bluuebunny/crossref_metadata_2025 huggingface dataset:
Oncogene Transformation can induce Tolerogenicity in Murine Macrophages after Down‐Regulation of Immunogenicity without altering Major Histocompatibility Complex Antigen Expression
Authors: WOTTGE H.‐U. , ECKSTEIN V. , SELIGER B. , MÜLLER‐RUCHHOLTZ W.
Published: July 2006

In vitro studies on cell lines may allow analyses of the mechanisms of immunogenicity and tolerogenicity in cells. We used a model of oncogenic transformation of an established murine macrophage cell line and report here that one v‐mos‐transformed clone expressing unaltered high amounts of MHC class I and II antigens does not induce proliferation of unprimed T cells in primary mixed lymphocyte reactions, in sharp contrast to its non‐transformed parental cells. Interestingly, this clone induces specific unrespon‐siveness, as revealed by the lack of responsiveness of MHC‐specific T cells when subsequently exposed to the pertinent MHC alloantigens in immunogenic form but unaltered MHC‐third party responsiveness of the naive spleen T cells. Furthermore, the accessory function of this clone is strongly reduced. These functional defects could be overcome by the addition of exogenous interleukin‐1α (IL‐1α). Analysis of mRNA expression showed a significant and selective reduction of IL‐1α mRNA levels when compared with parental cells.
Adoptive Transfer of Low Numbers of CD4+ T Cells into SCID Mice chronically treated with Soluble IL‐4 Receptor does not prevent Engraftment of IL‐4‐Producing T Cells
Authors: RUDOLPHI A. , ENBLE K.‐H. , CLAESSON M. H. , REIMANN J.
Published: July 2006

After intravenous injection of 105 purified, lymph node (LN)‐derived dm2 (H‐2d/Ld) CD4+ T cells into young C.B‐17 scidjscid (severe combined immunodeficiency, SCID) mice (H‐2d/Ld+), the transplanted Ld‐ T cells show a selective pattern of engraftment: they repopulate the spleen, the lamina propria of the small intestine and the mesenteric LN (but not other peripheral LN) of the immunodeficient host. CD4+ cells repopulating different lymphoid organs of the SCID recipient mice produce interleukin‐2 (IL‐2) and interleukin‐4 (IL‐4) in response to polyclonal stimulation in vitro. Some evidence has recently been provided that cytokines (e.g. IL‐4) present at the site of antigen stimulation in vivo decisively influence the pattern of cytokines expressed by T cells activated at these sites. We therefore asked if neutralization of IL‐4 by chronic treatment of SCID mice with high doses of recombinant soluble IL‐4 receptor (sIL‐4R) changes the IL‐4 or IL‐2 expression pattern of CD4+ T cells adoptively transferred into young SCID recipients. Transplanted SCID mice were chronically treated with two different, recombinant murine sIL‐4R proteins. The experimental series further included groups of transplanted SCID mice treated with a recombinant human sIL‐4R protein (which does not bind murine IL‐4), treated with the anti‐murine IL‐4 monoclonal antibody (MoAb) 11B11, or non‐treated. Transplanted SCID mice treated with the recombinant murine sIL‐4R protein preparations displayed detectable sIL‐4R serum levels, which demonstrates that the substitution therapy could maintain neutralizing serum levels of anti‐IL‐4 activity in SCID mice. By contrast, no serum sIL‐4R levels were detectable in the sensitive ELISA readout in transplanted SCID mice which were non‐treated, treated with the MoAb 11B11, or treated with the recombinant human sIL‐4R protein. The efficiency and the pattern of CD4+ T‐cell engraftment, and the lymphokine‐producing phenotype of the engrafted dm2 CD4+ cells, was not affected by the continuous IL‐4‐neutralizing treatment of mice with either the MoAb 11B11 or the soluble IL‐4R preparations. Hence, in contrast to the published evidence of the dramatic effect of IL‐4 on the lymphokine‐producing phenotype of CD4+ T cells stimulated in vitro or in vivo, the chronic suppression in vivo of IL‐4 activity (by either different sIL4‐R protein constructs, or by the anti‐IL‐4 MoAb 11B11) did not lead to preferential engraftment of Th1‐type CD4+ T cells after adoptive transfer of CD4+ T‐cell populations into an immunodeficient recipient.
Antibody Response of Restricted Isotype to Heat Shock Proteins in Juvenile Chronic Arthritis
Authors: NUALLÁIN E. M. Ó , MONAGHAN H. , REEN D. J.
Published: July 2006

Synovial fluid and peripheral blood mononuclear cells from juvenile chronic arthritis (JCA) patients have previously been shown to exhibit substantial proliferative responses to both human and mycobacterial heat shock protein (hsp) 65. We investigated the nature of the antibody response to mycobacterial and E. coli hsp 65 and human and E. coli hsp 70 in 56 JCA patients using an ELISA.

Elevated levels of antibodies to both human and E. coli hsp 70 were demonstrated. With hsp 65, raised levels of antibodies to the mycobacterial but not the E. coli protein were detected. Overall, 48% of patient serum samples contained antibodies of at least one isotype to mycobacterial hsp 65. These antibodies were predominantly of IgG and IgM isotype, a finding in contrast to adult rheumatoid arthritis, where IgA and IgG isotypes are most often detected.
Reactivity of Human γδ T Cells to Staphylococcal Enterotoxins: a Restricted Reaction Pattern mediated by Two Distinct Recognition Pathways
Authors: RUST C. , ORSINI D. , KOOY Y. , KONING F.
Published: July 2006

