significantly increased the risk of thrombosis in patients with other thrombophilic defects. Significantly higher PAI‐1 levels were observed in DVT patients than in the controls. Our results also showed significant differences in the plasma levels of PAI‐1 antigen (ag) and PAI‐1 act among the 4G/5G genotypes in DVT patients. A multivariate analysis revealed that, in the DVT group, PAI‐1 ag levels were influenced by the 4G allele dosage, triglyceride levels and body mass index (BMI). The influence of the 4G allele dosage on PAI‐1 levels was independent of the triglyceride levels and BMI. In the control group, no significant correlation between PAI‐1 levels and 4G allele dosage was observed. In conclusion, the PAI‐1 promoter polymorphism was found to have an influence on PAI‐1 levels in DVT patients and on the risk of venous thrombosis in subjects with other genetic thrombophilic defects.
The use of real‐time quantitative polymerase chain reaction and comparative genomic hybridization to identify amplification of the REL gene in follicular lymphoma
Authors: Goff Lindsey K. , Neat Michael J. , Crawley Charles R. , Jones Louise , Jones Emma , Lister T. Andrew , Gupta Rajnish K.
Published: July 2010

Using comparative genomic hybridization (CGH), aberrations in DNA copy number were studied before and after transformation of follicular lymphoma to diffuse large B‐cell lymphoma in six patients (15 lymph node biopsies in total). The most common and also the most discrete and intense amplification occurring in four out of 15 biopsies from three different patients was of 2p13–16. Using real‐time quantitative polymerase chain reaction (RQ‐PCR), REL amplification was found to be implicated at this locus. This technique also identified amplified REL in a further two biopsies, presumably below the detection level of CGH. REL amplification was quantified by comparing it, in most cases, with three endogenous reference genes, albumin, β2‐microglobulin and CD8α, that lie close to REL on 2p. There was no correlation apparent between 2p13–16 amplification or REL amplification and transformation. This study shows the usefulness of coupling CGH, for detecting recurring abnormalities, with the real‐time PCR technique for rapid gene dosage quantification and confirms that the REL gene is a potential candidate in the pathogenesis of a particular subset of follicular lymphomas.
Testing Sokal's and the new prognostic score for chronic myeloid leukaemia treated with α‐interferon
Authors:   ITALIAN COOPERATIVE STUDY GROUP ON CHRONIC MYELOID LEUKAEMIA *Writing committee:, Bonifazi Francesca , De Vivo Antonio , Rosti Gianantonio , Tiribelli Mario , Russo Domenico , Trabacchi Elena , Fiacchini Mauro , Montefusco Enrico , Baccarani Michele
Published: July 2010

As it has been shown that α‐interferon (αIFN) treatment modifies the survival of chronic myeloid leukaemia (CML) patients in comparison with conventional chemotherapy, a new prognostic score was devised with the aim of providing a treatment‐adapted risk evaluation. We have tested the new prognostic score (the Euro score) in an independent series of 272 patients less than 56 years old with previously untreated, chronic phase, Philadelphia (Ph)‐positive CML who had been assigned prospectively to αIFN treatment between 1989 and 1991. The Sokal score system was used as a reference. The new Euro score predicted the response to αIFN as the Sokal score. The median survival of low‐risk, intermediate‐risk and high‐risk patients was similar using the Euro score (105, 65 and 45 months) and Sokal score (105, 76 and 45 months) but, by multivariate analysis, the Euro was more potent than Sokal for predicting survival time. The new Euro score identified more low‐risk cases (59% vs. 48%) and fewer high‐risk cases (9% vs. 23%) than the Sokal score. The main differences between the Euro and Sokal scores concerned age (it is more important in the Euro than in Sokal), spleen size and the percentage of myeloblasts in peripheral blood (more important in Sokal than in Euro). We conclude that the new Euro score marks an improvement in the prognostic evaluation of CML treated with αIFN. By comparison with the Sokal score, the Euro was more potent and identified more low‐risk patients but left only a small number of cases in the high‐risk group.
Production of soluble CD34 by human myeloid cells
Authors: Fernández Mireya , Simon Valeska , Minguell José J.
Published: July 2010

CD34, a glycophosphoprotein present in lymphohaematopoietic stem and progenitor cells, as well as in other cell types, exists in both transmembrane and intracytoplasmic forms. Transmembrane CD34 expression, which is high in the earliest haematopoietic precursors, decreases as cells mature. However, to our knowledge, there is no information on whether a decrease in transmembrane CD34 can also predict a release of the molecule from the cell membrane into the extracellular fluid. To investigate the above possibility, we studied conditions (incubation time, cell density and proliferative status) in human myeloid cells (lines KG‐1a, KG‐1 and cord blood‐derived cells) that may cause a decrease in surface CD34 and the generation of a soluble form of the molecule. The latter, as demonstrated by Western blot analysis, adds more complexity to the proposed structural features and functional properties of CD34 in myeloid cells.
Absence of mutations in the granulocyte colony‐stimulating factor (G‐CSF) receptor gene in patients with myelodysplastic syndrome/acute myeloblastic leukaemia occurring after treatment of aplastic anaemia with G‐CSF
Authors: Kudo Kazuko , Nagai Hirokazu , Numata Shin‐ichiro , Ichihara Masatoshi , Kinoshita Tomohiro , Horibe Keizo , Kato Koji , Matsuyama Takaharu , Kodera Yoshihisa , Kojima Seiji
Published: July 2010

