bluuebunny/crossref_metadata_2025 huggingface dataset:
Prolonged In Vivo IL‐4 Treatment Inhibits Antigen‐Specific IgG1 and IgE Formation
Authors: VAN OMMEN R. , VREDENDAAL A. E. C. M. , SAVELKOUL H. F. J.
Published: July 2006

IL‐4 is obligatory for primary IgE responses, whereas primary IgG, and secondary IgE responses are partially IL‐4 independent. To investigate the effect of IL‐4 on the antigen‐specific memory formation for these isotypes, BALB/c mice were treated after primary TNP‐KLH immunization with recombinant IL‐4 for a period of 4 months. This prolonged presence of a high IL‐4 level resulted in increased serum levels of total IgG1 and IgE, whereas total IgG2a did not change. The expression of CD23, but not J‐Ad, increased on the splenic B cells. IL‐4 treatment did not affect the IL‐4 production by Con A stimulated spleen cells, whereas it did decrease the IFN‐γ production. In the same mice the TNP‐specific IgG1 and IgE serum levels, however, were decreased. Similar results were found when the antigen was continuously present during the IL‐4 treatment. Furthermore, it was shown that IL‐4 decreased the formation of IgG1, and IgE memory cells. These results point to different effects of IL‐4 in regulating antigen‐specific and bystander responses.
Affinity Maturation and Isotype Switch in Clonally Related Anti‐Erythrocyte Autoantibodies
Authors: SCOTT B. B , SADIGH S. , ANDREW E. M. , MAINI R. N. , MAGEED R. A.
Published: July 2006

The NZB mouse is genetically predisposed to develop, at approximately 6 months of age, a spontaneous and severe autoimmune anaemia caused by production of pathogenic anti‐mouse erythrocyte auto‐antibodies. Molecular analysis of a panel of five anti‐erythrocyte monoclonal antibodies (MoAb) derived from splenocytes of unimmunized NZB mice revealed that these autoantibodies all had functionally rearranged genes from the VH J558 family of immunoglobulin genes with closest homology to germline genes H10 and H30. Owing to clustering of nucleotide differences within the CDRs, compared with the germline, it was concluded that these antibodies were most likely generated by an antigen‐driven mechanism.

We report here further molecular analysis of two (4.16.1 and B4.13.2) of the panel of five anti‐mouse erythrocyte producing hybridomas which are apparently clonally related. Nucleotide analysis of the light chain cDNA indicated that both antibodies had closest homology to germline gene Vk24 and use Jk2 gene. Determination of the functional affinities of the MoAb reveal that B4.13.2 (IgG2a) has a > 10‐fold higher affinity for mouse erythrocytes when compared to 4.16.1 (IgGl). This finding supports the view that these two autoantibodies are generated by an antigen‐driven mechanism. The proposed mechanism would involve the selection and expansion of a small population of B‐lymphocytes by antigen leading to isotype switch, somatic mutation and increased affinity. Our data also point to the possibility that some framework residues may be involved in the binding to antigen.
Cytokine Dependence of Human to Mouse Graft‐versus‐Host Disease
Authors: HUPPES W. , FICKENSCHER H. , HART B. A. , FLECKENSTEIN B.
Published: July 2006

Human peripheral blood leucocytes (PBL) induce chronic graft versus‐host disease (GvHD) in non‐conditioned severe combined immunodeficient mice. Chronic GvHD was observed in such animals after transplantation of 6 × 107 human PBL per g body weight. However, acute xenogeneic GvHD results from grafting at least 2 × 107 human PBL per g body weight to heavily conditioned murine hosts. The large numbers of human PBL were thought to be required fo produce above threshold amounts of certain cytokines. We show that treatment of the recipient mice with human interleukin 2 reduces the number of cells to inflict acute GvHD by a factor of ten. Human T cells and not B cells or macrophages, were previously shown to generate acute xenogeneic GvHD, when selected cell types from peripheral blood were grafted. Most of the infiltrating cells had the CD4+ phenotype. We demonstrate that CD4+ T cells are the main mediator, as the disease is abrogated by treating the mice with cytotoxic CD4 antibodies, but not with CD8 antibodies. A survival pattern, similar to that seen in GvHD, was induced by transplantation of a Herpesvirus saimiri transformed human CD4+ clonal T cell line in conjunction with daily interleukin 2 injections. Herpesvirus saimiri transformed human T cells allow easily reproducible graft properties in chimeric mouse models for human diseases.
Intravenous Immunoglobulins Suppress Immunoglobulin Productions by Suppressing Ca2+ ‐Dependent Signal Transduction Through Fc γ Receptors in B Lymphocytes
Authors: KONDO N. , KASAHARA K. , KAMEYAMA T. , SUZUKI Y. , SHIMOZAWA N. , TOMATSU S. , NAKASHIMA Y. , HORI T. , YAMAGISHI A. , OGAWA T. , IWATA H. , TAKAHASHI Y. , TAKENAKA R. , WATANABE K. , HAGA M. , ORII T.
Published: July 2006

A high dose intravenous immunoglobulin (IVIG) therapy is used in the treatment of a wide range of autoimmune disorders. However, the mechanisms of the action of IVIGs remain poorly understood. To analyse the mechanisms of effects of IVIGs on immunoglobulin (Ig) production of B cells, the effects of IVIGs on B lymphoblastoid cell lines transformed by Epstein‐Barr virus (LCLs) were investigated. The productions of IgG or IgM of LCLs were dose‐dependently suppressed by polyethylene glycol (PEG)‐treated IVIG or pH 4‐treated 1VIG though the productions were not or only slightly suppressed by pepsin‐treated IVIG. The suppression by IVIGs was blocked by anti‐human IgG Fc or anti‐Fc γ RII. C μ gene expression and μ s C terminal gene expression of LCLs were suppressed by PEG‐treated IVIG, whereas neither C μ gene expression nor μ s C terminal gene expression of LCLs were suppressed by pepsin‐treated IVIG. Although the increase in intracellular calcium concentration in LCLs was not suppressed by pepsin‐treated IVIG, the increase was suppressed by PEG‐treated IVIG. This suppressing effect of PEG‐treated IVIG on intracellular calcium concentration of LCLs was blocked by anti‐human IgG Fc or anti‐ Fc γ RII. Our results suggest that IVIGs suppressed the Ca2+ ‐dependent signal transduction through Fc γ R on B‐cell membrane, consequently, the transcription of C γ mRNA, especially secreted γ mRNA was suppressed in the B cells.
Evolutionary Conservation of a Human Function‐Associated Molecule on Murine Natural Killer Cells: Expression and Function
Authors: KAPUR R. , EVANS D. L. , HARRIS D. T.
Published: July 2006

