bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytoplasmic Calcium Fluxes Induced in Cytotoxic Effector Cells by Engagement of Fcγ Receptors I, II, and III
Authors: CURNOW S. J. , GLENNIE M. J. , STEVENSON G. T.
Published: June 2006

Changes in the intracellular calcium ion concentration ([Ca2+]i) of monocytes. granulocytes. and NK eells have been studied following either (1) independent cross‐linking of Fcγ receptors (FcγR) I, H, or III, with F(abγ')2 fragments of monoclonal antibodies: or (2) linking ofa selected FcγR to a tumour cell target with bispecilic F(ab'γ)2 antibodies. Upon cross‐linking each FcγR with antibody an increase in the ([Ca2+]i), was observed, although all receptors apart from FcγRIII on NK cells required additional cross‐linking with an anti‐mouse Fab', These results indicale that each type of receptor can transduce signals to the cell independently, Bispecific antibodies (anti‐FcyR X anti‐target) linking cytotoxic FcyR‐bearing effector cells to tumour target cells also mediated increases in ([Ca2+]i) for all FcγR tested except for FcγRIII on granulocytes. The failure to transduce a signal via ihis receptor may be related to the GPI link, which is in contrast to the transmembrane link of Fcγ RIII on NK cells. Significant lysis of tumour cell targets occurred when bispecific antibodies recruited NK cells or monocytes. bul not granulocytes, via FcγR. Chelation of intracellular Ca2+i in the effector cells redueed the observed lysis, suggesting a role for Ca2+i in the pathways leading to cytotoxicity.
Adhesion of Activated T Lymphocytes to Vascular Smooth Muscle Cells and Dermal Fibroblasts is Mediated by β1‐ and β2‐Integrins
Authors: STEMME S. , PATARROYO M. , HANSSON G. K.
Published: June 2006

Several studies during recent years have demonstraled the potential for vascular smooth musele cells (SMC) and dermal fibroblasts to participate in immune interactions such as antigen presentation and allorcactivity. The molecular interactions mediating lymphocyte adhesion to these mesenchymal cells have, however, not previously been eharacterized in detail. In the present study we demonstrate ICAM‐1 (CD54) expression by cultured human SMC and its up‐regulation by IL‐1. IFN‐γ, and bacterial lipopolysaccharide. Monoclonal antibodies were used to define ihe molecular interactions in the adhesion of 51Cr‐labelled T lymphoblasts to adherent SMC and fibroblasts. ICAM‐1 appeared lo mediate adhesion ofT lymphocytes by binding to the β2 integrin CDlIa/CD18 (LFA‐1) expressed by ihe lymphoblasts. We present evidence for the involvement of at least three different mechanisms in ihe adhesion of activated T lymphocytes to cultured fibroblasts. It was found that β2‐integrin‐mediated interaction could only account for less than half of the binding activity. The remaining adhesion was partly mediated by β2‐integrins, presumably via VLA‐5 since an anti‐VLA‐5 antibody and an RGD‐containing peptide blocked adhesion to the same degree. However, antibodies to β1‐, β2, and β3‐integrin subunits added together only inhibited adhesion by approximately 50% The residual adhesion could be blocked by inhibition of cell metabolism an d was increased by stimulation of the lymphocytes with phorbol ester, suggesting involvement of other, as yet undefined, adhesion molecules. The molecular interactions between lymphocytes and mesenchymal cells demonstraled in this study may have implicalions in several inflammatory conditions such as vaseulitis, atherosclerosis, and connective tissue diseases.
The LFA‐3 Adhesion Pathway is Differently Utilized by Superantigen‐Activated Human CD4+ T‐Cell Subsets
Authors: GJÖRLOFF A. , HEDLUND G. , KALLAND T. , SANSOM D. , FISCHER H. , TROWSDALE J. , SJÖGREN H. O. , DOHLSTEN M.
Published: June 2006

The superantigen SFA binds to MHC class II molecules and activates a large fraction off cells as a result of iteraction with particular TCR‐Vβ sequences. MHC class II transfected CHO cells induce a marginal CD4+ T‐cell proliferation in the presence of SEA. CHO cells transfected with both MHC class II and LFA‐3 a (HLA‐DR4/LFA‐3 double transfectants) supported a vigorous T‐cell proliferation and required 1000‐fold lower SEA concentration than DR4‐transfected eells. DR4/LFA‐3 double transfectants presenting SEA to CD4+ T cells induced large amounts of IFN‐γ, while single DR4 transfectants failed to elicit IFN‐γ production. CD4+ 45RA+ naive T cells proliferated much more strongly compared with CD4/45R0+ memory T cells when SEA was presented by the DR4/ LFA‐3‐transfected cells. In contrast, IFN‐γ production was only detected in CD4+ 45R0+ memory cells. The enhanced proliferation by the CD4+ 45RA+ naive T‐cells was not due to a stronger binding lo the aecessory DR4/LFA‐3 cells. Human CD4+ T‐cell lines medialed a low level of SFA‐dependent cell‐mediated cytotoxicity (SDCC) against DR4 target cells, whereas a strong SDCC was mediated against DR4/LFA‐3‐expressing target cells. These results demonstrate that superantigen‐activated human CD4+ T cells require the adhesion molecule LFA‐3 for optimal stimulation and thai Ihe CD4+ naive and memory T‐helper cells are different in their response to LFA‐3 as an accessory molecule.
Control of Trypanosoma cruzi Infection in Mice Deprived of T‐Cell Help
Authors: ROTTENBERG M. E. , RODRIGUEZ D. A. , ÖRN A.
Published: June 2006

The role of CD4+ T lymphocytes in the resistance of BALB/c mice to Trypanosoma cruzi was examined by in vivo depletion using monoclonal anti‐CD4 antibodies (MoAbs)<. When the administration of MoAbs was initiated 2 days before, or 5 to 12 days after the infeetion (dpi) with 50 bloodstream‐from trypomastigotes of the Tulahun strain, mice showed an enhanced susceptibility to the parasite. Speeific IgM. but not IgM responses, were inhibited in anti‐CD4‐treated and infected mice