The development of myelodysplastic syndrome/acute myeloblastic leukaemia (MDS/AML) has been reported in patients with aplastic anaemia (AA) after administration of recombinant human granulocyte colony‐stimulating factor (rhG‐CSF). Similarly, patients with severe congenital neutropenia (SCN) have an increased risk of developing MDS/AML after treatment with rhG‐CSF. Point mutations in the G‐CSF receptor gene are found in about 20% of SCN patients who are predisposed to MDS/AML. We investigated the occurrence of mutations in the G‐CSF receptor in eight patients with AA who developed MDS/AML. No mutations were detected around the cytoplasmic domain of the gene in our patients, indicating that the mechanisms of clonal evolution to MDS/AML in patients with AA might be different from those with SCN.
Induction of MTG8‐specific cytotoxic T‐cell lines: MTG8 is probably a tumour antigen that is recognized by cytotoxic T cells in AML1–MTG8‐fused gene‐positive acute myelogenous leukaemia
Authors: Maeda Motoi , Otsuka Teruhisa , Kimura Nobuhiro , Kozu Tomoko , Fukuyama Tomofusa , Uchida Naoyuki , Sugio Yasuhiro , Itoh Yoshikiyo , Iino Tadafumi , Inaba Shoichi , Niho Yoshiyuki
Published: July 2010

Several reports have demonstrated the persistent detection of AML1–MTG8 fusion products, representing minimal residual disease (MRD), in patients with t(8;21) acute myelogenous leukaemia (AML) who are in long‐term remission. It is probable that immune‐mediated mechanisms that are able to suppress the expansion of MRD may result in the continuance of remission. It was previously shown that some t(8;21) AML patients had high anti‐MTG8 antibody titres. MTG8 expression in normal adult tissues is limited to the brain or heart in which human leucocyte antigen (HLA) class I cell‐surface antigens are either not or are only faintly detectable. We hypothesized that the overexpression of the MTG8 gene in t(8;21) AML cells could act as a possible tumour antigen, which might be able to induce the immune‐mediated suppression of the expansion of MRD. We were able to induce HLA‐A0201‐restricted cytotoxic T‐lymphocyte (CTL) lines against an MTG8 peptide (MTG8b amino acids 182–191) using monocyte‐derived dendritic cells from a healthy donor. T‐cell receptor (TCR)Vα17, TCRVβ14 and 15, and TCRJβ2.1 and 2.3 are predominantly used in these CTL lines. Our data, which suggest that the MTG8 protein could be one of the tumour antigens recognized by CTLs, may be helpful in further investigations of TCR analysis in t(8;21) AML patients with HLA‐A0201 who are in long‐term remission.
Q1311X: a novel nonsense mutation of putative ancient origin in the von Willebrand factor gene
Authors: Casaña Pilar , Martínez Francisco , Haya Saturnino , Lorenzo J. Ignacio , Espinós Carmen , Aznar José A.
Published: July 2010

Type 3 von Willebrand disease, a recessive autosomally inherited bleeding disorder, refers to complete deficiency of von Willebrand factor (VWF). The novel Q1311X mutation was detected in the homozygous state in four Spanish patients from two apparently unrelated families of gypsy origin. The lack of specific amplification of platelet VWF cDNA from two of the patients indicates reduced levels of mutated gene expression. The similar haplotype linked to mutated alleles suggests a common origin. On the basis of the two instabilities observed and the estimated mutation rate of the microsatellites of intron 40 of the VWF gene, we can estimate that this mutation could have arisen about 2300 years ago.
High levels of human herpesvirus 6 DNA in peripheral blood leucocytes are correlated to platelet engraftment and disease in allogeneic stem cell transplant patients
Authors: Ljungman P. , Wang F.‐Z. , Clark D. A. , Emery V. C. , Remberger M. , Ringdén O. , Linde A.
Published: July 2010

The aim of this study was to correlate human herpesvirus (HHV)‐6 viral load with clinical symptoms in allogeneic stem cell transplant (SCT) patients. Seventy‐four patients were monitored during the first 3 months after SCT using a qualitative polymerase chain reaction (PCR) for HHV‐6 DNA. HHV‐6 was detected in 181 out of 494 samples (36%) from 58 (78%) patients. These 181 samples were analysed using a quantitative competitive PCR. DNA could be quantified from 146 out of 181 samples (80·6%). The HHV‐6 viral load was highest at 4 weeks compared with 8 weeks (P < 0·001) and 12 weeks (P = 0·01) after SCT. Three patients had HHV‐6 encephalitis and one patient had hepatitis. The HHV‐6 DNA levels were higher in patients with HHV‐6 than in those without HHV‐6 (P = 0·01). Patients who received grafts from unrelated or HLA‐mismatched family donors had significantly higher HHV‐6 DNA levels than patients who received grafts from matched sibling donors (P < 0·001). In a multiple regression model, unrelated donor grafts (P < 0·001) and use of intravenous immunoglobulin prophylaxis (P = 0·04) influenced HHV‐6 DNA levels. HHV‐6 viral load was significantly correlated with delayed platelet engraftment in both univariate (P < 0·01) and multivariate analysis, and to the number of platelet transfusions.
Persistence of clonal T‐cell expansions following high‐dose chemotherapy and autologous peripheral blood progenitor cell rescue
Authors: Protheroe Andrew S. , Pickard Christopher , Johnson Peter W. M. , Craddock Tina , Shefta Jahan , Short Kath , Lancaster Fiona , Selby Peter J. , Henwood Judy , Boylston Arthur W.
Published: July 2010