Using a novel anti‐natural killer (NK) cell monoclonal antibody (MoAb), we have recently identified an evolutionary conserved function‐associated molecule (FAM) present on fish, rat and human NK cells. This molecule is involved in NK cell function as anti‐FAM MoAbs inhibit cytotoxicity, stimulate lymphokine secretion and inhibit conjugate formation between effector cells and target cells. We now have examined murine NK cells for the presence of this structure. It was observed by two‐colour flow cytometric analysis that the anti‐FAM MoAb 5C6 specifically bound to a subpopulation of nylon wool non‐adherent splenic lymphocytes (19–20%). The expression of the FAM molecule was restricted to NK cells that expressed the NK1.1 antigen. Neither T cells, B cells, nor macrophages reacted with the anti‐FAM MoAb. Analysis of FAM expression in various lymphoid tissues revealed that splenocytes expressed the greatest numbers of MoAb(+) cells. Generation of lymphokine‐activated killer (LAK) cells and adherent tymphokine‐activated killer (ALAK) cells resulted in higher levels of FAM expression. The anti‐FAM MoAb 5C6 also detected the presence of FAM on fresh SCID NK cells. It was demonstrated that the anti‐FAM MoAb 5C6 inhibited the lysis of target cells by endogenous NK cells, activated NK cells, 5d LAK cells, ALAK cells and SCID NK cells. Moreover, conjugate assays demonstrated involvement of this molecule in recognition between NK cells and target cells.
Real‐Time Biospecific Interaction Analysis of a Natural Human Polyreactive Monoclonal IgM Antibody and its Fab and scFv Fragments with Several Antigens
Authors: ROGGENBUCK D. , KÖNIG H. , NIEMANN B. , SCHOENHERR G. , JAHN S. , PORSTMANN T.
Published: July 2006

Surface plasmon resonance (SPR) was used to investigate the kinetics of interactions between the human monoclonal polyreactive IgM antibody CB03, its Fab as well as its single‐chain variable fragment (scFv) and different antigens. From these experiments apparent binding constants were determined and compared with binding constants obtained by ELISA experiments. In SPR studies with the complete antibody, the polyreactivity of the CB03 antibody as derived from ELISA experiments was confirmed.

Interaction of scFv with k casein and human myoglobin is strong evidence for the location of polyreactivity within the variable domains of the antibody.

Apparent binding constants of the complete antibody to immobilized K casein (9.2 × 107 M−1) and to human myoglobin (1.6 × 107 M −1) are up to 83 times higher than those of Fab. The binding constants of the scFv to the above mentioned antigens are again about 10 times lower when compared with Fab, which is mainly due to the lower association rates of the complexes formed by the scFv.
Cloning and Expression of the Cervine Interleukin 4 Gene
Authors: HOOK S. M. , CRAWFORD A. M. , CHINN D. N. , GRIFFIN J. F. T. , BUCHAN G. S.
Published: July 2006

Interleukin 4 (IL‐4), a cytokine produced by the T helper 2 subset of T lymphocytes is involved in up regulating antibody responses. A source of recombinant cervine IL‐4 would be useful for studying the immune response of deer to tuberculosis. We report here the cloning, sequencing and expression of recombinant cervine IL‐4. To achieve this mRNA was isolated from red deer (Cervus elaphus) mononuclear leucocytes. First strand cDNA was synthesized from the mRNA and the IL‐4 cDNA was amplified, cloned and sequenced. The IL‐4 cDNA is 408 bp in length and the deduced amino acid sequence is 92% homologous with the published bovine IL‐4 amino acid sequence. IL‐4 was expressed using the baculovirus expression system in Spodoptera frugiperda Sf9 cells. Northern blot, SDS‐PAGE analysis and bioassay were used to confirm the expression of IL‐4.
Autoantibodies Against Ganglioside GM3 Represent a Portion of Anti‐Lymphocyte Antibodies in AIDS Patients
Authors: GRIGGI T. , BAUER R. , GAROFALO T. , KUKEL S. , LENTI L. , MASSETTI A. P. , MÜLLER C. , SORICE M. , PONTIERI G. M.
Published: July 2006

In this study we analysed the relationship between anti‐lymphocytic ganglioside antibodies and anti‐lymphocyte antibodies in AIDS patients. Anti‐lymphocytic ganglioside antibodies were detected by thin layer chromatography (TLC) immunostaining; three colour flow cytometry was used to analyse circulating antibodies against different lymphocyte subsets.

Anti‐lymphocytic ganglioside antibodies were detected in 23 out of 49 AIDS patient sera (46.9%). All positive sera reacted selectively with the GM3 comigrating band from AIDS lymphocytes. Twenty two out of the 23 anti‐lymphocytic GM3 positive sera also had antibodies against CD4+ T cells, versus 17/26 anti‐GM3 negative. Furthermore, patients with lymphocytic GM3 antibodies showed a significantly higher antibody reactivity against CD4+ T cells than patients in which these antibodies were not detected. The absorption tests revealed that preincubation of positive sera with GM3 was followed by a decrease in the reaction with target lymphocytes.