However, when anti‐CD4 treatment of mice was delayed until the 8th week of infection. neither a reactivation of the infection as determined by mortality or parasitaemia. nor a modulation of the titre of anti‐ T. cruzi IgG antibodies was detected. Furthermore, mice chronically infeeted with T. cruzi and deprived of CD4 or T cells resisted the challenge with 50,000 trypomastigotes (approximately 1000 LD50)

Secondary antibody responses against parasite antigens were inhibited after in vitro depletion of CD4+ cells in chronically infected mice before boosting with T. cruzi antigens. However, recipients of CD4 or T‐cell‐depleled spleen cells from mice chronieally infected with T. cruzi were protected when challenged with the parasite

The possibility that the parasite control is maintained by long‐lived B cells capable of rapid differentiation into IgG‐secreting plasma cells in the absence of T helper cells is discussed considering the present data
Identification of Human T Cells that Require Zinc for Growth
Authors: MILLER G. G. , STRITTMATTER W. J.
Published: June 2006

Zinc is an essential trace clement required for normal tunction of Ihe immune system. Deficiency of zinc results in marked thymic atrophy in experimental animals, and in man immunodeficiency is a recognized complication of zinc deprivation. Althoug numerous proteins require zinc as a cofactor, its precise functions in the immune system remain unknown. The mechanism by which metals stimulate lymphocytes, whether all T cells are responsive, and the relationship to zinc requirements have not been determined. We unexpectedly isolated a number of human T‐cell lines that have a highly specific requirement for zinc. The ability to respond to zinc resides in only a subset of T cells since antigen‐specific clones are not stimulated by zinc. Although proliferation requires the presence of antigen‐presenting cells and is restricted by class II MHC antigens, antigen‐presenting cells could not be pulsed with zinc to induce T‐cell activation. Our results suggest that zinc‐dependent T cells are a subset of CD4+ cells present in all normal individuals and that zinc stimulates their growth by novel mechanisms.
Aberrations in Titre and Avidity of Serum IgM and IgG Antibodies to Microbial and Food Antigens in IgA Deficiency
Authors: CARDINALE F. , FRIMAN F. , CARLSSON B. , BJÖRLKANDER J. , ARMENIO L , HANSON L. Å.
Published: June 2006

The antibody levels and relative avidity of serum IgM and IgG aniibodies against E. coli O antigens, poliovirus type 1 and /J‐lactoglobulin were determined with enzyme‐linked immunosor‐bent techniques in IgA deficient (IgAd) patients with frequent respiratory tract infections an d healthy IgAd individuals. Heallhy individuals with normal tmmunoglobulin levels served as controls.

The IgM antibody levels against the bacterial, viral and food antigens and the IgG antibody levels against the bacterial antigens were significantly higher in the IgAd group with recurrent infections than in the group of healthy IgAd individuals. The symptomatic IgAd group had significantly higher levels of the IgG antibodies against the bacterial antigen, also when eompared with controls.

In contrast the healthy IgAd individuals had the highesi avidities of IgM antibodies to the viral and food antigens. The high avidities of aniibodies could be a compensatory host defence mechanism in IgAd.

These aberrations may appear as a consequence of increased mucosal exposure in IgAd to antigens such as E. coti or/f‐lactoglobulin. but presumably not to poliovirus which is only exceptionally present in the milieux. They could also be a result of the previously suggested dysregulation of antibody responses in IgAd.
Coding Region Polymorphisms of Human T‐Cell Receptor Vβ6.9 and Vβ21.4
Authors: HANSEN T. , RØNNINGEN K. S. , PLOSKI R. , KIMURA A. , THORSBY E.
Published: June 2006

Two new TCRVβ coding region polymorphisms were identified: Vβ6.9a/b and Vβ21.4a/b. In both cases, a single nucleotide difference gives rise to an amino acid exchange. Genomic typing by the PCR/sequence‐specific oligonucleotide probing technique was performed to study a possible contribution of these two new polymorphisms in susceptibility to autoimmune diseases. However, there was no association with insulin‐dependent diabetes mellitus, rheumatoid arthritis, juvenile rheumatoid arthritis, multiple sclerosis, myasthenia gravis or coeliac disease. On the other hand, significant differences were found between Caucasoid and Oriental populations in frequencies of the Vβ6.9 and Vβ21.4 alleles.
Treatment of Rheumatoid Arthritis with Single Dose or Weekly Pulses of Chimaeric Anti‐CD4 Monoclonal Antibody
Authors: CHOY E. H. S. , CHIKANZA I. C. , KINGSLEY G. H. , CORRIGALL V. , PANAYI G. S.
Published: June 2006

The aetiotogy of rheumatoid arthritis is unknown but CD4+ T eells are known to be involved in its pathogenesis. Because of this, anti‐CD4 monoelonal antibody has been used in open studies with clinical benefit in up to 60% of patients. We have used a ehimaeric anti‐CD4 monoclonal antibody (cM‐T4l2, Centocor) in a randomized, double‐blinded, placebo controlled trial as treatment for rheumatoid arthritis. Nine patients with active rheumatoid arthritis resistant to traditional disease‐modifying drugs were recruited. Four received an intravenous 50 mg bolus of antibody, and three received 50 mg weekly for four consecutive weeks. Two patients received placebo. Despite a marked reduction (P<0.001) in peripheral blood CD4+ lymphocytes, there was no significant clinical improvement in any of these patienis. The decrease in CD4+ lymphocyte number lasted one week after a single 50 mg dose of cM‐T412 but was more prolonged in the patients who reeeived four infusions. CD8+ T cells. CD16+ cytoloxic cells and CD14+ monoeytes showed only a transient reduction. It may be concluded that the therapeutic efficacy of anti‐CD4 therapy is not direetly related to CD4 T‐cell lymphopenia.
A Family of Cross‐Reacting Proteins Secreted by Mycobacterium tuberculosis
Authors: WIKER H. G. , NAGAI S. , HARBOE M. , LJUNGQVIST L.
Published: June 2006

Cross‐reactions between live proteins actively secreted by Mycohacierium tuberculosis were studied by crossed immunoelectrophoresis, SDS ‐PAGE with immuiioblotting. and ELISA using polyelonal rabbit aniisera and mouse monoclonal aniibodies to the purified proteins. The monoclonal antibody HBT4 was demonstrated to react with the MPT5I protein. The 85A, 85B and 85C constituents of the M. tuberculosis and Mycobacterium bovis BCG antigen 85 complex cross‐react extensively, each of the components containing component‐specific as well as cross‐reacting epilopes. These components also cross‐reacted with MPT5I and MPT64.