Analysing the regeneration of T lymphocytes after high‐dose chemotherapy with autologous peripheral blood progenitor cell rescue (PBPCR) may help elucidate the mechanisms of immune recovery. The T‐cell receptor variable beta chain (TCRBV) repertoire of adult patients undergoing high‐dose chemotherapy was analysed by flow cytometry, before and after treatment. Four patients were found to have a stable expansion present (TCRBV3, 17, 21 and 22) ranging from 8% to 42% of the CD4+ or CD8+ repertoire. We demonstrated that, in these patients, following high‐dose chemotherapy and autologous stem cell transplantation, the clonal expansions reappeared in peripheral blood and returned to pretransplant levels. Three expansions (CD3+CD8+TCRBV3+, CD3+CD4+TCRBV21+ and CD3+CD8+TCRBV22+) were further defined by sequence analysis of the complementarity‐determining region (CDR)3 portion within the TCR rearrangements. These were shown to be predominantly clonal, with the same sequences being identified in peripheral blood before and after PBPCR, providing evidence that the overwhelming majority of T cells in these expansions arise from mature lymphocytes. This study demonstrated that patients undergoing autologous PBPCR for high‐dose chemotherapy regenerate clonal expansions, consistent with pretreatment levels. They also regenerate T‐cell repertoires with each TCRBV family represented to a similar level as that prior to high‐dose chemotherapy.
A novel two base pair deletion in the factor V gene associated with severe factor V deficiency
Authors: Montefusco Maria Claudia , Duga Stefano , Asselta Rosanna , Santagostino Elena , Mancuso Giacomo , Malcovati Massimo , Mannucci Pier Mannuccio , Tenchini Maria Luisa
Published: July 2010

We studied a family in which the proband, a 13‐year‐old boy, had unmeasurable plasma levels of coagulation factor V antigen and activity. Clinical symptoms were severe, with several episodes of haemorrhages in the mucosal tracts (gastrointestinal, nose and urinary) and recurrent haemarthroses that caused permanent arthropathy. Sequence analysis of the factor V gene demonstrated the presence of a novel 2 base pair (bp) homozygous deletion in exon 13 at positions 2833–2834. This mutation, present in the heterozygous state in the asymptomatic mother and absent in the healthy brother, introduced a frameshift and a premature stop at codon 900. This would predict the synthesis of a truncated factor V molecule, lacking part of the B domain and the complete light chain. Because of the existence of a surveillance mechanism that selectively recognizes and degrades mRNA molecules carrying premature termination codons, we analysed the relative abundance of mutant vs. wild‐type mRNA molecules in the platelets of the heterozygous proband's mother. The mutant mRNA was significantly reduced in amount (mutant/wild‐type ratio 0·35). This is the first reported mutation in the factor V gene causing severe factor V deficiency, the effect of which was quantitatively analysed at mRNA level.
Systemic inflammation increases shear stress‐induced platelet plug formation measured by the PFA‐100
Authors: Homoncik Monika , Blann Andrew D. , Hollenstein Ursula , Pernerstorfer Thomas , Eichler Hans‐Georg , Jilma Bernd
Published: July 2010

The PFA‐100 measures platelet plug formation under shear stress and is strongly dependent on von Willebrand Factor (VWF) levels in plasma. We therefore hypothesized that elevated VWF levels, possibly as a result of acute inflammation, adversely influence PFA‐100 results. Healthy volunteers received either 2 ng/kg endotoxin or placebo in a randomized controlled trial. Four hours after endotoxin (but not placebo) infusion VWF levels increased by 85%, collagen epinephrine‐induced closure time (CT) decreased by 47% and collagen ADP‐CT decreased by 38% (P < 0·0001) respectively. In conclusion, systemic inflammation has a major impact on the results obtained by PFA‐100 and may confound interpretation of platelet function.
Mechanisms of bone marrow progenitor cell apoptosis in aplastic anaemia and the effect of anti‐thymocyte globulin: examination of the role of the Fas–Fas‐L interaction
Authors: Killick S. B. , Cox C. V. , Marsh J. C. W. , Gordon‐Smith E. C. , Gibson F. M.
Published: July 2010

The mechanism of action of anti‐thymocyte globulin (ATG) in aplastic anaemia (AA) is complex. Bone marrow (BM) CD34+ cells in AA have been shown to be more apoptotic and have a higher expression of Fas antigen (Fas‐ag) than in normal donors. The aims of this study were to delineate further the mechanism for increased bone marrow progenitor cell apoptosis in AA and investigate the effects of ATG on apoptosis and Fas‐ag expression. BM was obtained from six normal donors and 10 untreated AA patients. We confirmed that AA BM CD34+ cells were more apoptotic than normal donor cells (P = 0·002). Following treatment with ATG, the mean percentage reduction of apoptosis was 34% (9·2–65·9%). BM from 30 AA and 10 normal donors was then stained for CD34, Fas‐ag and 7‐AminoActinomycin D. The proportion of CD34+ Fas+ cells was higher in untreated AA (P = 0·0001) than in normal donors. Results also showed that the majority of CD34+ Fas+ cells were apoptotic/dead in normal donors (mean 81%) and AA (88%), indicating that Fas is involved in apoptosis of CD34+ cells. In contrast, the majority of CD34+ Fas− cells in normal donors were live (mean 91%), while two patterns emerged in untreated AA. In seven patients, the majority of cells were live, however, in the remaining eight patients, the majority of cells were apoptotic/dead, suggesting an alternative mechanism for apoptosis in addition to Fas‐ag. Finally, we have shown that in vivo ATG treatment reduced the expression of Fas‐ag on AA BM CD34+ cells.
High nerve growth factor receptor (p75NTR) expression is a favourable prognostic factor in paediatric B cell precursor‐acute lymphoblastic leukaemia
Authors: Troeger Anja , Gudowius Sonja , Escherich Gabriele , Den Boer Monique L. , Glouchkova Ludmila , Ackermann Birgit , Meisel Roland , Laws Hans‐Juergen , Groeger Marketa , Wessalowski Ruediger , Willers Reinhart , Harbott Jochen , Pieters Rob , Goebel Ulrich , Janka‐Schaub Gritta E. , Hanenberg Helmut , Dilloo Dagmar
Published: July 2010