These findings suggest that anti‐GM3 antibodies are a portion, but not the majority, of antibodies reacting with CD4+ T cells.
Natural Killer Cell Stimulatory Factor (NKSF)/IL‐12 and Cytolytic Activities of PBL/NK Cells from Human Immunodeficiency Virus Type‐1 Infected Patients
Authors: SIRIANN M. C. , ANSOTEGUI I. J. , AIUTI F. , WIGZELL H.
Published: July 2006

The aim of this study was to assess the effect of natural killer stimulatory factor (NKSF/IL‐12) on the cytolytic potential of natural killer (NK) cells from normal donors or HIV‐1 infected individuals, at different stages of disease. The results obtained confirm the powerful ability of 1L‐12 to enhance PBLs cytotoxic activity to tumour cells in normals and in those HIV‐1 infected patients, with maintained cytotoxic activity. IL‐12 was however poorly effective in enhancing the cytolytic potential of any donors. Similar results were obtained adding IL‐2, even at doses as high as 100 IU/ml in patients with CD4 number below 200/mm3. These results should be taken into account when designing therapeutic trials of HIV‐1 infection based on cytokines.
Responses of Human T Cells to Dominant Discrete Protein Antigens of Escherichia coli and Pseudomonas aeruginosa
Authors: KERSTEN C. M. , MCCLUSKEY R. T. , WARREN H. SHAW , KURNICK J. T.
Published: July 2006

Normal human beings have circulating T lymphocytes that proliferate in response to Escherichia coli and Pseudomonas aeruginosa. We performed the present study to characterize the nature of the responding T cells and to determine whether distinct or shared conventional antigens, superantigens or polylonal activators account for T cell proliferation. Long term antigen‐specific T cell lines were generated by repeated stimulation of PBMC from four donors with soluble antigen preparations of E. coli or P. aeruginosa. This resulted in the emergence of distinct T cell populations, which responded to strains of either E. coli or P. aeruginosa, but not to both. Trypsin treatment of the bacterial preparations largely eliminated their ability to stimulate the T cells. The T cell lines were predominantly CD4+ and their proliferation to bacterial antigens was optimal using autologous APC. E. coli T cell lines proliferated not only in response to the E. coli strain with which they were initially selected, but also to four different strains of E. coli, as well as to several related Gram‐negative species. P. aeruginosa selected T cells exhibited proliferative responses to six different P. aeruginosa strains, but not to the other Gram‐negative species. The finding that repeated stimulation of PBMC with E. coli or P. aeruginosa leads to CD4+ T cells highly reactive with conventional protein antigens specific either for E. coli or P. aeruginosa indicates that these bacteria possess separate dominant protein antigens that drive the proliferation of peripheral blood T cells.
Partial Amino Acid Sequence of a Novel Protozoan Parasite Antigen that Inhibits Non‐Specific Cytotoxic Cell Activity
Authors: LEARY J. H. , EVANS D. L. , JASO‐FRIEDMANN L.
Published: July 2006

Monoclonal antibody (MoAb) 18C2, prepared against a human EBV transformed lymphoblastic cell line (NC‐37) is specific for a target cell ligand recognized by fish NCC and by mammalian NK cells. MoAb 18C2 inhibits the lysis of a variety of transformed murine and human cells (e. g. NC‐37, YAC‐1, K562, etc.). This MoAb also recognizes a determinant on the fish protozoan parasite Tetrahymena pyriformis. In the present study, we used MoAb 18C2 to identify a target antigen in detergent lysates of T. pyriformis. MoAb 18C2 recognized a 46–50 kDa target antigen (NKTag) by Western blot analysis of both crude and ammonium sulphate (AS) fractionated (25–40% saturation) T. pyriformis lysates. AS fractionated or purified soluble NKTag inhibited NCC mediated lysis of IM‐9 target cells in a dose dependent fashion. AS fractionated NKTag also inhibited NCC lysis of a variety of human and murine transformed targets (e. g. HL‐60, MOLT‐4, DAUDI, NC‐37, U‐937, YAC‐1, EL‐4). Inhibition was specific for NCC and inhibition could be removed by adsorption of AS fractionated NKTag with MoAb 18C2 hybridoma cells. NKTag was prepared for amino acid sequencing by preparative SDS PAGE of whole cell detergent (CHAPS) lysate followed by Western transfer to nitrocellulose. The MoAb 18C2 recognized NKTag was excised and submitted for microsequencc analysis. Direct N‐terminal analysis yielded a 12 residue sequence. Additional sequences, obtained from in situ trypsin digests of the NKTag on nitrocellulose yielded four additional peptides of 10, 13, 16 and 21 residues. None of the sequences examined had significant homology to known sequences (Swiss‐Prot protein sequence database). These data indicate that MoAb 18C2 recognized a novel protein on T. pyriformis which may be involved in target cell recognition/lysis by NCC. Further, these data extend our previous observation that a common target determinant exists between higher and lower eukaryotic cells, and its expression may provide an explanation for the susceptibility of both protozoan parasites and transformed tumour cells to NK/NCC lysis.
Interleukin‐6 and Interleukin‐6 Receptor are Expressed by Cultured Glomerular Epithelial Cells
Authors: MOUTABARRIK A. , NAKANISHI I. , ISHIBASHI M.
Published: July 2006