N‐terminal sequence studies revealed striking homology at the amino acid level between 85A, 85B, 85C and MPT51, MPT64 showed less homology. In addition, striking homology was demonslraled between two diflerent stretches within the K5B sequence and indicated between three siretches within the MPT64 molecule. Thus, a family of at least four secreted proteins with common structural features has been demonstrated in mycobacteria, MPT64 may also belong to this family
Analysis of HLA Genotypes and Susceptibility to Insulin‐Dependent Diabetes Mellitus: Association Maps Telomeric to HLA‐DP
Authors: BAISCH J. M. , CAPRA J. D.
Published: June 2006

There is convincing evidence that certain combinations of alleles within the human leucocyte antigen (HLA) complex, particularly within HLA‐DQ, are associated with either resistance or susceptibility to insulin‐dependent diabetes mellitus (IDDM). A previous study conducted on a large, well‐defined grou p of patients demonstrated that DQB 1*0302 (DQw8) conferred ‘dominant susceptibility’ to IDDM while DQB 1*0602 (DQwl.2) conferred ‘dominant protection’. The availability of this population enabled us to further assess susceptibility associated with other class II alleles in an effort to map an outside HL A boundary of disease association. Using a group‐specific polymerase chain reaction protocol and a series of oligonucleotide probes which define over twenty DPβ alleles, we studied 286 unrelated Caucasian patients with IDDM and 184 normal subjects.

We found that while several alleles are increased (DPB1*0201, DPB1*0301, DPB1*0402) or decreased (DPB1*0101, DPB1*0202) in the diabetic population compared with the normal subjects, the HL A association with IDDM is considerably weaker at the DP locus. These data define the centromeric boundary for the HLA‐associated susceptibility gene in IDDM, localizing susceptibility to the region telomeric to HLA‐DP up to and including HLA‐DQ
Peptide Binding to Class I Molecules of the Major Histocompatibility Complex on the Surface of Living Target Cells
Authors: KUBITSCHECK U. , LEVI R. , HORWITZ R. J. , ARNON R. , PECHT I.
Published: June 2006

Molecules encoded by the class I major histocompatibility genes bind short (nonameric) peptides produced by inlracellular proleolysis of antigens. These complexes formed intraeellularly are then expressed on membranes of target eells and recognized by the anligen receptor of eytolylic T cells. No binding of externally added peptides could so far be monitored directly on the antigen presenting cells, although cytotoxicity experiments and indirect binding assays provided evidence for its existence. Here we report experiments where specific binding to class I molecules, of externally added peptides, has been monitored on living cells, N‐terminal biottn‐labelled Kd‐restricted peptides (residues 147‐155, residues 147‐158, and an analogue lacking the arginine at position 156. derived from the sequence of the influenza A virus nucleoprotein) were incubated with murine H‐2Kd mastocytoma cells (line P815)dt 4°C. The binding on surface of hve, intact cells was then demonstrated fluoro metrically via ihe inieraction ofa streptavidin‐phycoerythrin conjugate with the biotin‐labelled peptides. Thus, this binding does not involve processing, and its specificity in terms of peptide structure was established by competition with the respective unmodified peptides. The specificity ofbinding to class I molecules was demonstrated by blocking experiments u.sing monoclonal antibodies specific for H‐2Kd. Finally, a correlation was observed between the results of peptide binding measurements and those ofcytotoxieity assays.
Nucleotide Sequence Analysis of Rheumatoid Factors and Polyreactive Antibodies derived from Patients with Rheumatoid Arthritis Reveals Diverse Use of VH and VL Gene Segments and Extensive Variability in CDR‐3
Authors: PASCUAL V. , VICTOR K. , RANDEN I. , THOMPSON K. , STEINITZJ M. , FØRRE Ø. , FU S.M. , NATVIG J. B. , CAPRA J. D.
Published: June 2006

The heavy and light chain nucleotide sequences of 17 monoreactive and polyreactive rheumatoid factors largely derived from the inflamed synovial tissue of two patients with rheumatoid arthritis are described. Some of these sequences have been the subject of a previous report from our laboratories. Additionally, a few rheumatoid factors from the peripheral blood of patients with systemic lupus erythematosus and Sjogren's syndrome as well as a normal individual are included. A review of our previous results as well as the new data provided within this paper lead to the following major conclusions: (1) Rheumatoid factors and polyreactive antibodies derive from a diverse array of VH and VL gene segments; (2) While many rheumatoid factors and polyreactive antibodies are direct or nearly direct copies of germline genes, some show clear evidence of somatic mutation; (3) The CDR3 of all of these antibodies is extraordinarily diverse in length and composition. Certain ‘restrictions’ do appear in this very large sample: (a) the polyreactive antibodies are exclusively lambda, and (b) there seems to be a preponderance of a particular subset of VH3 genes beyond that one would expect based on random utilization.
Hypothesis for the Control of Clotting Factor VIII Inhibitory Antibodies by Decreasing Potency of Helper T‐Cell‐Recognized Epitopes in Factor VIII
Authors: Tiarks C. , Humphreys R. E. , Anderson J. , Mole J. , Pechet L.
Published: June 2006