Nerve growth factor (NGF) plays a pivotal role in cellular survival/death decisions with the low affinity receptor p75NTR predominately transmitting anti‐proliferative signals. In spite of its established role in B‐cell function and identification as a prognostically favourable marker in a number of malignancies, little is known about the expression pattern and prognostic significance of p75NTR in B cell precursor‐acute lymphoblastic leukaemia (BCP‐ALL). p75NTR expression was prospectively studied on primary ALL‐blasts in a cohort of paediatric patients with common ALL (n = 86) and preB‐ALL (n = 34) treated within the Co‐operative study group for childhood acute lymphoblastic leukaemia (CoALL) protocol, CoALL06‐97. Flow cytometric analysis showed that almost half of the patients expressed no or negligible amounts of p75NTR (<10%). The median expression in patients expressing p75NTR beyond that threshold was 49% (range 11–100%). In patients classified as low‐risk at diagnosis, p75NTR expression was significantly higher than in high‐risk patients (P = 0·001). Of note, p75NTR expression was lower in the 21 patients who subsequently developed relapse compared with those remaining in remission (P = 0·038). Accordingly, relapse‐free survival was significantly better in patients expressing high surface p75NTR (P = 0·041). Thus, in this prospective analysis, high p75NTR expression was a strong prognostic marker that identified a group of paediatric ALL patients with favourable outcome.
Genetic modifiers of the β‐haemoglobinopathies
Authors: Thein Swee Lay
Published: July 2010

Identification of the molecular basis of the β‐thalassaemias and sickle cell disease (SCD) has made it clear that patients with the same β‐globin genotypes can have very variable patterns of clinical expression. Extensive biochemical and pathophysiological studies over the last 50 years have derived two major modifiers – innate ability to produce fetal haemoglobin and co‐inheritance of α‐thalassaemia, subsequently validated by family and population studies. However, these two modifiers do not explain the full clinical spectrum. Genetic studies have been successful in identifying modifiers if the loci have a major clinical effect and if the genetic variants are common. It is possible that additional modifiers could be uncovered using genetic approaches but success will depend on large sample sizes of well‐characterised patients with well‐defined phenotypes. Since some of the complications, such as overt stroke in SCD, are relatively rare events, intermediate end‐points that contribute to the phenotype, such as Transcranial Doppler velocity (a major predictor of stroke in SCD), could be integrated within the genetic analysis. Integrating multiplex genetic testing with clinical and laboratory data to generate predictive models shows potential, but such genetic approaches also require large datasets.
Soluble interleukin-2 receptor, interleukin-2 and interleukin-4 in sera and supernatants from patients with progressive systemic sclerosis
Authors: FAMULARO G , PROCOPIO A , GIACOMELLI R , DANESE C , SACCHETTI S , PEREGO M A , SANTONI A , TONIETTI G
Published: July 2010

We studied the sera of patients with progressive systemic sclerosis (PSS) for elevated levels of soluble interleukin-2 receptor (sIL-2R), interleukin-2 (IL-2) and interleukin-4 (IL-4). We also measured IL-2, IL-4 and B cell growth factor (BCGF) activity in supernatants of peripheral blood mononuclear cells from the same patients. The finding of elevated serum sIL-2R and IL-2, and the increased levels of IL-2, IL-4 and BCGF activity in culture supernatants indicates that T lymphocyte hyperactivity likely play a major role in PSS. The failure to detect under our experimental conditions a direct proliferative effect of recombinant IL-2 on enriched normal B cells might suggest that IL-4 is the cytokine mainly responsible of the BCGF activity recovered in PSS supernatants.
Do infections induce monoclonal immunoglobulin components?
Authors: HAAS H , ANDERS S , BORNKAMM G W , MANNWEILER E , SCHMITZ H , RADL J , SCHLAAK M
Published: July 2010

The incidental finding of monoclonal immunoglobulin components (MC) in some infections prompted us to study this phenomenon more systematically. Using isoelectric focusing with immunoblotting (detection limit for MC 0.1 mg/ml), the following infections were studied for the presence of MC: visceral leishmaniasis, cytomegalovirus (CMV) infection, echinococcosis and infectious mononucleosis. MC were found in 16 of 20 leishmania patients and in eight of 18 CMV patients, but in only one of 20 echinococcosis patients and in none of 30 infectious mononucleosis patients. The MC were mostly transient, where tested. A minority of the MC found in the leishmaniasis patients was shown to bind to leishmania antigens. The specificity of the majority of the MC remains unknown. Further study is required to explain the high incidence of MC in CMV infection and visceral leishmaniasis.
Autoantibodies to endothelial cells and neutrophil cytoplasmic antigens in systemic vasculitis
Authors: FRAMPTON G , JAYNE D R W , PERRY G J , LOCKWOOD C M , CAMERON J S
Published: July 2010