Staphylococcal enterotoxins (SEs) are known superantigens for T cells expressing the αβ T‐cell receptor (TCR). They bind to MHC class II molecules on antigen‐presenting cells and can subsequently trigger T‐cell responses by binding to Vβ‐gene products. The reactivity of γδ T cells with enterotoxins is less well defined although both proliferative and cytotoxic responses have been described. In the present study we have tested the cytotoxic reactivity of a panel of 41 γδ T‐cell clones against target cells coated with the enterotoxins SEA, SEB, SEC1, SEC2, SEC3, SED, SEE or TSST. Three reaction patterns were observed with the γδ T‐cell clones: (1) clones that specifically lysed SEA‐coated target cells only; (2) clones that specifically lysed SEE‐coated target cells only, and (3) clones that specifically lysed SEA‐coated target cells only in the presence of certain human sera. The presence of SEA‐specific antibodies in such human sera could be demonstrated. Moreover, γδ T‐ cell clones of this third category expressed the IgG FcRIII (CD16) which indicates that these clones are capable of mediating antibody‐dependent cellular cytotoxicity towards SEA‐coated target cells. Thus, the cytotoxic response of γδ T cells to SEs is mediated by two distinct pathways: an antibody‐independent and an antibody‐dependent pathway. The antibody‐independent reactivity of γδ T cells was directed to either SEA or SEE, whereas antibody‐dependent reactivity was found only towards SEA. The capacity of γδ T‐cell clones to respond to stimulation with SEs, combined with their high cytolytic capacity in vitro, suggests that these cells can be involved in SE‐directed immune responses and efficiently kill SE‐coated target cells in vivo.
The Specificity of Alternative Complement Pathway‐Mediated Lysis of Erythrocytes: A Survey of Complement and Target Cells from 25 Species
Authors: ISH C. , ONG G. L. , DESAI N. , MATTES M. J.
Published: July 2006

Sera from 20 species of mammals were tested for their ability to lyse erythrocytes from 18 species of mammals and birds by the alternative complement pathway. Erythrocytes were not lysed by homologous complement, with one minor exception, but all erythrocytes tested were lysed by at least one complement source, and all sera tested except that of the horse lysed at least one type of erythrocyte. Control experiments indicated that lysis was via the alternative complement pathway and that antibodies were not involved. Complement from the various species could be ranked from most active to least active, and erythrocytes could be ranked from most susceptible to least susceptible. There was an inverse correlation between complement activity and erythrocyte susceptibility. The ranking of the orders of placental mammals, from strongest to weakest complement, was carnivore>oartiodactyl (ruminants and swine) > primate = armadillo > rodent > rabbit > horse. Opossum serum had activity that placed it in the centre of this range. Ferret complement, the most potent tested, lysed all erythrocytes tested except for homologous erythrocytcs, with APCH50 titres as high as 4000. Although the overall reactivity pattern was clear, there were several striking exceptions. For example, the only complement source which lysed ferret erythrocytes was sera of the mouse. The amount of sialic acid present on erythrocytes of 14 mammals was determined, and was, in general, directly correlated with resistance to alternative complement pathway lysis, although there were prominent exceptions to this correlation, involving crythrocytes of the horse, burro and human. All 20 types of complement were also tested for their ability to lyse antibody‐coated human tumour cells, under conditions in which both the classical and alternative complement pathways were functional. The data obtained suggest that alternative pathway activation is, in some cases, a major factor determining the effectiveness of a particular complement source in the lysis of xenogeneic tumour cells.
Interleukin‐6 is a Mediator of TNF‐α Regulation of LAK Cell Function
Authors: IHO S. , GOLUB S. H. , SHAU H.
Published: July 2006

TNF‐α at 50–100 U/ml synergizes with IL‐2 in enhancing LAK activity and IL‐6 production in low‐dose IL‐2 (1–10 U/ml) culture of human PBL. High‐dose TNF‐α (≥200 U/ml) has less effect and even sometimes resulted in lowering of both LAK activity and IL‐6 production below control levels. TNF‐α‐mediated regulation of low‐dose IL‐2 activation occurs even at late stages (effector phase) of LAK development. IL‐6, as previously reported, acts at late stages of low‐dose IL‐2 culture to enhance LAK, but does not stimulate TNF‐α production. The combined addition of TNF‐α and IL‐6 to late stages of IL‐2 culture does not produce any additive or synergistic effect on LAK. We tested for the relative roles of TNF‐α and IL‐6 in late stage regulation of LAK development with antibodies (Abs) to these cytokines. Anti‐IL‐6 Ab abrogates late phase LAK enhancement by TNF‐α, while anti‐TNF‐α Ab has no effect on IL‐6 augmentation of LAK cytotoxicity, IL‐2 added to PBL culture at doses greater than 10 U/ml induces production of both TNF‐α and IL‐6. Addition ofanti‐TNF‐α Ab at late stages of high‐dose IL‐2(≥20U/ ml) culture decreases both LAK cytotoxicity and IL‐6 production, and the inhibition of LAK is reversed by the addition of IL‐6. By contrast. anti‐IL‐6 Ab decreases LAK cytotoxicity, but does not alter TNF‐α production, and the inhibition of LAK is not reversed by addition of TNF‐α. These data indicate that TNF‐α is important for both LAK development and IL‐6 secretion in PBL, and that IL‐6 is the proximate mediator in TNF‐α regulation of these cytotoxic cell functions.
Development of B Cells secreting Endogenous or Transgene‐Encoded Immunoglobulins in H‐Chain Transgenic Mice
Authors: GRANDIEN A. , COUTINHO A. , ANDERSSON J.
Published: July 2006

The development of splenic B cells secreting transgene‐encoded or endogenous immunoglobulin (Ig) was analysed in the μ heavy (H‐)chain transgenic mouse line M54. The results show that cells secreting endogenous Ig are not detectable during the perinatal period, even after lipopolysaccharide stimulation in vitro. At this time, transgene‐secreting cells are readily detectable and keep increasing with age of the animals. After a few weeks of age cells secreting endogenous Ig appear in the spleen and keep increasing with age, reaching numbers comparable to non‐transgenic littermates by 5 weeks of age. Thereafter, the proportion of transgene‐secreting B cells decreases. We conclude that the preferential expression of endogenous Igs by secreting B cells in the adult does not result from peculiar genetic features of those cells, but from age‐dependent cellular selection operating on all B cells.
Lactoferrin Triggers In Vitro Proliferation of T Cells of Lewis Rats Submitted to Mycobacteria‐Induced Adjuvant Arthritis
Authors: ESAGUY N. , FREIRE O. , VAN EMBDEN J. D. A. , ÁGUAS A. P.
Published: July 2006