The study of the immunobiology of FVIII inhibitors may lead to new therapies for this potentially severe complication of haemophilia A and to new principles for the use of therapeutic proteins. In order to characterize the idiotype‐anti‐idiotype networks regulating FVIII inhibitors, we developed rabbit antiidiotypic sera to 7 murine inhibitors and found at least 12 independent FVIII loci to which inhibitors could be raised. Rabbit antisera to the FVIII peptide. Ser1687‐Thr1695 characterized one functional site to which about 46% of patients' inhibitor sera reacted. The multiplicity of inhibitor‐recognized epitopes in FVIII makes it impractical, at the present time, to develop clinically useful specific anti‐idiotypic therapies for FVIII inhibitors. Alternatively, one might induce genomic mutations in recombinant FVIII molecules to decrease immunogenicity of epitopes recognized by T helper cells. Methods to design such altered therapeutic proteins are presented, based on changing the longitudinal hydrophobic strip of‐helix which is in or near many T‐cell‐presented epitopes.
Adhesion Molecules Involved in the Interaction of LGL/NK Cells and Human Endothelial Cells Stimulated with Salmonella Bacteria
Authors: PINOLA M. , SAKSELA E.
Published: June 2006

Previously we showed that pretreatment of LGL/NK or HUVE cells with Salmonella bacteria augments the adhesion of LGL/NK cells to endothelium. Here we analyse the roles of HUVEC adhesion molecules VCAM‐l, ICAM‐I and E‐selectin, and the counter‐receptors VLA‐4, LFA‐1 and SLe3 in the increase of LGL/NK adhesion to HUVEC, stimulated with Salmonella Minnesota mR595 bacteria. LPS or TNF‐α.

On salmonella‐stimulated HUVEC, VCAM‐l and ICAM‐1 were the major binding structures involved, and their effect was additive in monoclonal antibody inhibition experiments. We could demonstrate the induction of both structures on cultured HUVEC after 24 h of Salmonella stimulation in flow cytometric analysis. For salmonella‐stimulaled LGL/NK, the principal binding structure was LFA‐1, Stimulation of LGL/NK cells did not alter the expression of the adhesion structures (subunits CD11a/CD18, CD49d/CD29). as determined by flow cytometric analysis, and thus the increased adherence is presumably produced by an increased avidity of the receptors on LGL/NK cells.

Pretreatment of endothelium or lymphocytes with various stimuli, including Salmonella bacteria or LPS, leads to an activation state which provides for characteristic anchorage sites for the increased migration of LGL/NK cells towards the site of inflammation.
T‐Cell Receptor V‐Gene Usage in Synovial Fluid and Synovial Tissue from RA Patients
Authors: GUDMUNDSSON S. , RÖNNELID J , KARLSSON‐PARRA A. , LYSHOLM J. , GUDBJÖRNSSON B. , WIDENFALK B. , JANSON C. H. , KLARESKOG L.
Published: June 2006

The question of whether there is a preferential use of certain V genes in T cells entering an inflamed joint has hitherto been studied mainly using unfractionated cells from synovial fluid and tissue respectively, and no clear answer to the question has yet been provided. Concomitantly. evidence has been provided that the use of V genes may differ considerably between CD4+ and CD8+ T cells, and consequently that detection of biased V‐gene expression within an inflammatory lesion may require separate analysis of the two T‐cell subsets.

In this paper we have therefore studied T‐cell receptor V‐gene expression in rheumatoid arthritis by means of double stainings of synovial fluid and blood for available anti‐TCR monoclonal antibodies and antibodies to CD4 and CD8. respectively. Double stainings were also performed with anti‐TCR antibodies and antibodies to activation markers HLA‐DR and 1L‐2R. A certain bias towards the preferential use of certain V genes was seen particularly in the synovial fluid samples within both the CD4+ and CD8+ T‐cell populations, but no uniform pattern was evident among the 35 patients investigated.
Isoelectrophoretic Characterization of Protein Antigens Present in Mycobacterial Culture Filtrates and Recognized by Monoclonal Antibodies Directed Against the Mycobacterium bovis BCG Antigen 85 Complex
Authors: DROWART A. , DE BRUYN J. , HUYGEN K. , DAMIANI G. , GODFREY H. P. , STELANDRE M. , YERNAULT J.‐C. , VAN VOOREN J.‐P.
Published: June 2006

Nine monoclonal antibodies (MoAbs) were produced againsi Mycobacterium bovis BCG aniigen 85 complex. Using isoelectric focusing combined with Western (immunoblot) blot analysis, antigenically related proteins could be identified in culture filtrates from M. tuberculosis, M. bovis. M. kansasii, M. aviumi, M. xenopi, M. gordonde, M. fortuitum, M. phlei M.smegmatis, Most of the MoAbs were found to be broadly cross‐reactive between the various mycobacterial species, albeit some minor differences were observed. These MoAbs reacted generally, in each species, with different components. One MoAb (VIDl‐14) was found to be specifically directed only against antigen 85B from M. bovis. M. tuberculosis and M. kunsasii.
The Role of BCG/PPD‐Activated Macrophages in Resistance against Systemic Candidiasis in Mice
Authors: VAN't WOUT J. W. , POELL R. , VAN FURTH R.
Published: June 2006

The main conclusions of this study are that BCG/PPD‐activated macrophages, in contrast to macrophages from control mice, exhibit an increased PMA‐induced production of H2O2. kill about one‐third of the phagocytosed Candida albicans, and cause more than 5O% inhibition of the intracellular formation of germ tubes by C albicans. Peritoneal macrophages from mice that were colonized post‐natally with C. albicans do not show increased produciion of H2O2: upon stimulation with PMA and the intracellular outgrowth of germ tubes is inhibited to only a limited degree. These macrophages are capable of killing about 2O% of the ingested C, albicans. In vivo, the number of Candida in the kidney, spleen and liver after intravenous injection of Candida albicans is significantly lower in BCG‐treated mice than in control mice. Post‐natal colonization with C. albicans has only a limited effect on the outgrowth of intravenously injected C albicans the spleen and liver but does not influence growth in the kidney. These results indicate that acquired immunity against a systemic Candida infection involves both oxidative and nonoxidative mechanisms of intracellular killing and that these mechanisms may have different effects on the yeast and hyphal forms of C. albicans.
Antigen‐Activated T Cells Inhibit Cartilage Proteoglycan Synthesis Independently of T‐Cell Proliferation
Authors: WILBRINK B. , HOLEWIJN M. , BIJLSMA J. W. J. , VAN ROY J. L. A. M. , VAN DER ZEE R. , BOOG C. J. P. , DEN OTTER W. , VAN EDEN W.
Published: June 2006