The interaction of circulating autoantibodies with the endothelium may be an important mechanism in the pathogenesis of systemic vasculitis. In a prospective study, we looked for circulating anti-endothclial cell autoantibodies (AECA) and anti-neutrophil cytoplasm autoantibodies (ANCA) in 80 patients with suspected systemic vasculitis. AECA were measured using an isotype-specific cellular ELISA incorporating human umbilical vein endothelial cells. ANCA activity was determined by indirect immunofluorescence and radioimmunoassay. Sequential studies were performed on sera from four cases with dual positivity, where autoantibody binding was compared with von Willebrand factor (vWF) concentration and disease activity. IgG AECA were significantly higher in the 27 ANCA-positive sera as compared with normal controls (P= 0027) with IgG (P= 0 009) and IgA (P = 0046) AECA isotypes correlating with ANCA positivity; in contrast, no differences were found between AECA levels in the ANCA-negativc sera and the normal controls. Cross-inhibition studies pointed to the co-existencc of two autoantibody populations. An association between autoantibody binding, disease activity and vWF concentration was found for both ANCA and AECA. Some patients with systemic vasculitis have detectable AECA that recognize different epitopes to ANCA and like ANCA. their titre correlates with disease activity and thus they may have a pathogenetic role in these conditions.
Effect of macrophage-derived factor on hypoferraemia induced by Japanese encephalitis virus in mice
Authors: BHARADWAJ M , KHANNA N , MATHUR A , CHATURVEDI U C
Published: July 2010

Depression of serum iron following Japanese encephalitis virus (JEV) infection was observed in mice. The hypoferraemia was associated with the accumulation of iron in reticulo-endothelial cells in the spleen. Splenectomy (compared with sham-operation) prevented the depression in serum iron concentration after JEV infection. It also prevented the rise in levels of liver iron. The effect of JEV-stimulaled. splenic macrophage-derived factor (MDF) was evaluated in causing hypoferraemia. MDF produced a rapid reduction in the serum iron levels with accumulation of iron in spleen. These observations suggest that MDF plays a key role in the regulation of iron metabolism during JEV infection.
Vascular damage in Wegener’s granulomatosis and microscopic polyarteritis: presence of anti-endothelial cell antibodies and their relation to anti-neutrophil cytoplasm antibodies
Authors: SAVAGE C O S , POTTINGER B E , GASKIN G , LOCKWOOD C M , PUSEY C D , PEARSON J D
Published: July 2010

To define mechanisms of vascular injury in Wegener’s granulomatosis and microscopic potyarteritis, anti-endothelial cell antibodies (AECA) were sought in serum from 168 patients, all of whom had anti-neutrophil cytoplasm antibodies (ANCA) detectable by indirect immunofluorescence. Using an ELISA with human umbilical vein endothelial cells (HUVEC), IgG AECA were demonstrated in 59% and IgM AECA in 68% of patients. Pretreatment of HUVEC with tumour necrosis factor (TNF). IL-1 or interferon-gamma (IFN-γ) led to increased binding. Adsorption of AECA/ANCA-containing serum with HUVEC or neutrophils demonstrated that AECA and ANCA recognized different targets, von Willebrand factor (vWf) antigen levels in the patient samples were markedly elevated, with a mean of 3-10+ 1-89 U/ml (control population mean 1-04 + 0-36 U/ml). suggesting widespread endothelial cell damage. Studies using an’ In-labelled HUVEC release assay with 29 AECA-containing sera did not demonstrate complement-mediated cytotoxicity. even following activation of HUVEC with TNF. Four out of 16 AECA-containing sera tested showed antibody-dependent cellular cytotoxicity with unfractionated peripheral blood mononuclear cells. These data suggest that patients with Wegener’s granulomatosis or microscopic polyarteritis can develop AECA to constitutively expressed but cytokine modulated determinants on HUVEC. These antibodies do not appear to support complement-mediated cytotoxicity. but a proportion can support antibody-dependent cellular cytotoxicity, suggesting that they may contribute to vascular injury.
Alveolar macrophages from patients with bronchogenic carcinoma and sarcoidosis similarly express monocyte antigens
Authors: BARBOSA I L , GANT V A , HAMBLIN A S
Published: July 2010

It has been shown that bronchoalveolar lavages (BALs) from patients with sarcoidosis and otlher interstitial lung diseases contain abnormally increased numbers of alveolar macrophages (AM) expressing antigens found on monocytes. The aim of this study was to compare the phenotype of AM from patients wilh sarcoidosis with those from patients with non-interstitial lung disease, namely carcinoma. Using a panel of monoclonal antibodies against cells of the mononuclcar phagocytic series and immunohistochemieal staining, we have compared the expression of antigens on AM recovered at BAI, and peripheral blood monocytes from patients wilh sarcoidosis. with similar cell preparations from bronchogenic carcinoma patients and normal volunteers. We have shown that CD14, CR1 (CD35) and CR3 (CD11b, CD18) are expressed on the majority of monocytes from all subjects, but on only a minority of normal AM. In both patients with sarcoidosis and patients with bronchogenic carcinoma increased proportions of AM expressed these monocyte-associated antigcns. Since BALs from the carcinoma patients were derived from lung lobes which were radiologically free of tumour, the accumulation of AM expressing monocytic antigens is not a local response to the tumour. We conclude that infiltration of the lung wilh monocytes is a more general response to lung disease than has hitherto been reported.
Month-related variability in immunological test results; implications for immunological follow-up studies
Authors: VAN ROOD Y , GOULMY E , BLOKLAND E , POOL J , VAN ROOD J , VAN HOUWELINGEN H
Published: July 2010