Interleukin‐6 (IL‐6) has been extensively studied in mesangial cells but little is known about the expression of this cytokine and its receptor in glomerular epithelial cells (GEC). IL‐6 was detected in the culture supernatants of human GEC and its production was enhanced in time and dose dependent manner by lipopolysaccharide (LPS), interleukin‐1β (IL‐Iβ) and tumour necrosis alpha (TNF‐α). Quiescent, serum‐starved GEC did not express clearly IL‐6 mRNA. Stimulation of cells with LPS, TNF‐α or IL‐1β resulted in an increase of detectable IL‐6 mRNA. Interestingly, it was found that IL‐6 induced its own mRNA attesting that this cytokine was secreted in autocrine fashion by GEC. GEC expressed IL‐6 receptor (IL‐6R) as demonstrated directly by the existence of IL‐6R mRNA detected by northern blotting. Stimulation of GEC by pro‐inflammatory mediators such as LPS increased the expression of IL‐6R mRNA. The soluble form of IL‐6 receptor (sIL‐6R) was not detectable in the culture supernatants harvested from untreated or cytokine‐treated cells. We investigated further, whether IL‐6 may influence growth of cultured GEC. Incubation of GEC with recombinant (r) IL‐6 resulted in a dose dependent increase in 3H thymidine incorporation indicating that IL‐6 acts as an autocrine growth factor for GEC. We conclude that GEC are a potent source of IL‐6, the local excessive expression of IL‐6 and its receptor may play a substantive role in the regulation of processes which appear critical to the initiation of progressive glomerular disease such as cell proliferation.
Cyclic AMP Sensitive Signalling by the CD28 Marker Requires Concomitant Stimulation by the T‐Cell Antigen Receptor (TCR/CD3) Complex
Authors: SKÅLHEGG B. S. , RASMUSSEN A. M. , TASKEN K. , HANSSON V. , JAHNSEN T. , LEAF T.
Published: July 2006

We have previously demonstrated that activation of cAMP‐dependent protein kinase (cAK) type I (cAKI, RIα2‐Cβ2) mediates the inhibitory effects of cAMP on T‐cell replication induced through the TCR/CD3 complex. In the present study we have investigated the effect of cAMP on T‐cell DNA synthesis, tyrosine phosphorylation of a 100 kDa protein (pp100) and IL2 mRNA expression, induced through stimulation of the TCR/CD3‐ and/or the CD28 molecules. Our results demonstrate that tyrosine phosphorylation of pp100 stimulated by anti‐CD3 is inhibited by cAMP both in the presence and absence of the phorbol ester PMA, and reflects the changes seen in IL2 mRNA expression and T‐cell replication. Combined stimulation with anti‐CD3 and anti‐CD28, which gives a synergistic response in T‐cell replication, gave pp100 phosphorylation and IL2 mRNA expression sensitive to cAMP‐dependent inhibition. When PMA was added in addition to anli‐CD3 and anti‐CD28, the inhibitory effect of cAMP on both T‐cell replication and pp100 phosphorylation was completely abolished. The fact that pp100 phosphorylation in response to TCR/CD3‐, CD28‐ and PMA stimulation and cAMP mediated inhibition are identical to the effects of the same stimuli on T‐cell proliferation, makes this protein an interesting candidate in downstream signalling from these receptors. In addition, our results are compatible with a model where cAMP, through activation of cAKI, eliminates both the PTK and PKC activating capability of the T‐cell receptor at a site(s) proximal to PKC activation. Furthermore, the CD28 molecule which activates PTKs, enters the PTK cascade at a point distal to the target(s) for cAKI action. Therefore, during CD28 signalling PKC activation can be achieved either by TCR/CD3 stimulation (inhibited by cAMP), or directly by PMA (not inhibited by cAMP)
γδ and αβ T Cells are Equally Susceptible to Apoptosis
Authors: ARSTILA T. P. , TOIVANEN P. , VAINIO O. , LASSILA O.
Published: July 2006

Little is known about the role of apoptosis in the regulation of γδ T cell development and function. We have used chicken as a model to study apoptosis of γδ T cells at different stages of their development. Apoptosis was measured with electrophoretic analysis of DNA fragmentation and flow cytometric determination of DNA content combined with immunofluorescence staining of cell surface molecules. In vitro culture, dexamethasone, and γ‐irradiation induced apoptosis of both γδ TCR thymocytes and peripheral γδ T cells. Apoptosis could be induced even in the earliest thymic γδ thymocytes on embryonic day 13. Resting peripheral blood γδ T cells were more resistant to apoptosis than thymocytes and spleen cells. Following polyclonal activation of splenic γδ T cells by Con A, the proportion of the CD8+γδ T cell blasts decreased significantly when recultured without further stimulation. These results indicate that γδ T cells are susceptible to apoptosis in a manner similar o γδ T cells, and suggest that apoptosis plays an important role in the regulation of the development and function of both thymic and peripheral γδ T cells
Anti‐Idiotypic B Lymphocytes in Patients with Monoclonal Gammopathies
Authors: BERGENBRANT S. , YI Q. , ÖSBY E. , ÖSTERBORG A. , ÖSTMAN R. , BJÖRKHOLM M. , HOLM G. , LEFVERT A. K.
Published: July 2006

The prevalence of peripheral blood B cells secreting antibodies reacting with the F(ab')2 fragment of monoclonal IgG was studied in five patients with multiple myeloma (MM), nine patients with monoclonal gammopathy of undetermined significance (MGUS) and six healthy controls. An enzyme‐linked immunospot assay allowed direct visualization of antibody producing B cells. All patients had B cells producing antibodies to autologous or ailogeneic monoclonal IgG. Autoreactive cells were found more frequently than alloreactive cells in seven out of nine patients with MGUS and three out of four patients with MM. The same frequency of alloreactive cells in the patient groups was detected in healthy individuals. These findings show the existence of B cells producing anti‐idiotypic antibodies which could be a part of an idiotypic network in monoclonal gammopathies
Lipopolysaccharide‐stimulated events in B Cell Activation
Authors: HU H. , MÖLLER G.
Published: July 2006