We have recently reported antigenic (B‐ccll) cross‐reactivity between the mycobacterial 65 kDa heat shock protein (hsp65) and human lactoferrin (LF) and we suggested that this cross‐reactivity might have a role in mycobacteria‐associated autoimmune disease. Here, we have searched for anti‐LFT‐cell reactivity in Lewis rats submitted to a mycobacteria‐triggered autoaggressive disorder (adjuvant arthritis, A A), an autoimmune disorder characterized by high anti‐hsp65 reactivity. We have quantified the in vitro proliferate response to LF of lymph node and spleen cells of Lewis rats killed 9, 14 and 21 days after the immunization with the AA‐triggering, mycobacteria‐containing adjuvant (complete Freund's adjuvant. CFA). We found that LF induced significant proliferation of lymph node T cells of rats undergoing AA. This T‐cell proliferation was not as marked as the one provoked by hsp65; it was, nevertheless, significantly higher (P < 0.05) than that produced by a non‐arthritogenic antigen (i.e. albumin). T cells from naive or mineral oil (incomplete Frcund's adjuvant, IFA) injected rats did not respond to LF or hsp65. These data indicate that LF may work as an accessory stimulatory factor of the T‐cell autoreactivity associated with mycobacteria‐induced arthritis.
Lymphocyte‐Endothelial Interactions in Inflamed Synovia:Involvement of Several Adhesion Molecules and Integrin Epitopes
Authors: FISCHER C. , THIELE H.‐G. , HAMANN A.
Published: July 2006

The role of several adhesion molecules for lymphocyte endothelial interactions in the synovia of rheumatoid arthritis patients was studied using the frozen section assay. Partial inhibition of lymphocyte binding to endothelium of synovial sections could be observed with antibodies against CD44, L‐selectin, and β1‐ and β2‐integrins, pointing to the participation of several adhesion molecules in the regulation of lymphocyte immigration into inflamed synovia rather than the presence of a unique homing receptor. Different degrees of inhibition were found within a series of antibodies against α4 and β1‐integrins known to have functional effects in other interaction systems. In addition, increased binding to endothelial cells was induced when lymphocytes were pretreated with TS2/16 anti‐β1 IgG, whereas binding to non‐endothelial components of synovia was increased after treatment with HP 2/4 (anti‐α4) Fab. The data suggest a multifunctional role of α/β1‐integrins in directly mediating adhesion as well as regulating adhesive interactions in the rheumatoid synovia.
T‐Cell Stimulation with Purified Mycobacterial Antigens in Patients and Healthy Subjects infected with Mycobacterium leprae: Secreted Antigen 85 is Another Immunodominant Antigen
Authors: LAUNOIS P. , NIANG M'B N'DIAYE , SARTHOU J.‐L. , RIVIER F. , DROWART A. , VOOREN J.‐P. VAN , MILLAN J. , HUYGEN K.
Published: July 2006

Peripheral blood leucocytes from 9 paucibacillary and 12 multibacillary leprosy patients, from 18 healthy controls and from 34 healthy leprosy contacts were stimulated with three mycobacterial heat shock proteins with respective molecular weights of 70,65 and 18 kDa and with the secreted 30–32 kDa protein, also called antigen 85. Antigen 85 was found to be the most powerful T‐cell antigen (as measured by lymphoproliferation and IFN‐γ secretion), eliciting a positive response in all (100%) paucibacillary patients and in all lepromin‐positive controls and contacts. The three heat shock proteins (hsp) were less active T‐cell stimuli. Reactivity to the 70 kDa hsp was found in only 44% of the paucibacillary patients, in 80% of the lepromin‐positive controls and in 60% of the lepromin‐positive leprosy contacts. The 65 kDa hsp stimulated T cells in 89% of the paucibacillary patients and in 80% of the lepromin‐positive controls and contacts. Responsiveness to the 18 kDa hsp, finally, was clearly more frequent in tuberculoid leprosy patients (78%) than in lepromin‐positive controls (40%) or lepromin‐positive leprosy contacts (4%). T‐cell reactivity of 8 lepromin‐negative controls, of 9 lepromin‐negative contacts and of 12 multibacillary leprosy patients was low to all the antigens tested. Although proliferative and IFN‐γ responses were generally closely related, some subjects demonstrated a dissociation of these two immune parameters. Our data confirm previous findings on the powerful T‐cell stimulatory properties of antigen 85 during M. leprae infection and suggest that this antigen is indeed a potentially protective T‐cell immunogen.
Immunoassay for Transthyretin Variants associated with Amyloid Neuropathy
Authors: COSTA P. M. P. , TEIXEIRA A. , SARAIVA M. J. M. , COSTA P. P.
Published: July 2006

An anti‐transthyretin (TTR) mouse monoclonal antibody (88.6.FD6)of IgG1 subclass, obtained using as immunogen TTR from the serum of a patient with familial amyloidotic polyneuropathy, was found to bind to sera from carriers of several amyloidogenic TTR variants associated with peripheral neuropathy, but not to normal sera or sera from carriers of non‐pathogenic or cardiomyopathic variants, in an ELISA performed under special conditions. Further characterization suggests that it recognizes an epitope near the N‐terminal side of the TTR monomer. We propose that this epitope is exposed in amyloid and could be implicated in fibril deposition in the peripheral nervous system.
Evidence for the Role of CR1 (CD35), in addition to CR2 (CD21), in Facilitating Infection of Human T Cells with Opsonized HIV
Authors: DELIBRIAS C.‐C. , KAZATCHKINE M. D. , FISCHER E.
Published: July 2006