This longitudinal study was originally designed to detect changes in the in vitro immune response of healthy subjects as a result of a psychological intervention. In this study a significant proportion, about 70%, of the immunological variability in the test results was accounted for by the differences in immunological response levels of the subjects. Apart from this between-subject-effect, a significant proportion of the variability in test results was related to the month of data sampling. The month-effect was computed in such a way that the between-subject variation was taken into account. This resulted in a more accurate estimation of the month-effect. Even after correction for the intervention, i.e. the defence of the PhD thesis, the effect of month of data sampling remains significant for mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, percentage of CD4 and CD8 cells, and for the response to the mitogens phytohaemagglutinin, pokeweed mitogen and concanavalin A as well as the results for the mixed lymphocyte culture for one pool out of three. In contrast, no significant month-effect was observed for the whole blood cell counts, for the differential white blood cell counts as determined by monoclonal antibody staining for cell surface markers CD3, GD16, TAG and OKM1, nor for the immunoglobulin IgM and IgG serum levels. Likewise the cell-mediated lympholysis activities measured against three pools of stimulator cells remained unaltered. We discuss the implications for future immunological follow-up studies of the observation that a significant proportion of the variability in immunological test results is related to differences between subjects and to the month of data sampling.
Structural properties of the glycoplasmanylinositol anchor phospholipid of the complement membrane attack complex inhibitor CD59
Authors: RATNOFF W D , KNEZ J J , PRINCE G M , OKADA H , LACHMANN P J , MEDOF M E
Published: July 2010

CD59, the membrane regulator of aulologous C5b-9 channel formation, exhibits variable sensitivity to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that releases glyco-inositolphospholipid (GPI)-anchored proteins from cell surfaces. To determine whether the GPI-anchor phospholipid of CD59 is similar to that of decay-accelerating factor (DAF) and whether variation in its structure underlies its variable enzyme susceptibility, the GPI anchors of the two proteins expressed on erythrocytes, polymorphonuclear and mononuclear leucocytes were compared in situ and after purification. Flow cytometric analyses of PI-PLC-treated cells showed parallel cell type specific release of both proteins as a function of enzyme concentration. Non-denaturing PAGE analyses of alkaline/hydroxylamine-treated proteins (affinity-purified from [125I]-surface-labelled cells) provided evidence for (i) comparable proportions of GPI-anchor acylation, and (ii) alkali-resistant rather than alkali-sensitive lipid substituents in erythrocytes. These findings argue that the differential C5b-9 sensitivity that distinguishes paroxysmal nocturnal haemoglobinuria II and III erythrocytes does not derive from expression of CD59 molecules with alternative GPI-anchor phospholipid structures.
Hyper-response of serum IgG1 toStaphylococcus aureuspeptidoglycan in patients with hyper-IgE syndrome
Authors: ISHIZAKA A , KOJIMA K , SAKIYAMA Y , MATSUMOTO S , KUWAJIMA K , WAGATSUMA Y , SHIBATA R , JOH K
Published: July 2010

The hyper-IgE (HIE) syndrome is characterized by high IgE scrum levels, chronic dermatitis and recurrent infections. To determine whether an impairment of the antibody response to Staphylococcus aureus contributes to infections in this syndrome we measured total serum IgG subclass, specific IgG1 and IgG2 levels against peptidoglycan (PG), the immunodominant cell wall component of S. aureus and serum opsonic activity lo PG. Of the 14 patients with HIE syndrome, nine had increased level of serum IgG1 and six had IgG2 subclass deficiency. In regard to specific response of IgG1 and IgG2 antibodies to PG. patients were divided into live groups related to ages and compared with 10 control subjects for each age cohort. Patients with HIE syndrome had significant high levels of serum-specific IgG1 to PG and significant decreased levels of serum-specific lgG2 lo PG in all five groups. Additionally, serum opsonic activity in patients was significantly higher than that in normal control subjects. It is concluded that IgG2 deficiency or poor IgG2 antibody response to S. aureus is not the explanation of (he abnormal susceptibility to S. aureus infections of HIE patients.
Effect of lymphocytic infiltration on the blood-retinal barrier in experimental autoimmune uveoretinitis
Authors: LIGHTMAN S , GREENWOOD J
Published: July 2010

Using an experimental model of autoimmune uveoretinitis, we have examined the relationship of T cell infiltration in the retina to blood-retinal barrier (BRB) breakdown. Sensitive quantitative in vivo techniques were used to examine BRB permeability to sucrose, a low mol. wt non-transported solute. Electron microscopy was also used to localize extravasated horseradish peroxidase, a macromolecular visual tracer, from the retinal vasculature and to identify the route by which any leakage was occurring. No increase in BRB permeability was found prior to lymphocytic infiltration. By day 10 of the disease inflammatory cells could be seen within the structurally intact retina, which was shortly followed by an increase in the permeability of the BRB to sucrose. Only later in the disease process, when damage to the photoreceptor layer became apparent, did extravasation of the macromolecule HRP occur. At no stage of the disease process was there any detectable damage to inter-endothelial tight junctions. The size-dependency of tracer extravasation in the initial stages of the disease is indicative of a paracellular route being responsible for the increase in BRB permeability. In later stages of the disease some evidence of horseradish peroxidase filled ‘vesicle-like’ profiles was observed. We suggest that the devastating complication of BRB breakdown in ocular inflammation is a direct consequence of lymphocytic infiltration.
Monoclonal antibody epitopes of mycobacterial 65-kD heat-shock protein defined by epitope scanning
Authors: HAJEER A H , WORTHINGTON J , MORGAN K , BERNSTEIN R M
Published: July 2010