High concentrations of thymus‐independent (T1) antigens are capable of inducing polyclonal B cell activation by their intrinsic mitogenic properties, irrespective of the specificity of the Ig receptors. Due to a genetic defect on the 4th chromosome, B cells from C3H/HeJ mice do not respond to lipopoly‐saccharide (LPS). In order to define at which step the mutation affects the signalling pathway, we compared B cells from C3H/HeJ and CBA mice with regard to changes of three events, namely cell size, and MHC class I and II antigen expression after LPS stimulation. We found that cell size and expression of MHC antigens increase in B cells from CBA mice after LPS stimulation, whereas B cells from C3H/HeJ mice do not respond at all. This suggests that the defect in C3H/HeJ mice interferes with early events in the signalling pathway, either due to the absence of a LPS receptor on B cell surface or the lack of an initial component necessary for effective signal transmission subsequent to LPS receptor binding. Our results also have shown that stimulation of anti‐Ig antibodies and LPS differ in some signalling events and have different final effects on B cells, which suggests that they may function differently via distinct signalling pathways.
Short‐Term Kinetics of the Humoral Anti‐Clq Response in SLE Using the ELISPOT Method: Fast Decline in Production in Response to Steroids
Authors: RÖNNELID J. , HUANG Y. H. , NORRLANDER T. , ROGBERG S. , NILSSON B. , GUSTAFSSON R. , KLARESKOG L.
Published: July 2006

Twenty four systemic lupus erythaematosus patients and 17 patients with other diagnoses were investigated regarding the presence of cells producing Clq reactive antibodies in peripheral blood mononuclear cells using the ELISPOT technique. These results were then compared with parallel serum levels of Clq reactive antibodies. Current production of anti‐CIq was almost entirely confined to the systemic lupus erythaematosus group. Longitudinal analysis of anti‐Clq ELISPOT positive patients showed rapid changes in the number of anti‐Clq producing cells, but only slowly changing serum levels of the corresponding antibodies in response to glucocorticoids. In one systemic lupus erythaematosus patient prednisolone treatment had a selective effect on this autoantibody production, as the production of anti‐Clq spot forming cells rapidly dropped to zero, at the same time as the number of total spotforming cells showed only less change. In another patient, self‐limiting connective tissue disease was associated with temporal occurrence of IgM anti‐Clq. We believe, from these data, that the ELISPOT method for determination of current antibody production may be of particular value in longitudinal evaluation of disease course and therapeutic effects in systemic lupus erythaematosus and other rheumatic diseases.
Antibodies Directed Against Monomorphic and Evolutionary Conserved Self Epitopes may be Generated in ‘Knock‐Out’ Mice. Development of Monoclonal Antibodies Directed Against Monomorphic MHC Class I Determinants
Authors: CLAESSON M. H. , ENDEL B. , ULRIK J. , PEDERSEN L. Ø. , SKOV S. , BUDS S.
Published: July 2006

Beta‐2 microglobulin (β2m) gene ‘knock‐out’ mice (C1D) were primed wilh purified H‐2Kb and H‐2Db molecules and spleen cells from immunized mice were used to generate monoclonal antibody secreting B‐cell hybridomas. Approximately 0.2% of the Ig‐secreting primary microcultures contained H‐2b binding antibodies. Three stable anti‐MHC class I (MHC‐I) antibody secreting hybridoma clones were established and subcloned. All three MoAbs precipitated radiolabelled H‐2 molecules as analysed by SDS PAGE, and all three MoAbs stained H‐2b, H‐2d, as well as H‐2k cells by FACS analysis. The MoAbs stained to two β2m loss mutant cell lines, C4.4‐25‐ and R1E, suggesting that some MHC‐I heavy chain is exported to the cell surface even in the absence of endogenous β2m. Staining of murine cell lines kept under serum‐free culture conditions was strongly influenced by the addition of bovine or human serum as a source of exogenous β2m suggesting that xenogeneic β2m affects the conformation of class I molecules. Furthermore, all three MoAbs strongly stained the peptide transporter deficient cell line, RMA‐S, when cultured at 26°C, however, staining was reduced five‐fold when RMA‐S cells were cultured at 37°C. In total, these observations suggest that the MoAbs recognize conformational, presumably β2m and peptide dependent, self epitopes on MHC‐class I. One of the three MoAbs stained rat blood mononuclear blood cells (BMC), all three MoAbs stained hamster BMC, whereas two of the MoAbs stained human cells. These data suggest that the MoAbs recognize determinants which are conserved between species. All three antibodies strongly inhibited the development of CTLs generated in an allogeneic one‐way MLC, provided that the MoAbs were present during the first 24 h of culture. It is concluded that MoAbs reacting with monomorphic self epilopes may be generated using animals deleted of the gene of interest. The implications may be far reaching since such MoAbs potentially identify evolutionary conserved and physiologically important epitopes.
The Fifth Serum Amyloid A‐Related Gene Sequence, GSAA4, is SAA3
Authors: WATSON G. , FAULKES D. J. , WOO P.
Published: July 2006

The human serum amyloid A (SAA) gene family consists of two acute phase genes, SAA1 and SAA2; a pseudogene, SAA3 and a fourth gene, SAA4. The existence of SAA4 was first described in this laboratory. Subsequently, Sack & Talbol isolated a clone, designated GSAA4, which had homology to SAA3. The clone was described as the same as SAA4 and was characterized as a pseudogene. However, our restriction site and sequence analyses of SAA4 demonstrated that SAA4 and GSAA4 are separate entities. SAA4 encodes a functional, constitutively expressed protein.

Recently, the GSAA4 clone has been reported by Sellar & Whitehead as constituting a fifth SAA‐ related sequence. However, we have demonstrated that GSAA4 has striking homology with the 3′ terminus of SAA3 in the reverse orientation. Furthermore, computer analyses strongly indicate that the GSAA4 or fifth SAA‐related sequence is in fact, a clone from the SAA3 locus. We have amplified, isolated and sequenced fragments from genomic DNA which demonstrate that the GSAA4 sequence is the correct sequence of the 3′ region of SAA3. The SAA family must therefore still be viewed as consisting of four SAA loci at present.
T Cell Receptor Vα Gene Usage in Human T Cells Stimulated with SEE and SED
Authors: LIU J. L. , HODARA V. L. , WIGZELL H.
Published: July 2006

Vα gene usage in human T cells stimulated with SEE and SED was investigated by using polymerase chain reaction with specific primers. Vβ8 T cells from normal blood donors PBMC were sorted at day 5 after stimulation with SEE and analysed for TCR‐Vα gene usage. Whole T cells stimulated with anti‐CD3 MoAb or SED were also analysed and compared at different time points after stimulation. There was no biased Vα gene usage found as a response to either of the two superantigens. These results show that Vα gene usage of human T cells stimulated with SEE or SED is normal.
Lymphocyte Transformation Test in Adult Patients with Minimal Change Nephrotic Syndrome
Authors: AL‐AZZAWI Y. H. M. , AL‐TAWIL N. G. , AL‐SHAMAA I. A.
Published: July 2006

The lymphocyte transformation test was used to evaluate the cellular immune response in 31 adult patients with MCNS in comparison with 30 normal control and 49 patient (CRF, FSGN. MGN) control groups. The results showed that the stimulation indices of the lymphocytes in MCNS group were significantly lower than those of the normal control and patient control groups with the exception of CRF group.