Previously we have shown that blood mononuclear cells (MNC) obtained from patients with rheumatoid arthritis (RA) have the capacity to induce depletion of proteoglycans (PG) in human cartilage explants. This was observed especially after stimulating MNC with mycobacterial antigens, rather than with the miiogen Concanavalin A (Con A). We have now co‐cultured cartilage explants in the presence of T‐cell clone A2b obtained from the rat model of adjuvant arthritis (A A). We show that inhibition of the cartilage PG synthesis is a consequence of antigenspecific T‐cell activation and that it is mediated by a humoral factor. This seems to be a cytokine rather than an enzyme. Moreover, at the level of polyclonally responding T cells. inhibition of PG synthesis due to T‐cell activation by mycobacterial antigens was shown to depend on prior mycobacterial immunization. Arthritogenic T‐cell clone A2b also showed PG synthesis inhibitory effects when co‐cultured with cartilage alone. The inhibitory activity was shown to be unrelated to the degree of T‐cell proliferation. We conclude that antigen‐specific T‐cell activation may be one of the initiating events leading to cartilage damage in arthritic processes. The measurement of T‐cell‐mediated PG synthesis inhibition may be a more sensitive and relevant assay for the detection of pathogenic T cells than T‐cell proliferation.
Tumour Necrosis Factor‐β Gene RFLP Alleles in Finnish IDDM Hapiotypes
Authors: ILONEN J. , MERIVUORI H. , REIJONEN H. , KNIP M. , ÅKERBLOM H. K. , POCIOT F. , NERUP J. ,   the Childhood Diabetes in Finland (DiMe) Study Group
Published: June 2006

The genes located between class II and class I HLA genes including polymorphic tumour necrosis factor (TNF) genes may contribute to the disease susceptibility in IDDM. Restriction fragment polymorphisms of the TNF‐β gene have been found to be fixed in the major IDDM susceptibility haplotypes, the B62,DR4 haplotype being associated with the 10.5‐kb fragment and the B8,DR3 haplotype with a 5.5‐kb fragment. We studied this TNF polymorphism in a sample of diabetic families. In all IDDM‐associated haplotypes (n= 129) the 5.5‐kb allele was more frequent than in haplotypes found only in healthy family members (n=112) (58.1% versus 40.2%, P<0.01). Among IDDM haplotypes the B62,DR4 haplotype was characterized by the 10.5‐kb TNF fragment, whereas two other common Finnish IDDM‐associated DR4 haplotypes‐A24,B39,DR4 and A2,B56,DR4‐had the 5.5‐kb TNF fragment. Both IDDM‐associated and non‐associated DR3 positive haplotypes were linked to the 5.5‐kb fragment. The distribution of various combinations of TNF alleles in IDDM probands (n= 63) did not differ from that expected according to the Hardy‐Weinberg distribution. Our results indicate that the 10.5‐kb allele of TNF‐β gene as such is not a risk factor contributing to DR4/DQ8‐associated susceptibility. Alternatively, there may be heterogeneity in pathogenetic effector mechanisms.
Dopamine‐Induced Lymphoma Cell Death by Inhibition of Hormone Release
Authors: BRAESCH‐ANDERSEN S. , PAULIE S. , STAMENKOVIC I.
Published: June 2006

Dopamine inhibits prolactin release from pituitary cells and seems to affect the release of several other hormones as well. We report here that dopamine may have similar effects on human B lymphoma cells leading to inhibition of production or release of endogenous factors required for cell viability and proliferation. Thus, addition of dopamine to serum‐free cultures of Burkitt lymphoma cells (Raji, Namalwa, Daudi and Jijoye) resulted in rapid and extensive cell death while a myeloma cell line, SKO, appeared to be refractory to this treatment. The addition of FCS or supernatant from serum‐free cultures of Raji or T24 bladder carcinoma cells could, to a variable degree, counteract the effect of dopamine, suggesting that dopamine acts by inhibiting the production of essential autocrine factors. When two of the hormones known to be under dopamine control, i.e. prolactin (PRL) and thyrotropin (TSH), were tested, they were able to prevent dopamine‐induced cell death if combined with heparin. We further observed that the reducing agent 2‐mercaptoethanol (2‐ME), which is known to inhibit the binding of TSH to its receptor, displayed similar effects to those of dopamine and was strongly inhibitory for Burkitt lymphoma but not for myeloma cells. As expected from its blocking activity at the receptor level, the effect of 2‐ME could not be reversed by adding exogenous factors. Contrary to its effect on B lymphoma cells, 2‐ME is essential for growth of the murine T‐cell lymphoma line CTLL. However, we show here that dopamine can fully compensate for 2‐ME, suggesting that TSH or another factor under dopamine control is intimately involved in the regulation of T‐cell growth. This study lends further support to the notion of an active interplay between the neuroendocrine and immune systems and emphasizes PRL and TSH as important regulators of lymphoid cell function. It also shows that these hormones may contribute to the autonomous growth pattern of B lymphoma cells and suggests a potential role for dopamine in the treatment of B‐cell tumours.
Autoantibodies to the Nuclear Sp100 Protein in Primary Biliary Cirrhosis and Associated Diseases: Epitope Specificity and Immunoglobulin Class Distribution
Authors: SZOSTECKI C. , WILL H. , NETTER H. J. , GULDNER H. H.
Published: June 2006

