bluuebunny/crossref_metadata_2025 huggingface dataset:
Human IgG Rheumatoid Factors and RF‐Like Immune Complexes Induce IgGl Rheumatoid Factor Production in Mice
Authors: ABEDI‐VALUGERDI M. , RIDDERSTAD A. , AL‐BALAGHI S. , MÖLLER E.
Published: July 2006

The synovial fluid of patients with rheumatoid arthritis (RA) was found to contain IgG and /or IgG‐containing immune complexes (ICs) that stimulated an intense antibody formation when injected into mice of certain strains, notably of NZ background. The response was characterized by high and sustained levels of IgG1 antibodies with rheumatoid factor (RF) activity. In the study described, we investigated whether it is the antibodies with RF activity in the synovial fluid, that are responsible for stimulation of mouse RF in vivo. Different mouse strains were injected with synovial fluid from a seropositive RA patient (RA‐SF), with human monoclonal antibodies with RF activity, with a human non‐RF monoclonal antibody or with different preformed RF‐like antibody antibody (Ab‐Ab) ICs. The experimental mice were monitored subsequently for IgGl RF production. IgGl RF antibodies were found in all strains (NZB, BALB/c and CBA) injected with Ab‐Ab ICs formed at equivalence, but only in NZB using RA‐SF or human monoclonal antibodies with RF activity. Optimal production of IgG I RF by Ab‐Ab ICs required the integrity of Fc and F(ab)'2 portions respectively of the antibodies; soluble and truncated ICs were less effective. Further studies demonstrated that the IgGl RF response was not simply the result of a specific immune response against human IgG, since humoral immunity against human IgG was induced only when combined with an efficient adjuvant. During a typical adjuvant‐associated primary response specific antibodies of IgM, IgG1 and IgG2a isotypes were found, i. e. quite different from the selective IgGl response induced by RF‐like containing immune complexes. This conclusion is substantiated further by the clear differences in responses to IgG containing fraction obtained from RA‐SF in NZ mice compared to other strains. Our findings argue for a different type of reaction leading to the selective IgGl response and might aid in elucidating the mechanisms for chronic production of antibodies with RF activity in patients with RA.
Molecular Analyses of Anti‐DNA Antibodies Induced by Polyomavirus BK in BALB/c Mice
Authors: REKVIG O. P. , FREDRIKSEN K. , HOKLAND K. , MOENS U. , TRAAVIK T. , KRISHNAN M. R. , MARION T.
Published: July 2006

In the present experiments, two groups of BALB/c mice (five individuals in each group) were hyperimmunized through four consecutive immunizations with either BK virus (Group 1) or BK dsDNA complexed with methylated BSA (Group 2). All immune sera taken after the fourth immunization from both groups reacted strongly with polyomavirus BK dsDNA as well as with calf thymus dsDNA, and all sera contained antibodies that bound in the Crithidia luciliae assay. This indicates that polyomavirus BK was able to induce antibodies with binding characteristics similar to SLE anti‐DNA antibodies. To further characterize these induced anti‐DNA responses, 10 monoclonal anti‐DNA antibodies (four from Group 1, and six from Group 2) were generated and selected for reactivity with Sl‐nuclease digested CT dsDNA. Their specificity for BK and CT dsDNA molecules, as well as their light and heavy chain variable region cDNA nucleotide sequences were analysed to compare them with known SLE derived anti‐DNA antibodies. All of the 10 antibodies bound strongly to BK dsDNA, while seven also bound to CT dsDNA in competitive ELISA experiments. V‐region analysis revealed that the induced antibodies resembled anti‐DNA antibodies characteristic for murine SLE, and all but one contained arginine in the VH CDR3 region. The arginines present in the monoclonal antibodies originated either from an RF shift from RF1RF3 of the D‐genes or from N‐sequence additions. Taken together, the data demonstrate that anti‐DNA antibodies in response to hyperimmun‐ization with polyomavirus BK have the same characteristics as of those occuring spontaneously in SLE. As virus infection/replication in vivo implies expression of immunogenic (non‐self) DNA‐binding proteins that may render DNA immunogenic, the present results may therefore suggest one physiological mechanism for production of SLE‐related anti‐DNA antibodies.
The Involvement of CD 14 in Stimulation of TNF Production from Peripheral Mononuclear Cells Isolated from PNH Patients†
Authors: SUNDAN A. , RYAN L. , BRINCH L. , ESPEVIK T. , WAAGE A.
Published: July 2006