In cross studies, lymphocytes obtained from normal individuals were incubated in homologous serum obtaitied from MCNS patients in relapse, and lymphocytes from MCNS patients in relapse were incubated in normal homologous AB serum. In both circumstances there was a depressed lymphocyte function which indicates that there is a defect in lymphocyte itself as well as a serum inhibitory factor(s) during the active stage of the MCNS. This depressed cellular immune response returned to normal level during remission of the patients with MCNS.
Sustained and Distinctive Patterns of Gene Activation in Synovial Fibroblasts and Whole Synovial Tissue Obtained from Inflammatory Synovitis
Authors: RITCHLIN C. , DWYER E. , BUCALA R. , WINCHESTER R.
Published: July 2006

Fibroblastoid synovial lining cells isolated from rheumatoid and other chronic inflammatory synovial tissue exhibit distinctive and sustained alterations in serial culture not commonly found in similarly cultured cells from osteoarthritic synovium. These are demonstrable using a multi‐gene dot blot assay by labelling reverse transcribed fibroblast cDNA which is hybridized to plasmids containing relevant target gene inserts. Cultured synovial fibroblastoid cells from patients with chronic inflammatory synovitis expressed significantly higher levels of stromelysin, vimentin and TIMP‐1 mRNA and lower levels of c‐myc compared to cells isolated from osteoarthritis synovium although with considerable variation. Early fetal synovial lining cells were similar to cells from osteoarthritis synovium but vimentin expression was higher. Marked differences in patterns of gene expression between cell lines persisted through 10 serial passages over 6–8 months. In whole synovia, the average level of mRNA for stromelysin, vimentin, IL‐4, IL‐6, TIMP‐1, cathepsin D, gelatinase, TGFα, c‐fms and DRβ were preferentially expressed in inflammatory tissue while c‐myc expression was higher in osteoarthritis synovium. Inflammatory synovium also expressed TNFα, IL‐1α, IL‐1β, IL‐2, c‐sis, tissue plasminogen activator, CSF‐1, and GM‐CSF. This pattern resembles, in part, that found in cultured inflammatory fibroblasts but, in addition., gene products apparently reflecting the presence of activated monocytes and lymphocytes were detected. These results provide evidence that profiles of certain gene activation in cells from patients with inflammatory synovitis differ from those with non‐inflammatory disease and suggest that the fibroblastoid cells are responsible for a considerable proportion of the altered phenotypic expression pattern in whole tissue. Furthermore, this modulated pattern of gene activation appears to be an intrinsic pro‐inflammatory characteristic of the fibroblastoid cells initiated in response to chronic inflammation and persists for a prolonged period in the absence of other inflammatory cells.
The Role of B Lymphocytes in Experimental Herpes Simplex Viral Retinitis
Authors: ARRUNATEGUI‐CORREA V. , DUTT J. , FOSTER C. S.
Published: July 2006

The purpose of this study was to examine B cell participation in experimental herpes simplex virus (HSV) retinitis. Passive immunization with anti‐herpes antibody protects BALB/c mice from herpes simplex retinitis (HSR). Using anti‐Mu antibody treatment, we modified the B cell population of C. B‐17 mice, normally resistant to HSR, in order to test the hypothesis that such treatment would render them susceptible to HSR by impairing their early antibody response to anterior chamber (AC) inoculation with HSV. We analysed the effect of anti‐Mu treatment on their susceptibility to HSR and then employed Polymerase Chain Reaction (PCR) and ELISA techniques to study the patterns of immunoglobulin gene and protein expression, and the T‐cell receptor α/β (TCR α/β) gene expression after AC inoculation of HSV. Immunohistopathologic analysis revealed that 100% of the B cell deficient mice (B−) developed contralateral retinitis following AC inoculation, confirming the hypothesis that anti‐Mu antibody treatment would convert HSR‐resistant mice into HSR‐susceptible ones. Transfer of B cells from naive congenic donor mice resulted in 67% of recipient B− mice developing contralateral retinitis. Transfer of anti‐HSV antibody conferred nearly complete protection, with only 11% of mice developing retinitis (P <0.005). PCR and ELISA analysis showed that both untreated and B− C. B‐17 mice showed similar dynamic patterns of mRNA IgG isotype expression and of anti‐HSV IgG isotypic antibody response following AC inoculation. Thus, we were forced to reject the hypothesis that an impaired early antibody response is primarily responsible for the increased HSR susceptibility seen in B− mice. In contrast, PCR analysis of TCR α/β mRNA expression revealed dramatic differences between susceptible and resistant mice, suggesting that TCR Vβ selection and usage may be a critical factor influencing HSR‐sensitivity in this murine model, and that B cells (and immunoglobulin isotype) may play a role in TCR Vβ selection and usage after ocular encounter with HSV.
Inhibitory Activity of Soluble IL‐2R in Sera, Ascites and Culture Supernatants from Murine Leukaemic Cells
Authors: WALDNER C. , MONGINI C. , ALVAREZ E. , ROIG I. , HAJOS S. E.
Published: July 2006

The effect of sera from mice bearing a T cell lymphoid leukaemia (LB) and the supernatants from short term cultures of the tumour cells were studied on cell proliferation using syngeneic and allogeneic normal and tumour cells. An inhibitory activity was demonstrated in 24–48 h supernatants of LB cells in culture and disappeared after 4 days of culture. Inhibitory activity was cytostatic but not cytotoxic and was non‐specific since it inhibited the growth of both syngeneic and allogeneic normal and tumour cells. Such activity was found in the 105–1.3 × 105 Mr serum fraction after a Sephacryl S200 chromatography. Though sensitive to protease, trypsine or neuraminidase treatment, which indicated its glycoprotein nature, it remained stable after heating or freezing‐thawing cycles as well as after alkaline, acid or hyaluronidase treatment. Addition of exogenous IL‐2 abrogated inhibitory activity. ELISA showed the presence of soluble IL‐2R both in LB conditioned medium and in above serum fraction.