Sp100, a protein with a dot‐like intranuclear localization in immunofluorescence microscopy, is a major target for patient autoantibodies in primary biliary cirrhosis (PBC) and occasionally in rheumatic disorders. The human Sp 100 cDNA has recently been cloned, and the deduced amino acid sequence was found to contain sequence similarities with an MHC class I domain and several transacting regulatory proteins, including HIV‐1 nef proteins. In this study, recombinant Sp100 fusion proteins were used to differentiate the immunoglobulin isotypes and to map the epitopes involved in the anti‐Sp100 autoimmune response. PBC patients developed IgG as well as IgM and/or IgA class anti‐Sp100 autoantibodies whereas most patients with rheumatic diseases developed IgG class autoantibodies only. For epitope mapping, truncated versions of the Sp100 protein were probed for immunoreactivity in ELISA and immunoblotting. With 55 sera, 17 different reaction patterns were obtained, and at least three non‐overlapping major autoantigenic domains were recognized by the majority of sera. One domain, which contains the sequence similarity with HIV nef proteins, was recognized by all anti‐Sp100 sera and harbours multiple, in part discontinuous, epitopes. These data demonstrate a heterogeneous and patient‐specific anti‐Sp100 autoimmune response which is antigen‐driven and, at least in terms of isotype composition, different in PBC and non‐PBC patients.
Interleukin 4 and Tumour Necrosis Factor α Induce Different Adhesion Pathways in Endothelial Cells for the Binding of Peripheral Blood Lymphocytes
Authors: GALÉA P. , LEBRANCHU Y. , THIBAULT G. , BARDOS P.
Published: June 2006

We demonstrated that tumour necrosis factor α (TNF‐α) and interleukin 4 (IL‐4) increased endothelial cell (EC) adhesiveness for peripheral blood lymphocytes (PBL) by promoting transcription and protein synthesis. Theo different kinetics observed with TNF‐α and IL‐4 suggest the involvement of different adhesion molecules.

Blocking adhesion assays and immunofluorescence analysis showed that PBL adhesion to endothelial cells involves different pair adhesion molecules. Whereas IL‐4 promoted an LFA‐1‐dependent/ICAM‐1‐independent adhesion pathway on EC, TNF‐α stimulated an LFA‐1‐dependent/ICAM‐1‐dependent adhesion pathway on EC. In conttast. VLA‐4/VCAM‐1 molecules were involved in PBL adhesion both to IL‐4 and to TNF‐α‐stimulated EC. Finally, we found that a CD2‐dependent/LFA‐3‐independent adhesion pathway was mainly involved in IL‐4‐stimulated EC.
Analysis of Mature Guinea Pig T Cells with a MOnoclonal Antibody directed against a Framework Determinant of the T‐Cell Receptor for Antigen
Authors: SCHÄFER H. , BURGER R.
Published: June 2006

Rats were immunized with guinea pig T lymphocytes and the spleen cells were fused with cells of a mouse myeloma line. The resulting hybrids were screened for the production of antibodies selectively reacting with guinea pig T cells. The monoclonal antibody (MoAb) H159 was analysed in detail because it bound to T lymphocytes, but not to B lymphocytes or macrophages. Cellular ELISA cytofluorometry and immunohistology revealed that the antigen detected by H159 is selectively expressed on the majority of peripheral mature T lymphocytes (about 95%). In contrast, it stained only a minor population of thymocytes in FACS analysis. H159 precipitated from NP40 lysate of T cells a protein with a molecular weight of about 90 kDa when separated under non‐reducing conditions in SDS‐PAGE. Under reducing conditions bands with molecular weights of about 50 kDa were found. After binding to anti‐rat Ig coated beads, the MoAb H159 had a mitogenic effect for guinea pig T lymphocytes whereas soluble MoAb H159 in the presence or absence of macrophages was not mitogenic. The cellular expression and molecular characeristics of the H 159 antigen together with the mitogenic activity of the antibody for T cells indicate that the MoAb H159 recognizes the guinea pig T‐cell receptor for antigen via a constant region determinant.
Productin of Basic Fetoprotein in Human Peripheral Lymphocytes during Blastic Transformation
Authors: FUJITA Y. , FUJITA N. , NAKAMURA T. , GOTO T. , FUJII T. , HIRAMORI N. , KIDA T. , SAKAI M. , NAKAGAKI Y. , IWASAKI Y. , NAKAGAWA M.
Published: June 2006

To clarify the significance of basic fetoprotein (BFP) in lymphocytes, we investigated whether BFP is produced in lymphocytes during blastic transformation. Peripheral blood lymphocytes obtained from 14 adults were cultured under the stimulation of lectins. The concentration of BFP in the culture medium (extracellular BFP) was estimated serially. The incorporation of [6‐3 H] thymidine was assayed simultaneously. The intracellular BFP was measured by dual flow cytometry for DNA and BFP. A lymph node was studies immunohistochemically. Serum BFP was measured in four cases of lumphocytic leukaemia. In two cases, dual staining was performed. The intracellular BFP of the mitogen‐stimulated lymphocytes was increased within 24 h. The extracellular BFP was increased exponentially from 72 h. The extracellular BFP at 96 h did not correlate with the [3H]‐thymidine incorporation. The intracellular BFP increase began in G1 phase. Immunostaining showed that the B cells also produced BFP. The

serum BFP level in leukaemia was high in 1 of 4 cases and the leukaemic cells in two cases showed high intracellular BFP content. These observations indicate that BFP is produced in activated human lymphocytes and in lymphocytic leukaemic cells. The production of BFP during blastic transformation will be a useful new in vitro model for studying the biological role of BFP, and BFP labelling may offer some new possibilities for study of lymphocytes.
Use of an Anti‐Idiotypic Monoclonal Antibody in Studying Amyloidogenic Light Chains in Cells, Urine and Fibrils: Pathophysiology and Clinical Implications
Authors: BELLOTTI V. , STOPPINI M. , PERFETTI V. , ZORZOLI I. , MARINONE G. , INVERNIZZI R. , ZAMBELLl L. M. , ARBUSTINU E. , GRASSO M. , FERRI G. , ASCARI E. , MERLINI G.
Published: June 2006