The binding sites for MoAbs to the 65-kD heat-shock protein (hsp65) of mycobacteria have been investigated by epitope scanning. Five hundred and twenty-six 8-mer peptides representing the complete sequence of Mycobacterium tuberculosis hsp65 were synthesised in duplicate using the Epitope Scanning Kit (CRB Ltd.). The epitopes of six MoAbs raised to the hsp65 of M. tuberculosis or M. leprae were investigated. We have identified the epitope of a new MoAb (DC 16); this epitope is continuous, hydrophilic in nature and 11 amino acids long. We have also confirmed the location of the epitopes of three MoAbs (IIH9, ML30 and IIC8). Thus the epitope scanning technique has proved suitable for the detection of continuous epitopes of hsp65.
Suppression of autoimmune thyroid disease by FK 506: influence on thyroid-infiltrating cells, adhesion molecule expression and anti-thyroglobulin antibody production
Authors: TAMURA K , WOO J , MURASE N , CARRIERI G , NALESNIK M A , THOMSON A W
Published: July 2010

Autoimmune thyroid disease was induced in female PVG/c rats by neonatal thymectomy, followed by sublelhal, whole body x-irradiation. Disease development, assessed by histological evidence of lymphocytic thyroiditis and circulating levels of anti-thyroglobulin antibodies, was reduced significantly by a 3-week course of FK 506 (0.5 or 1.5 mg/kg per day) commencing after the detection of autoantibody production. Thyroid-infiltrating mononuclcar cells (MNC) in untreated rats stained predominantly for CD4+ and MHC class II antigen which was expressed widely on dendritic cells. Fewer infiltrating cells expressed TCR α/β, CD5, CD8 or LFA- 1β. Intercellular adhesion molecule-1 (ICAM-1) was observed on MNC, vascular endothelial cells and a minoritiy of residual thyroid epithelial cells. FK 506 administration reduced markedly the incidence of infiltrating TCRα/β+, CD5+, CD4+, CD8+, and LFA-1β+ cells and the expression both of MHC class II antigens and ICAM-I on MNC, endothelial cells and thyrocytes. Compared with normal PVG/c rats, there were reduced incidences of CD4+ CD8− and CD4− CD8+ lymphocytes and an elevation in the CD4+/ CD8+ cell ratio in the spleens of animals with autoimmune thyroiditis. These changes were partially reversed by FK 506. Systemic drug levels estimated by enzyme immunosorbent assay were in excess of those known to blockade cytokine production by CD4+ T lymphocytes in vitro and some evidence of minor renal dysfunction was observed. The results are consistent with a therapeutic effect of FK 506 mediated via interference with CD4+ T lymphocyte function and adhesion molecule-dependent cytotoxic effector mechanisms.
Impact of 15-deoxyspergualin on effector cells in experimental autoimmune diseases of the nervous system in the Lewis rat
Authors: JUNG S , TOYKA K V , HARTUNG H-P
Published: July 2010

The influence of the immunosuppressive antibiotic agent 15-deoxyspergualin (DSG) on macro-phages and autoreactive T helper lymphocytes from Lewis rats was analysed in vitro and in vivo. DSG did not inhibit antigen- or mitogen-induced proliferation of encephalitogenic or neuritogenic T helper cell lines in vitro. However, the presence of DSG during in vitro activation of the T cells strongly suppressed or completely abrogated their capacity to induce encephalitis (EAE) or neuritis (EAN) after adoptive transfer to naive rats, although expression of activation markers or adhesion molecules on the T line blasts was not down-regulated by DSG, Like activation-induced T cell proliferation, IL-2-dependent growth of CD4+ T line cells was not affected by DSG, Preincubation of CD4+ T line cells in DSG during IL-2-driven proliferation for 48 h, however, inhibited the subsequent antigen- but not mitogen-induced activation of these T cells, although neither density of T eel) receptors nor other surface molecules involved in antigen recognition were lowered on the cells exposed to DSG. Similar to its effect in vitro, in vivo administration of DSG for 10 days even at a concentration with cumulative toxicity did not suppress in vitro proliferation of spleen cells induced by mitogen or a mitogenic combination of anti-CD2 antibodies. Furthermore, spleen cell and peripheral blood lymphocyte (PBL) surface antigens, particularly MHC molecules, were not altered by long-term treatment with DSG for 30 days. While there was a slight reduction in the number of polymorphonuclear cells in both populations, the proportion of the different leucocyte subpopulations remained unchanged. In contrast to the strong functional impact of DSG on autoreactive T helper cells, the drug did not inhibit the oxidative burst of macrophages or their MHC antigen expression. This study demonstrates a clear inhibitory effect of DSG on CD4+ T lymphocytes, but not macrophages. It provides an explanation for recent observations of a strong immunosuppressive in vivo effect of DSG on transplantation rejection and experimental autoimmune diseases, despite a normal mitogen response of T cells exposed to DSG in vivo and in vitro.
Uveitis and anti-neutrophil cytoplasmic antibodies
Authors: HAGEN E C , VAN DE VIJVER-REENALDA H , DE KEIZER R J W , KULSTRA A , VAN ES L A , DAHA M R , VAN DER WOUDE F J
Published: July 2010