It is demonstrated that the inhibitory factor, soluble IL‐2R, is produced by LB leukaemia cells, then secreted into blood and ascitic fluid or released into culture supernatants. Soluble IL‐2R exerts inhibitory activity blocking cell proliferation and modulating immune response by binding to free IL‐2.
Circulating Myeloperoxidase May Cause False Negative Findings in the Analysis of Myeloperoxidase Antibodies in Systemic Vasculitis
Authors: HAAPALA A.‐M. , SOPPI E. , HYÖTY H.
Published: July 2006

In systemic vasculitis reliable detection of myeloperoxidase antibodies (MPO‐Abs) is of great clinical importance in the diagnosis and follow‐up of patients. We have studied whether circulating myeloperoxidase (MPO) could have an effect on MPO‐Ab findings. Serum MPO and MPO‐Abs were measured in 50 healthy individuals, 35 patients and in the follow‐up samples from two patients with Wegener's granulomatosis.

Heating the sera at 56°C for 30min reduced the concentration of immunoreactive MPO both in control and patient sera. In 71% of the patient sera heating made initially negative MPO‐Abs detectable. In a few cases with severe vasculitis the antibody findings remained totally negative. These results, together with the data from the follow‐up samples from two patients with Wegener's granulomatosis, revealed that the serological diagnosis of vasculitis may be considerably delayed if only native samples are analysed for MPO‐Abs. These findings are of considerable clinical significance for the interpretation of MPO‐Ab results.

Circulating myeloperoxidase affects MPO‐Ab measurements, causing false negative findings in MPO‐Ab assays. Therefore, it is recommended to denaturate circulating MPO by heating the sera before the analysis of MPO‐Abs and to re‐evaluate the cut off‐values.
A Highly Conserved Phenylalanine in the α, β‐T Cell Receptor (TCR) Constant Region Determines the Integrity of TCR/CD3 Complexes
Authors: CASPAR‐BAUGUIL S. , ARNAUD J. , HUCHENQ A. , HEIN W. R. , GEISLER C. , RUBIN B.
Published: July 2006

In the present study, we have investigated the importance of a phenylalanine (phe195) in the Tcr‐Cα region on Tcr‐α, β/CD3 membrane expression. An exchange of phe195 with a tyrosine residue does not affect Tcr/CD3 membrane expression; however, exchange with aspartic acid, histidine or valine prohibit completely Tcr/CD3 membrane expression. This seems to be due to a lack of interaction between mutated Tcr‐α, β/CD3‐γɛ, δɛ complexes and ζ2 homodimers. The Tcr‐Cα region around phe195 seems together with the same region in the Tcr‐Cβ region to constitute an interaction site for ζ2 homodimers. The presence of phe195 on both Tcr‐Cα and Tcr‐Cβ causes high avidity interaction with ζ2 homodimers, whereas his195 in both Tcr‐Cγ and Tcr‐Cδ results in an apparently lower avidity interaction with ζ2 homodimers. It is suggested that the phe195 region (on β‐strand F) and eventually adjacent aromatic amino acid residues on β‐strand B region may play an important role in Tcr‐α, β/CD3 membrane expression, in Tcr‐α, β/CD3 competition with Tcr‐γ, δ/CD3 complexes for ζ2 homodimers and in the control of formation of ‘mixed’ Tcr heterodimers.
Characterization of Purified Protein Derivative of Tuberculin by use of Monoclonal Antibodies: Isolation of a Delayed‐Type Hypersensitivity Reactive Component from M. tuberculosis Culture Filtrate
Authors: KLAUSEN J. , MAGNUSSON M. , ANDERSEN Å. BENGÅRD , KOCH C.
Published: July 2006

Nine monoclonal antibodies were raised against purified protein derivative (PPD) of tuberculin in mice previously treated with Bacilli Calmette Guérin (BCG). The antibodies also reacted with a culture filtrate from Mycobacterium tuberculosis strain H37Rv. In immunobtotting after SDS‐PAGE the reaction with PPD was seen as a diffuse smear, whereas ammonium sulphate‐precipitated proteins from H37Rv gave well‐defined bands ranging from 10 to 65kDa. Enzyme immunoassay showed that both PPD and H37Rv antigens were able to inhibit binding of the antibodies to PPD coated microtitre wells, suggesting that the antibodies reacted with continuous epitopes. A 12kDa protein purified by immunoaffinity chromatography from H37Rv antigens was tested intradermally in M. tuberculosis MNC3 sensitized guinea pigs and gave a delayed type hypersensitivity reaction.
Resistance to Secondary Amyloidosis in A/J Mice is Not Significantly Associated with Allelic Variants Linked to the Serum Amyloid A Gene Cluster
Authors: BUTLER A. , WHITEHEAD A. S.
Published: July 2006