Complement activation by HIV results in the binding of C3 fragments to the gp160 complex and enhanced infection of C3 receptor‐bearing target cells. We have studied complement‐mediated enhancement of infection of the human CD4‐positive T‐cell line HPB‐ALL which expresses the CR1 (CD35) and CR2 (CD21) receptors for C3. CR1 and CR2 are present on 15% and 40% of normal peripheral blood CD4‐positive T lymphocytes respectively. Opsonization of the virus with complement resulted in a 3‐ to 10‐fold enhancement of infection of HPB‐ALL cells, as assessed by measuring the release of p24 antigen in culture supernatants throughout the culture period. Blockade of CR2 with cross‐linked anti‐CR2 monoclonal antibodies decreased infection to the level observed with unopsonized virus. Blocking CR1 reduced complement‐mediated infection by 50–80%. Experiments using serum deficient in complement factor I demonstrated that CR1 mediates the interaction between opsonized virus and T cells in addition to its ability to serve as a cofactor for the cleavage of C3b into smaller fragments that interact with CR2. A requirement for CD4 in complement‐mediated enhancement of infection was observed with HIV‐1 Bru but not with HIV‐1 RF. Thus, CR1 and CR2 contribute in an independent and complementary fashion to penetration of opsonized virus into complement receptor‐expressing T cells. Involvement of CD4 in infection with opsonized virus depends on the viral strain.
Interferon‐γ Induces a Phospholipase D‐Dependent Release of Arachidonic Acid from Endothelial Cell Membranes: A Mechanism for Protein Kinase C Activation
Authors: MATTILA P. , USTINOV J. , RENKONEN R.
Published: July 2006

Interferon‐γ (IFN‐γ) induces MHC class II expression on endothelial cells in a protein kinase C (PKC)‐dependent manner. Here we show that IFN‐γ induces a sixfold arachidonic acid (AA) release from cultured rat microvascular endothelial cell membranes compared with non‐treated cells. Since this result suggests that AA could act as a second messenger for IFN‐γ, we analysed its capacity to directly activate PKC. We have previously shown that IFN‐γ induces a transient, multiphasic activation of PKC via the action of the phospholipase D (PLD) pathway. Here we show that AA is able to activate PKC. In an attempt to characterize the source of the liberated AA after IFN‐γ induction in endothelial cells we used a panel of enzyme inhibitors. The IFN‐γ‐induced release of AA could not be modified by interfering either with the phospholipase A2 (PLA2) pathway using bromophcnacyl bromide (BPB), or with the phospholipase C (PLC) pathway using neomycin. The phosphatidic acid phosphatase (PAPase) inhibitor propranolol, inhibiting the generation of diacylglycerol (DAG) and further AA from phosphatidic acid (PA), could totally down‐regulate the IFN‐γ‐induced release of AA. Since PA is produced solely by the action of PLD from phosphatidylcholine (PC) we conclude that the AA originated from the cell membrane‐associated PC. In summary, we show here that IFN‐γ causes the liberation of cell membrane‐associated, PC‐linked AA. This AA could directly activate PKC in a similar multiphasic manner to IFN‐γ, suggesting that it is a true second messenger for IFN‐γ in cultured endothelial cells.
A Rapid Method for Selecting Specific Hybridoma Clones using Paramagnetic Dynabeads®
Authors: BENNICK A. , BROSSTAD F.
Published: July 2006

This paper describes how specific hybridoma clones can be rapidly selected using paramagnetic beads coated with the antigen used for immunization. Spleen cells from a mouse immunized with fragment D dimer (DD) from plasminolysed fibrin were first fused with X‐63 mouse myeloma cells. Paramagnetic monodisperse beads (precoated with sheep anti‐mouse antibodies) were then coated with S4, a monoclonal antibody to DD, and subsequently with DD, Mixing such beads with the fused cells allowed selective harvesting of cells with membrane‐expressed anti‐DD gammaglobulins using a magnetic particle concentrator. Within 24 h, the cells spontaneously detached from the beads and were plated out on 96‐well plates. Supernatants from the clones obtained were tested by the ELISA technique. Antibodies specific for DD were produced by 40–79% of the tested clones. It is concluded that it is possible to use antigen‐coated paramagnetic beads to select, prior to cloning, hybridomas that produce specific antibodies. Implementation of this technique has significantly reduced costs and time in our efforts to obtain hybridoma clones of interest.
Effects of Growth Factors on Cytokine Production in Serum‐Free Cultures of Human Thymic Epithelial Cells
Authors: ANDERSEN A. , PEDERSEN H. , BENDTZEN K. , RÖPKE C.
Published: July 2006

Human thymic epithelial cells (TEC) of medullary phenotype were cultured for 14 days in a growth factor‐defined scrum‐free medium. The effects of added growth factors on cell numbers and the production of cytokines were investigated by separate exclusion of the various growth factors from the medium. We found that hydrocortisone stimulated cell proliferation but inhibited the differentiation of TEC and significantly reduced the production of interleukin‐lα, interleukin‐6 and granulocyte‐macrophage colony stimulating factor. Insulin was found to enhance the differentiation of TEC and the production of the three cytokines. Transfcrrin and choleratoxin were found to inhibit cell proliferation, but they did not affect produetion of the cytokines. Exclusion of epidermal growth factor, however, leads to cell death. We conclude that it is essential to exclude hydrocortisone from the medium to optimize production of cytokines, and that transferrin and choleratoxin seem to be unnecessary constituents in serum‐free cultures of human TEC.
Leucocyte Endothelial Cell Adhesion: a Study comparing Human Umbilical Vein Endothelial Cells and the Endothelial Cell Line EA‐hy‐926
Authors: THORNHILL M. H. , LI J. , HASKARD D. O.
Published: July 2006