A monoclonal anti‐idiotype antibody (IgG1k MoAb 3B11D4) raised against the amyloidogenic DEPλ chain dimer binds a conformational idiotope also present on the monoclonal DEP IgA immunoglobulin. MoAb 3B11D4 does not recognize the reduced and alkylated λ chain monomers, nor the 15‐17‐kDa fibrillar light chain fragments which have the same N‐terminal sequence of the urinary light chains. The lack of about 70 ammo acid residues ofthe C terminal of the protein prevents the formation of the self‐limiting dimer and may facilitate the deposition of the fragments into amyloid fibrils. MoAb 3B11D4 recognizes the plasma cell clone in bone marrow and 9% of circulating B lymphocytes. Panning experiments demonstrate that this antibody has the capability to selectively eliminate the idiotvpe positive cells from peripheral blood. Antibodies with these characteristics could become a useful tool for better understanding the pathogenesis of the disease and for new therapeutic options.
The Influence of Adjuvants on the Generation of Autoantibody and Specific Suppression in Rat Erythrocyte‐Immunized Mice
Authors: ELLIOTT J. I. , HUTCHINGS P. , MALKOVSKY M. , COOKE A.
Published: June 2006

A number of adjuvants were investigated for their ability to modulate either the autoimmune response induced in mice by immunization with rat erythrocytes (RRBC) or the ability of RRBC‐primed spleen cells to suppress the induction of anti‐red cell autoimmunity in recipient mice. The inability of the agents used to do so is discussed on the background of models used to explain the generation of suppression in this system.
The Influence of I‐E on the Generation of Autoantibody and Specific Suppression in Rat Erythrocyte‐Immunized Mice
Authors: ELLIOTT J. I. , COOKE A.
Published: June 2006

A portion of the antibodies produced by mice in response to the injection of rat erythrocytes (RRBC) cross‐react with autologus red cells. When spleen cells from mice so immunized are transferred to naive syngeneic recipients, the recipient mice produce high anti‐RRBC antibody titres but little or no autoantibody. This phenomenon has been attributed to the action of suppressor T cells. To date, the only mouse strain which consistently fails to demonstrate specific suppression of the autoantibody response is the SJL, which lacks the I‐E molecules suggested to be important in the generation of suppressor T cells.

The results presented here show that some, but not all, I‐E negative strains of mice are capable of exhibiting transferable suppression of RRBC‐induced antibodies.
Development of the Non‐Palindromic Adaptor Polymerase Chain Reaction (NPA‐PCR) for the Amplification of Alpha‐and Beta‐Chain T‐Cell Receptor cDNAs
Authors: CHEN P.‐F. , PLATSOUCAS C. D.
Published: June 2006

We have developed a highly efficient new method for the amplification of α‐ and β‐chain human T‐cell receptor (TCR) cDNAs. This method is designated non‐palindromic adaptor polymerase chain reaction (NPA‐PCR). cDNA was synthesized from total RNA isolated from mono‐nuclear leucocytes, using either an oligo (dT)15‐Not 1 or a Cα‐Not 1 or a Cβ‐Not 1 primer and RNase H‐negative reverse transcriptase. The double‐stranded cDNA was ligated with the non‐palindromic adaptors EcoRI‐Xmn I [d(ATTCGAACCCCTTCG)] and Xmn I G strand [d(pCGAAGGGGTTCG)] (phosphorylated), which resulted in the addition of the EcoR I‐Xmn I site in both 5′ and 3′ ends. These two non‐palindromic adaptors, EcoR I‐Xmn I and Xmn I G strand, are complementary to each other and both are required for ligation. The EcoRI‐Xmn I adaptor was removed from the 3′ end by treatment with Not I restriction nuclease, whereas it was retained al the 5′end. The non‐palindromic adaptor EcoR I‐ Xmn I was used as the 5′ amplitication primer. Cα or Cβ constant region primers were used as 3′ amplification primers. The amplified cDNAs were cloned and the plasmids were used to transform DH5α competent cells. Over 1000 white colonies per 0.1–0.25 μg of total RNA or per 10,000 to 50,000 human peripheral blood mononuclear cells were obtained after amplification of either the α‐ or the β‐chain TCR cDNAs. Between 40 and 62% of the colonies (range from live donors) were positive after screening with either a Cα or a Cβ probe, located 5′ to the Cα and Cβ amplification primers. A total of 50 amplified α‐ or β‐chain cDNA positive clones from two normal donors were randomly chosen and sequenced, and the sequences obtained were typical of αβ TCR. Two new Jα gene segments were identified. Approximately 30% of the α‐chain positive clones have 5′ untranslated region, and most of the remaining α‐ or β‐chain TCR clones started from the initiation codon or near the 5′ end. NPA‐PCR has several advantages over existing PCR methods for the amplification of cDNAs with unknown or variable 5′ end, such as the T‐cell antigen receptors and the immunoglobulins. Among these advantages is that only one 5′ end extension primer is required. Because of the large number of TCR Vα and Vβ families, a large number of different 5′ end primers are required for amplification of αβ TCR cDNAs by conventional PCR.
Expression of CD26 (Dipeptidyl Peptidase IV) on Memory and Naive T Lymphocytes
Authors: ULMER A. J. , MATTERN T. , FLAD H.‐D.
Published: June 2006