We have tested the hypothesis that structural allelic variants of serum amyloid A confer relative resistance to secondary amyloidosis in the A/J mouse. F2 mice, previously generated from amyloid‐resistant (A/J) and amyloid‐susceptible (C57BL/6J) strains and categorized with respect to amyloid susceptibility, were genotyped by polymerase chain reaction (PCR) amplification of the polymorphic D7Nds5 microsatellite. This microsatellite is closely linked to the SAA gene cluster and can discriminate between D7Nds5 alleles of A/J and C57BL/6 J origin. The distribution of D7Nds5 genotypes in relation to splenic amyloid load did not deviate significantly from that expected of a random distribution, indicating that A/J amyloid resistance is not determined by variants at, or close to, D7Nds5. Therefore, structural alleles in the tightly‐linked SAA gene cluster do not confer amyloid resistance in this mouse model.
Activation of the Classical Pathway of Complement by Non‐Immune Complexes of Immunoglobulins with Human Protein Fv (Fv Fragment‐Binding Protein)
Authors: RUFFET E. , PIRÈS R. , PILLOT J. , BOUVET J.‐P.
Published: July 2006

Protein Ev, a human sialoprotein recently described in the stools of patients suffering from liver diseases, binds the variable domain of the heavy chains of immunoglobulins. We show here that preincubation of this protein with monoclonal human IgG1 and IgM activates the complement cascade by forming nonimmune complexes, as evidenced by haemolysis inhibition of antibody‐coated sheep erythrocytes. As negative controls, no inhibition was observed after incubation either with immunoglobulins or with protein Fv alone, and with the protein Ev‐depleted medium. Activation was due to the binding of immunoglobulins with protein Fv, as shown by inhibition of protein Fv‐induced agglutination of the sensitized erythrocytes in the absence of complement. Activation of the classical pathway was demonstrated both by using a buman IgG4 or F(ab)2 fragments unable to activate C1q, and by Western blot analysis of the cleavage of C4 in human serum. These results confirm that protein Fv‐binding mimicks antigen‐antibody reactions, and suggest its involvement in hepatitis‐associated vasculitis and in local lesions of some infiammatory gut diseases.
Vδ1+ Gamma/Delta T Lymphocytes Infiltrating Human Lung Cancer Express the CD8α/α Homodimer
Authors: FERRARINI M. , HELTAI S. , CHIESA G. , SABBADINI M. G.
Published: July 2006

Murine γ/δ T lymphocytes localize to different epithelial tissues and are phenotypically distinct from peripheral γ/δ T cell‐populations in that they show limited TCR diversity, express the CD8 α/α homodimer and lack the CD8β chain. In humans, a compartmentalization of γ/δ cells sharing similar phenotypic features has been documented to date only in the case of intestinal epithelium. In the present study we show that about half of Vδ1+ (as well as Vδ1−Vδ2−) γ/δ lymphocytes, which can be selectively expanded from human lung cancers, coexpress the CD8α/α homodimer. The accumulation of intraepithelial CD8+γ/δ+ lymphocytes might then be a more general phenomenon, possibly as a result of common mechanisms operating at those sites.
Evolutionarily Conserved Function of CD28 in αβ T Cell Activation
Authors: ARSTILA T. P. , VAINIO O. , LASSILA O.
Published: July 2006

The functional role of the chicken homologue of CD28 was studied. It is expressed on all thymocytes, and both Vβ1‐ and Vβ2‐family expressing peripheral αβ T cells. Peripheral γβ T cells are CD28‐negative. Monoclonal antibody against CD28 had a costimulatory effect on T cells stimulated by phorbol myristate acetate (PMA), concanavalin A or MoAb against TCR. Vβl and Vβ2 expressing cells responded equally well to stimulation with anti‐CD28 in combination with PMA. These responses were resistant to cyclosporin A, but inhibited by herbimycin A, suggesting that CD28 employs a signalling pathway at least partly distinct from that triggered by TCR/CD3. These data indicate a striking conservation of the costimulatory function of CD28 and emphasize the importance of this costimulatory pathway.
Non‐Coordinated Biosynthesis of Early Complement Components in a Deficiency of Complement Proteins Clr and Cls
Authors: CHEVAILLER A. , DROUET C. , PONARD D. , ALIBEU C. , SURANITI S. , CARRERE F. , RENIER G. , HUREZ D. , COLOMB M. G.
Published: July 2006

We report on a 60‐year‐old woman with systemic lupus erythematosus and a total (95%) Clr and a partial (36%) Cls deficiency. The patient complained about cutaneous lesions on forearms and legs without other systemic involvement. Elevated anti‐nuclear, anti‐native DNA and anti‐SSA antibodies were present. The finding of persistently depressed levels of haemolytic complement activity (CH50) on both serum and plasma, associated with normal levels of C3, C4 and C2 components, and normal alternative pathway haemolytic activity showed a deficiency of an early component of the classical pathway. Indeed Clr component was below the limits of detection whereas Cls component was lowered (36%). The depressed CH50 was only corrected by purified Clr. Biosynthesis of Clr and Cls by patient's monocytes was spontaneously normal but not up‐regulated by interferon‐γ for Clr alone, whereas the biosynthesis of Cls, but also of interleukin‐6, was increased, indicating a specific disregulation of Clr. The deficiency was associated with a lupus syndrome and a fatal assumed septic shock. This is in agreement with other reported cases.
Molecular Characterization of Immunoglobulin Variable Regio from Murine Monoclonal Antibodies Specific for Simian Virus 40 Large Tumour Antigen
Authors: SHEARER M. H. , BRIGHT R. K , KENNEDY R. C.
Published: July 2006

In this report we compare the immunoglobulin variable (IgV) region function and structure relationships of murine monoclonal antibodies that recognize simian virus 40 (SV40) large tumour antigen (T ag). Comparison of monoclonal antibody V region function is based on SV40 T ag epitope recognition and idiotype (Id) expression. Structural comparisons are based on V region gene sequence determination. The data presented herein, suggests that a high degree of homology within both the Vk and VH regions, along with minor differences within Vk complementarity determining regions (CDR) may result in the detection of similar SV40 T ag epitopes by the monoclonal anti‐SV40 T ag preparations. The expression of a cross‐reactive Id also appears to be based on the high degree of homology within both VK and VH regions and depends on conformational interactions imparted by both regions.