EA‐hy‐926 is a cell line produced by hybridizing human umbilical vein endothelial cells (HUVEC) and the epithelial cell line A549. To establish whether EA‐hy‐926 could be used as a model for endothelial cells (EC) in leucocyte‐EC adhesion interactions, the effect of interleukin‐4 (IL‐4), tumour necrosis factor (TNF) or interferon‐γ (IFN) stimulation on their adhesiveness and expression of E‐selectin, vascular cell adhesion molecule‐1 (VCAM‐1) and intercellular adhesion molecule‐1 (ICAM‐1) was compared with that of HUVEC and A549. Although HUVEC exhibited increased adhesiveness and adhesion molecule expression with IL‐4, TNF or IFN, EA‐hy‐926 exhibited these responses only with TNF. CD11/CD 18‐dependent binding accounted for a significant component of basal binding to HUVEC and EA‐hy‐926, but did not account for the increased binding of T cells, JY, J6, ICH‐BJ or ICH‐KM cell lines to TNF‐stimulated monolayers. At least part of the CD1l/CD18‐independent adhesion was attributable to VCAM‐1 induction on HUVFC and FA‐hy‐926. TNF‐stimulation also induced F‐selectin expression on EA‐hy‐926 and HUVEC and an accompanying increase in neutrophil (PMN) binding. The EA‐hy‐926 cells used in this study, therefore, showed responses similar to HUVEC when stimulated with TNF but not when stimulated with IL‐4 or IFN.
Up‐Regulation by Cyclosporine (CsA) of the In Vitro Release of Soluble CD23 (sCD23) and of the In Vitro Production of IL‐6 and IgM
Authors: HORNUNG N. , DEGIANNIS D.
Published: July 2006

In the present study, we examined the effect of CsA on the in vitro production of Ig and on the in vitro production of molecules known to have B‐cell growth and differentiation activities, such as IL‐6 and sCD23. For the purpose of this study, we developed an experimental In vitro system closely resembling an In vivo model of ongoing B‐cell activation.

Pre‐activated B cells proliferated and produced IgM optimally when they were re‐cultured in the presence of IL‐2/IL‐6. CsA down‐regulated the IL‐2/IL‐6‐induced proliferative responses of pre activated B cells by at least 50%., but it up‐regulated IgM production in the same experiments. This up regulating effect was not cytokine‐related since it was also seen when cells were re‐cultured in the absence of any cytokines. Optimal release of sCD23 was observed when SAC‐pre‐activated B cells were re cultured in the presence of IL‐4 or IL‐4 plus IL‐2 and CsA up‐regulated significantly the release of this molecule in these cultures. Finally, CsA was shown to inhibit PHA‐induced cell proliferation of PBMC and to up‐rcgulate IL‐6 production in the same cultures.

We conclude that CsA can amplify in vitro both the production of Ig and the release of sCD23 by pre activated B cells. This finding, in combination with the CsA‐induced up‐regulation of lectin‐induced IL‐6 production, may have clinical implications in disease states with an ongoing immune activation, where prolonged administration of CsA might be anticipated.
Immunogenicity of HILDA/LIF either in a Soluble or in a Membrane Anchored Form expressed In Vivo by Recombinant Vaccinia Viruses
Authors: TAUPIN J.‐L. , ACRES B. , DOTT K. , SCHMITT D. , KIENY M.‐P. , GUALDE N. , MOREAU J.‐F‐.
Published: July 2006

Insertion of various cDN As in the genome of the vaccinia virus (VV) enables the in vivo and in vitro study of the functional role and/or the immunogenicity of the virally encoded recombinant proteins. We have prepared a recombinant VV expressing the cDNA of the human cytokine HILDA/LTF (human interleukin for DA cells/leukaemia inhibitory factor), and used this virus to immunize mice against this protein, which is very homologous to its murine counterpart (∼80% homology). We also constructed and expressed by the same system a chimeric gene encoding the HILDA/LIF protein fused to the 37 COOH‐terminal amino‐acids of the human decay accelerating factor (DAF). This sequence proved to be sufficient for the targeting of the fusion protein to the cell membrane, where it is linked to the phosphatidylinositols. Both recombinant VVs induced cytokine‐specific antibodies in mice as analysed with an FLISA where the recombinant HILDA/LIF was plastic‐coated and a cytofluorometric assay where the LIF‐DAF molecule was present at the cell surface of stably transfected PS15. In the latter case HILDA/LIF remained biologically active suggesting that it was expressed in its native form. The LIF‐DAF fusion protein was found to exhibit a better capacity to elicit an antibody response against the native form of the cytokine as detected in cytofluorometric assays. Whatever the recombinant virus used to immunize the mice, the MoAbs obtained were positive either in the ELISA or in the cytofluorometric assays but one, which suggested that the plastic coating induced a conformationa1 change of HILDA/LIF.
B‐Cell Abnormalities in AIDS: Stable and Clonally‐Restricted Antibody Response in HIV‐1 Infection
Authors: MÜLLER S. , WANG H. , SILVERMAN G. J. , BRAMLET G. , HAIGWOOD N. , KÜHLER H.
Published: July 2006

Antibodies in sera of HIV‐1 infected individuals against the HIV‐1 core protein (p24), HIV‐1 envelope gtycoprotein (gp120) and reverse transcriptase (RT) are characterized by a skewed light chain isotype expression. The kappa/lambda ratios of antibodies to p24 and gp120 in infected individuals were found to be unique in each individual, but constant over several years independently from disease progression. The oligoclonal nature of the anti‐HIV‐1 antibodies suggested by skewed kappa/lambda expression was confirmed with isoelectric focusing of affinity purified antibodies to p24 and gp120. Analysis of the utilization of V gene families in purified anti‐p24 and anti‐gp120 antibodies revealed a restricted and biased VH gene family usage. In contrast, the utilization of VK gene families appeared to be random.