Serum samples of 485 uveitis patients were screened for the presence of anti-neutrophil cytoplasmic antibodies using a standardized immunofluorescence test (IIF) on neutrophil granulocytes. Seventeen of these sera contained cytoplasmic (C)-ANCA antibodies, while two of the sera contained perinuclear (P)-ANCA antibodies (both antinuclear antibody (ANA)-positive, one anti-myeloperoxidase (MPO)-positive). None of the C-ANCA-positive sera reacted with proteinase-3 in ELISA using a highly purified proteinase-3 preparation. Four C-ANCA and one P-ANCA-positive serum reacted with MPO. The majority of the sera did react with azurophilic granules in ELISA. The implication of these results is that in patients with uveitis a positive C-ANCA test is not diagnostic for Wegener's granulomatosis, but is most probably caused by the presence of autoantibodies against as yet unknown constituents of azurophilic granules.
Enrichment and characterization of dendritic cells from human bronchoalveolar lavages
Authors: HAVENITH C E G , VAN HAARST J M W , BREEDIJK A J , BETJES M G H , HOOGSTEDEN H C , BEELEN R H J , HOEFSMIT E C M
Published: July 2010

In the present study about 0·3% to 1·6% of human bronchoalveolar lavage (BAL) cells were identified as typical dendritic cells (DC), having an irregular outline, lobulated nucleus, and clear distinguishable acid phosphatase activity or EBM11 (anti-CD68) reactivity in a spot near the nucleus. After DC enrichment, using transient adherence to plastic, FcR-panning, and a density metrizamide gradient, a population containing 7–8% typical DC was obtained. This DC-enriched low density fraction, containing the highest percentages of DC, very strongly induced T cell proliferation in an allogeneic mixed leucocyte reaction (MLR), which was significantly higher than that induced by other partly (un)fractionated BAL cells. These data indicate that DC seem to be the major accessory cells in the BAL fluid, and therefore may be important in the regulation of T cell immune responses in the lung.
Susceptibility to adjuvant arthritis: relative importance of adrenal activity and bacterial flora
Authors: VAN DE LANGERIJT A G M , VAN LENT P L E M , HERMUS A R M M , SWEEP C G J , COOLS A R , VAN DEN BERG W B
Published: July 2010

Previous studies on the regulation of bacterial-induced arthritis in rats have focused on endocrine aspects as well as differences in T cell immunity against bacterial epitopes. We analysed the role of both adrenal activity and bacterial flora in determining susceptibility to bacterial-induced arthritis. Outbred Wistar rats show a low incidence of adjuvant arthritis. Moderate sensitivity to adjuvant arthritis was found in a selected., stress-resist ant line of the Wistar rat, whereas no arthritis was found in a stress-susceptible Wistar line. Plasma corticosterone responses after IL-1α exposure were, however, identical in these two lines, excluding a direct correlation between susceptibility and corticosterone levels. In line with previous findings in germ-free (GF) F344 rats, GE Wistars also appeared highly susceptible to arthritis. We further analysed the corticosterone responses in GE and conventional (CV) rats. Administration of IL-1α induced identical corticosterone responses in both CV and GF E344 rats. In addition, plasma corticosterone levels were measured around the time of onset of arthritis. Whereas no rise was seen in the arthritis-resistant CV rats, a significant increase was observed from day 14 in GE rats, at the moment of onset of arthritis. Although this corticosterone response was insufficient to prevent arthritis, it may have ameliorated disease expression in the GK F344 rats. Our data indicate that the bacterial flora, and therefore T cell tolerance, is of prime importance in determining susceptibility, whereas the activity of the hypothalamus-pituitary-adrenal (HPA) axis may modulate disease severity.
Autoantibodies to cerebroside sulphate (sulphatide) in leprosy
Authors: WHEELER P R , RAYNES J G , MCADAM K P W J
Published: July 2010

Sera from 40 leprosy patients were screened for autoantibodies to cerebroside sulphate (sulphatide). Anti-sulphatide IgM in groups of patients with lepromatous (LL) and borderline (BL + BB + BT), but not with tuberculoid (TT) disease, were significantly elevated above the levels found in endemic control subjects. Eight-six percent (18 out of 21; mean 1·59 OD units) of LL, 33% (four out of 12; mean 1·08OD units) of borderline and 13% (one out of eight; mean 0·69 OD units) of tuberculoid patients had anti-sulphatide IgM in their sera above a cut-off value of 2 s.d. above the mean value (0·66 OD units) for control sera. Elevated anti-sulphatide IgG was detected in only one patient's serum, an individual with LL disease. The level of anti-sulphatide IgM was strongly correlated to expression of the TH3 idiotype, an idiotype previously defined by a human MoAb that bound Mycobacterium leprae phenolic glycolipid, Klebsiella capsular polysaccharide, polynucleotides and human tissues. The purified, TH3 MoAb was found in this study to bind sulphatide, but not cholesterol-3-sulphate or cerebroside. It is suggested that antisulphatide IgM is elevated in leprosy, in relation lo the bacterial load. Anti-sulphatide IgM fell at the onset of erythema nodosom leprosum (ENL) reaction, consistent with the deposition of serum antibodies, and thus may play a part in pathology during periods of inflammation, particularly in multibacillary patients.