It has been suggested that human CD26‐positive T lymphocytes represent the memory pool of the cellular immune system. For proof of this suggestion we analysed the responsiveness or CD26‐positive and CD26‐negative T lymphocytes after antigenic stimulation in limiting dilution experiments. After stimulation with tetanus toxoid (TT) the number of proliferating cells within the CD26‐posiiive subset was two‐ to sixfold higher than that within the CD26‐negative subset. These differences in responsiveness were also detectable between CD4+/CD26+ and CD4+ CD26‐ T cells. To further investigate the memory character of the cells, human peripheral blood mononuclear cells were stimulated with TT for 7 or 14 days. Thereafter, CD26+ and CD26‐ T cells were isolated and restimulated with TT in limiting dilution experiments. Responding cells were found not only within the CD26‐positive subset but also within the CD26‐negative subset, and their number increased with time. Surface marker analysis of freshly isolated human T lymphocytes or T‐cell clones indicated that CD26 is not a stable cell surface marker. Furthermore, CD26 is both absent and present on CD29‐positive or CD45RA‐positive cells, indicating no association of CD26 with surface markers for memory or naive T cells, respectively. These results strongly argue against the hypothesis that CD26‐positivc T cells represent the memory pool. We conclude that CD26 is an activation marker of T lymphocytes, which is associated with reactivity on naive and memory cells.
B Lymphoblasts show Oxidase Activity in Response to Cross‐Linking of Surface IgM and HLA‐DR
Authors: FURUKAWA K. , TENGLER R. , NAKAMURA M. , URWYLER A. , DE WECK A. L. , KANEGASAKI S. , MALY F.‐E.
Published: June 2006

Human B lymphocytes express components of the superoxide generating system of phagocytes, NADPH oxidase. We studied regulation of this ‘B‐cell oxidase’ during in vitro blast transformation, using Lucigenin‐amplified chemiluminescence (CL) to detect superoxide release. While freshly isolated tonsil B lymphocytes showed no CL responses, culture with phorbol myristate acetate (PMA) and ionomycin induced susceptibility to CL triggering by anti‐IgM and anti‐HLA‐DR. Maximal effects were observed after 3 days of culture with 0.4 ng/ml PMA+1μg/ml ionomycin. Cells from such B lymphoblast cultures showed no CL responses to opsonized zymosan. In contrast, peripheral blood mononuclear cells, where monocytes are the predominant oxidant source, showed CL responses to opsonized zymosan but not to anti‐IgM and anti‐HLA‐DR, either before or afterculture with PMA+ionomycin.

Culture of B cells with the surface immunoglobulin cross‐linking agent staphylococcus aureus Cowan 1 also led to emergence of a CL response to anti‐IgM, which was enhanced by interferon‐γ. Interestingly, markedly fewer B blasts than freshly isolated B lymphocytes expressed cytochrome b‐558 surface antigen. Thus, the B‐cell oxidase is up‐regulated during blast transformation and can be triggered via surface IgM and HLA‐DR; however, this appears to be restricted to a subset of B lymphoblasts.
Transplantation of Cultured Thymic Fragments in Congenitally Athymic and Euthymic Rats Culture with Deoxyguanosine or Cyclosporin A does not influence the Histologic Characteristics and Outcome after Transplantation in Syngeneic and Allogeneic Combinations
Authors: MARTíN‐FONTECHA A. , BROEKHUIZEN R. , DE HEER C. , ZAPATA A. , SCHUURMAN H.‐J.
Published: June 2006

Cultured thymic fragments (CTF) from WAG/CPB(RTU and DA/01a (RT1a rats were prepared in the presence or absence of 2′ deoxyguanosine or cyclosporine A, and subsequently transplanted under the kidney capsule of congenitally athymic and euthymic WAG CPB recipients. The rationale of the culture supplements was that these may affect the disappearance of medullary dendritic cells, with subsequent induction of allotolerance. However, the immunohistology of the CTF showed more RTI class II‐positive cells than keratin‐positive cells, indicative of the maintenance of dendritic cells. Grafts in athymic animals showed the recovery of the original thymic architecture within 6 weeks utter transplantation. The influx of host‐derived lymphocytes was accompanied by an influx of dendritic cells in the deculla‐like area and macrophages in the cortex. A similar recovery was observed for syngeneic CTF in euthymic recipients. In addition lymphocytic infiltration was seen in the connective tissue surrounding the epithelial areas. Allogeneic grafts in euthymic animals were rejected within 3 weeks after transplantation. This outcome of the transplanted CTF under different conditions was not affected by the supplementation of the thymic culture before transplantation with 2′ deoxyguanosine or cyclosporine A.

We conclude that there is no tolerance induction after transplantation in euthymic allogeneic rats of CTF prepared in the presence of 2′ deoxyguanosine. This conclusion is in contrast to date in the mouse, which may be explained by the maintenance of dendrite cells during culture. A chimaeric state of donor‐derived epithelium and host‐derived dendritic cells is obtained by transplantation of allografts in arthymic rats.
T Cells Cloned from Human Rheumatoid Synovial Membrane Functionally Represent the Th 1 Subset
Authors: MILTENBURG A. M. M. , VAN LAAR J. M. , DE KUIPER R. , DAHA M. R. , BREEDVELD F. C.
Published: June 2006

The presence of activated T cells in the synovial membrane of patients with rheumatoid arthritis (RA) suggests a role for these cells in the pathogenesis of the disease, Recent evidence indicates that human T cells may fall into functional categories dependent on their cytokine profile and cytotoxic capacity. The human Th 1 subset is cytolytic and produces high levels of IFN–gamma whereas the Th2 type of T cell produces IL–4. In order to investigate whether Th 1 or Th 2 type cells are present in the inflammatory synovial membrane in RA. a panel of synovial membrane derived T–cell clones (n=19) was generated and studied functionally. Anti–CD3–induced cytotoxicity assays were performed to demonstrate the cytotoxic potential of clones. Except for two, all clones were cytotytic in this lest. Clone cells were activated Io initiate cytokine production and assessment of the cytokine levels showed that all clones produced large amounts of IFN–gamma (18 out of 19 clones: over 50,000 pg/ ml) whereas IL–4 was absent or present in minimal amounts (17 out of 19 clones: less than 1000 pg/ml). The production of IL–1, IL–2 and IL–6 was variable.

The functional characteristics of the clones studied indicate that they may resemble the Th 1 subtype of T ceils. Our data suggest a relation between Th1–type functions and the chronic inflammation characteristic of RA.