The finding of stable and restricted antibody responses in infected individuals could be one of the causes for the failure to produce antibodies to HIV‐1 that are effective against escape virus variants.
A Subset of Vδ1+ T Cells Proliferates in Response to Epstein‐Barr Virus‐Transformed B Cell Lines In Vitro
Authors: ORSINI D. L. M. , RES P. C. M. , VAN LAAR J. M. , MULLER L. M. , SOPRANO A. E. L. , KOOY Y. M. C. , TAK P. P. , KONING F.
Published: July 2006

It has previously been shown that murine tissue derived T‐cells expressing the γδ T‐cell receptor can respond to autologous (stressed) cells implying the recognition of an autoantigen. Here we report that a large proportion of human synovial tissue and peripheral blood derived Vδ1+γδ T‐cell clones proliferate in response to stimulation with autologous and allogeneic EBV‐transformed B‐lymphobiastoid cell lines (LCL). In contrast, Vδ1‐γ/δ and α/β TCR+ T‐cell clones isolated from the same tissue samples did not display proliferation towards the LCL. The proliferative response of these Vδ1+ clones was dependent on contact between responder and stimulator cells and could be blocked by a MoAb to LFA‐1 and by antibodies to the γδ TCR/CD3 complex. Because the responses of these clones to LCL cells appear to be independent of the γ‐chain co‐expressed with the Vδ1‐chain these resemble a superantigen response. The capacity of this subset of Vδ1+ T‐cell clones to proliferate after stimulation with LCL may imply the recognition of an endogenous epitope. Moreover, since so far we have been able to isolate only LCL reactive γδ T‐cell clones from synovial tissue and peripheral blood of reactive arthritis patients and not from peripheral blood of healthy individuals, tbe frequency of such ‘autoreactive’γδ cells may be higher in these patients.
The Genetic Basis of Human Vh4 Gene Family‐Associated Cross‐Reactive Idiotype Expression in CD5+ and CD5‐ Cord Blood B‐Lymphocyte Clones
Authors: DEANE M. , MACKENZIE L. E. , STEVENSON F. K. , YOUINOU P. Y. , LYDYARD P. M. , MAGEED R. A.
Published: July 2006

In a recent study we have observed a high frequency expression of cross‐reactive idiotypes encoded by genes from the relatively small VH4 family of immunoglobulin heavy chain genes in cord blood B‐lymphocyte lines. Furthermore, we have demonstrated a selective pattern of expression of two VH4‐associated cross‐reactive idiotype (CRI) in B‐lytnphocyte lines established from CD5+ and CD5‐ cord blood B‐lymphocytes. There was a restricted expression of one CRI marker recognized by the 9G4 monoclonal antibody in lines established from CD5+ B‐lymphocytes but not in those established from the CD5‐ population. In the current study we examine the molecular basis for the selective pattern of CRI expression. Nucleotide‐sequence analysis of functional immunoglobulin heavy chain (IgH) gene rearrangements in three CD5 + lines expressing the CRI recognised by 9G4 reveal that all use a single gene from the Vh4 family, the V4.21 gene. However, all three lines have distinct third complementarity determining regions (CDR3) implying different clonal origins. In contrast, four cord blood cell lines (two established from CD5+ B‐lymphocytes) expressing the CRI recognized by MoAb Lcl have functional IgH gene rearrangements involving two ditferent genes from the Vh4 family, the V71–4, and V2–1 genes. Antigen specificity analysis reveals that all three 9G4‐reactive lines produce antibodies that react with the I and/or i red blood cell carbohydrate antigens. These data suggest that the distinction in VH4 gene use in CD5+ B‐lymphocytes in cord blood results from a selection process in vivo that shapes the repertoire of CD5+ B‐lymphocytes. This study extends recent observations that the monoclonal anti‐CRI antibodies 9G4and Lc1 are markers of two distinct subgroups of proteins encoded by two subsets of genes within the VH4 family. Furthermore, it appears that amino acid residues in framework region one and complementarity determining region two are critical for the expression of the cross reactive idiotypes and the serological distinction between the two subgroups of proteins.
Expression of a Mast Cell Tryptase in the Human Monocytic Cell Lines U‐937 and Mono Mac 6
Authors: HUANG R. , ÅBRINK M. , GOBL A. E. , NILSSON G. , AVESKOGH M. , LARSSON L. G. , NILSSON K. , HELLMAN L.
Published: July 2006

Expression of a mast cell tryptase mRNA was detected in two human monocytic cell lines, the U‐937 and the Mono Mac 6, and in normal human peripheral blood(PB) monocytes. In the U‐937 cell line but not in normal PB monocytes, the tryptase expression was upregulated 3–50 fold following phorbol ester (PMA)‐induced differentiation, but no such induction was seen after retinoic acid, interferon‐γ or vitamin D3 exposure.

The tryptases expressed in PMA‐induced and non‐induced U‐937 and in Mono Mac 6 were characterized by PCR amplification and nucleotide sequence analysis. The U‐937 cell line was found to express a tryptase identical to one of the previously cloned mast‐cell β tryptases (Tryptase I), and the tryptase expressed in Mono Mac 6 was found to be nearly identical to the previously cloned α tryptase. By northern blot analysis with oligonucleotide probes specific for the α and β tryptases both cell lines were found to express only one type of tryptase. Densitometric quantifications of tryptase mRN A levels, in the two cell lines, showed approximately 80 times higher mRNA levels in Mono Mac 6 compared to non‐induced U‐937. Immunohistochemical staining for tryptase showed a marked heterogeneity in the Mono Mac 6 cell line. Only one out of 10 cells were positive for the protein but the levels in these cells were very high, equivalent, or even higher than the levels seen in the human mast cell line HMC‐ 1. This shows that the expression of a single tryptase, in this case the a tryptase, is sufficient for the production of a stable protein and probably also a stable proteolytically active tetramer.

The family of human mast‐cell tryptases has been considered to represent a class of proteases specifically expressed in mast cells and basophilic leucocytes. The expression of tryptases in two monocytic cell lines and in normal PB monocytes indicate that in humans, the lineage specificity of these serine proteases is less restricted than earlier expected.

The cloning of a full length cDNA for the murine counterpart to the human mast cell tryptases, the MMCP‐6, is presented. No expression of the MMCP‐6 was detected in a panel of mouse monocyte or macrophage cell lines indicating a species difference in the lineage specificity of the ‘mast cell tryptases’.
Characterization of Monocyte‐Activating Tumour Cell Membrane Structures
Authors: WESTENFELDER U. , SCHRAVEN B. , MÄNNEL D. N.
Published: July 2006

Tumour cells are known to activate monocytes/macrophages and it has been shown that this stimulation was conferred by tumour‐cell membranes. In order to analyse the relevant structures for tumour cell‐specific TNF‐induction monocytes from healthy donors were cultured in the presence of plasma membrane preparations from Jurkat or K562 cells. Both tumour cell lines revealed a monocyte‐stimulating plasma membrane component of about 45 kDa. The TNF‐inducing factor exhibited characteristics of a glycoprotein with the carbohydrate moiety as the structure responsible for stimulation. CD2, a glycosylated T‐cell specific membrane component, was identified as being involved in monocyte activation in the case of the Jurkat cells whereas the identity of the activating structure on K562 cells is still unknown. From the data presented here indicating the importance of carbohydrate structures for monocyte activation we conclude that altered glycosylation of cell surface molecules of tumour cells might be responsible for tumour cell‐induced monocyte stimulation.
Human Cord Blood Contains an IGM Antibody to the 41KD Flagellar Antigen of Borrelia burgdorferi
Authors: COOKE W. D. , ORR A. S. , WISEMAN B. L. , ROUSE S. B. , MURRAY W. C. , RANCK S. G
Published: July 2006

Natural antibodies are the IgM products of fetal and neonatal B cells. These are germline encoded low affinity antibodies with multiple specificities to self and exogenous antigens. Lyme borreliosis is the disease resulting from infection with the spirochete, Borrelia burgdorferi. The humoral response to this organism is brisk, directed at multiple proteins, and persistant. Antibody to the 41 kd flagellar antigen is found early in disease, but may also be found in non‐exposed individuals. These properties suggest that the anti‐41kd antibody may be a natural antibody. We report here the finding of an IgM anti‐41kd reactivity in 29% of cord blood samples from patients in an area non‐endemic for Lyme disease. The results are consistent with the hypothesis that antibody to flagellin may be a germline encoded natural antibody, and could be important in the immunopathogenesis of Lyme arthritis and other arthritides.
Quantitation of Gm Allotypes
Authors: OXELIUS V.‐A. , CARLSSON A.‐M.
Published: July 2006

A method for quantitation of Gm allotypes is described. Alternative Gm allotypes of the three IgG subclasses, IgG1, IgG2 and IgG3, were investigated for the six most common Caucasian Gm phenotypes, Quantitation of Glm(a), Glm(f) of IgG1, G2m(n) of IgG2 and G3m(b) of IgG3 was performed with specific monoclonal antisera and purified myeloma proteins of different Gm allotypes. Mean±SD are given as percentage of a normal serum pool and in g/1 for the Gm allotypes Glm(a), Glm(f), G2m(n) and G3m(b), For homozygous individuals the G2m(″,″) values are equal to the IgG2 levels and the G3m(g,g) values equal to the IgG3 levels. For heterozygous individuals the value for G2m(″) is calculated as IgG2 minus G2m(n) and for G3m(g) as IgG3 minus G3m(b), Homozygous individuals have about double the amounts of the Gm allotype compared with heterozygous individuals. The gene activity of heterozygous individuals is given by quotients, mean±SD for G1 m(a)/G1 m(f) of IgG 1, G2m(n)/G2m(″) of IgG2 and G3m(b)/G3m(g) of IgG3 in different Gm phenotypes. Heterozygous individuals on all three IgG subclass loci have at least six different qualities of IgG molecules compared with three for homozygous individuals.
Serum IgG and IgG Subclass Contents in Different Gm Phenotypes
Authors: OXELIUS V.‐A.
Published: July 2006

Different serum IgG and IgG subclass levels were found among Gm phenotypes of a normal population. One hundred and fifty‐seven Caucasian blood donors were investigated for the reciprocal Gm allotypes on IgG subclass loci namely: for IgG1, Glm(f) and Glm(a); for IgG2, G2m(n) and G2m(″); and for IgG3, G3m(b) and G3m(g), and subgrouped in the seven most common Gm phenotypes. The frequencies of Gm phenotypes and haplotypes were given, including numbers of the previously little known G2m(n,″) heterozygous individuals. Mean serum quantities ±SD and range of IgG, IgG1, IgG2, IgG3 and IgG4 were given for different Gm phenotypes. The IgG content was significantly lower in the Gm(f,″,b/f,″,b) phenotype in which the IgG2 levels were also significantly lower, compared with values of the other phenotypes. IgG3 levels were significantly lower in the Gm(a,″,g/a,″,g) phenotype compared with other phenotypes. These data imply the importance of Gm(f,″,b/f,″,b) and Gm(a,″,g/a,″,g) phenotypes causing lower amounts of IgG antibodies. In evaluating IgG subclass deficiency, the range for the low responding Gm(f,″,b/f,″,b) and Gm(a,″,g/a,″,g) phenotypes should be considered.
Immunobiological Studies on Experimental Visceral Leishmaniasis. III. Cytokine‐Mediated Regulation of Parasite Replication
Authors: SAHA B. , BASAK S. K. , ROY S.
Published: July 2006

The role of T‐cell‐derived cytokines in the regulation ofLeishmania donovani replication was studied in a murine model. It was observed that in H‐2d mice at the early and later stages of the disease IFN‐γ‐secreting T cells predominate, whereas in between the above stages IL‐4–secreting T cells predominate. Possibly, IL‐4 abrogates the protective ability of IFN‐γ and thereby exponential parasite growth is ensured at the active stage of the disease. By contrast, H‐2b mice were possibly incapable of inducing IL‐4‐secreting T cells and therefore parasite replication remains under control at any point post infection.
Impact of HIV‐Positive Sera on the Functional Activity of Polymorphonuclear Neutrophils from Healthy Donors
Authors: GABRILOVICH D. I. , AVDEEVA L. A. , SEREBROVSKAYA L. V. , SHEPELEVA G. K. , SUVOROVA Z. K. , BURAVCOVA E. V. , OGANEZOV V. K. , PLATONOV A. E. , HABAROVA V. V. , POSLY I. M. , POKROVSKY V. V.
Published: July 2006

The impact of the sera from 64 HIV‐1‐infected patients on the functional activity of polymorphonuclear neutrophils (PMN) from 9 healthy donors was investigated. Augmentation of PMN oxygen radical production in the presence of sera from HIV‐infected patients (PMN chemiluminescence) was demonstrated. This enhancement was connected with intracellular generation of chemiluminescence (CL). A significant correlation between this enhancement and stages of HIV infection was not found. The presence of the sera from HIV‐infected patients in the incubation media led to a decrease of PMN phagocytosis. It was found that contrary impact of the sera on PMN CL and phagocytosis was connected with different factors. The decrease of PMN phagocytosis correlated with the level of complement and was independent of other factors. At the satne time the increase of PMN CL was not connected with the level of complement activity but correlated with the level of anti‐HIV antibody and circulating immune complexes. The reasons for this phenomenon are unclear. It was suggested that one of the serum factors which caused increase of PMN CL is HIV or HIV compounds.
A Trypanosoma brucei brucei‐Derived Factor that Triggers CD8+ Lymphocytes to Interferon‐γ Secretion: Purification, Characterization and Protective Effects In Vivo by Treatment with a Monoclonal Antibody against the Factor
Authors: BAKHIET M. , OLSSON T. , EDLUND C. , HÖJEBERG B. , HOLMBERG K. , LORENTZHN J. , KRISTENSSON K.
Published: July 2006

A protein factor that stimulates CD8+ lymphocytes to produce and secrete IFN‐γ has been purified from Trypanosoma brucei brucei (T.b. brucei). This was accomplished by raising monoclonal antibodies (MoAbs) against a fraction of T.h. brucei obtained by gel filtration, whieh contained high levels of material inducing rat mononuelear cells (MNC) to IEN‐y production. MoAbs from four hybridomas strongly inhibited trypanosome‐induced IFN‐γ production. One of them (MO1) was used for purification of the trypanosome‐derived lymphocyte triggering factor (TLTF) by affinity chromatography, SDS electrophoresis of the purified TLTF displayed a band of 42‐45 kDa MW. Gel filtration of homogenates of whole parasites yielded several peaks of IFN‐γ‐inducing activity with a lowest MW of 41 46 kDa. Bioactivity of all peaks was blocked by MO1. suggesting that a single molecule, or a single epitope of additional molecules, is responsible for the different peaks with IFN‐γ‐indueing activity. IFN‐γ released from MNC stimulates T.b. brucei growth. Blocking of TLTF in vitro with MO1 inhibited MNC‐supported growth of the parasites. To study the in vivo relevance of TLTF in the course of experimental African trypanosomiasis, MO1 was used to treat rats and mice at different times after infection. Treatments instituted at different time‐points after infection suppressed parasite growth, abrogated the IFN‐γ production by splenocytes indueed by the infection and prolonged survival of the animals. The data support the hypothesis that TLTF and IFN‐γ have a erueial regulatory function in the parasite host interactions and that these moleeules influence the disease eourse during experimental African trypanosomiasis.
Neutrophil Cytotoxicity Induced by Immune Complexes prepared with Cationized Antibodies
Authors: GEFFNER J. R. , TREVANI A. S. , MALCHIODI E. , SEREBRINSKY G. P. , ISTURIZ M. A.
Published: July 2006

Here we analyse the ability of soluble imtnune complexes (IC) prepared with cationized antibodies to induce cytotoxic responses mediated by neutrophils. While eationized IC induced high levels of cytotoxicity, control IC induced very low levels of response. Inhibition of cytotoxicity by catalase but not by three haemenzyme inhibitors suggests that oxygen‐dependent but myeloperoxidase‐independent mechanisms are responsible for cytolysis. While the response induced by control IC was enhanced by cytochalasin B and was not modified by colchicine, that induced by cationized IC was markedly inhibited by cytochalasin B and significantly enhanced by colchicine. Cytotoxicity induced by cationized IC was completely abrogated by monoclonal antibodies to FcγRII. Using control IC, a partial inhibition was observed employing either anti‐FcγRII or anti‐FcγRIII monoclonal antibodies. Treatment of neutrophils with ehemotrypsine or pronase significantly enhanced cytotoxicity induced by cationized IC but not by control IC. We also found that non‐specific absorptive mechanisms appear to play an important role in the binding of cationized IC, but not control IC, to the neutrophil surface. The significance of these results is discussed.
Purification of Amyloid‐Associated Heparan Sulphate Proteoglycans and Galactosaminoglycan Free Chains from Human Tissues
Authors: STENSTAD T. , MAGNUS J. H. , HUSBY G. , KOLSET S. O.
Published: July 2006

Basement membrane‐associated heparan sulphate proteoglycans have been demonstrated immunohistochemically in organs from patients afflicted with various types of amyloidosis. In a recent report, we were able to isolate and partly characterize a basement membrane‐associated heparan sulphate proteoglycan from human hepatic amyloid. In the present study proteoglycans were extracted with guanidine from human amyloid‐laden kidney, spleen and lymph nodes. All tissues extracted with guanidine contained both heparan sulphate proteoglycan (HSPG) and galactosaminoglycan (CS/DS) free chains.

Tissue staining using a monoclonal antibody against basement membrane HSPG revealed the presence of HSPG in amyloid deposits in kidney and spleen. Furthermore, following SDS‐PAGE of HSPG from kidney after deaminative cleavage of the HS chains, a 15‐kDa and 80‐kDa protein appeared, probably representing the core protein(s). In lymph node HSPG, three core proteins of 65, 30 and 25 kDa could be demonstrated on SDS‐PAGE, the first reacting with the anti‐basement membrane HSPG antibody when subjected to Western blotting subsequent to SDS‐PAGE. By immunohistochemistry, we failed to demonstrate any staining of the renal and splenic tissue sections employing an antibody against the decorin core protein.