Peripheral blood mononuclear ceils (PBMC) from six patients with paroxysmal nocturnal haemoglobinuria (PNH) were analysed by flow cytometry for expression of CD14 and for ability to respond to bacterial lipopolysaccharide and β 1–4 linked polymannuronic acid by TNF secretion. Expression of cell surface CD 14 could not be detected on cells from the PNH patients, whereas the levels of expression of other monocyte antigens, e. g. CD33 and CD13, were comparable to that of cells from healthy subjects. The cells from the patients with PNH responded with secretion of significantly less TNF after stimulation with LPS and polymannuronic acid than mononuclear cells from healthy subjects, suggesting an impaired ability in PNH to respond to bacterial infection by TNF secretion from monocytes. Soluble CD 14 appeared to be involved in the residual activation of CD14 negative PBMC, and the sera of these patients contained normal or slightly elevated levels of soluble CD 14. After allogeneic bone marrow transplantation in one patient the monocytes expressed CD 14 at normal levels and responded normally with respect to their ability to generate TNF upon stimulation.
Stimulation by T Cell Independent Antigens Can Relieve the Arrest of Differentiation of Immature Auto‐Reactive B Cells in the Bone Marrow*
Authors: ANDERSSON J. , MELCHERS F. , ROLINK A.
Published: July 2006

The pair of μH‐chain and kL‐chain transgenes encoding the Sp6 TNP/DN A‐specific IgM was bred onto the rearrangement‐deficient genetic background of RAG‐2T mice, and onto the kL‐chain expression‐deficient background of iEkT mice. Bone marrow of Sp6 transgenic RAG‐2T mice contained normal numbers of B220(CD45R)+ c‐kit+ pro/preB‐I‐like cells and normal numbers of B220(CD45R)+TAC+ preB‐II‐like cells. Most strikingly, the numbers of immature sIgM+ B cells in the bone marrow were at least five‐fold lower than normal, while mature B cells were almost undetectable in bone marrow as well as spleen. Hence, B cell development in these mice appears to be arrested at the transition from preB‐II to immature B cells. The contents of bone marrow and spleen of the different precursors, immature and mature B cell compartments in Sp6iEkT mice were found to be similar to those of normal mice except that all sIg+ cells expressed μL‐chains, of which 40% coexpressed the transgenic kL‐chain. It indicates that the repertoire of μL‐chain rearrangements and the μL‐chains expressed from it suffices to relieve the arrest of differentiation seen in Sp6RAG‐2T mice. The T cell‐independent antigen TNP‐Ficoll elicited within 5 days a response of the Sp6RAG‐2T mice to develop to IgM‐secreting cells and to fill the serum pool with the Sp6 transgenic IgM to 100 μg/ml, i. e. to normal serum levels of IgM in normal mice. TNP‐Ficoll appears to interfere with the arrest of differentiation. Two scenarios for this arrest of differentiation and its relief by the T‐independent antigen TNP‐Ficoll are discussed.
Engagement of MHC Class I Proteins on Natural Killer Cells Inhibits their Killing Capacity
Authors: PETERSSON M. G. E. , GRÖNBERG A. , KIESSLING R. , FERM M. T.
Published: July 2006

We have studied whether engagement of MHC class I (MHC—I) molecules on natural killer (NK) cells can influence the NK killing activity. Human NK effector cells, enriched by nylon wool passage, were incubated with monoclonal antibodies (MoAb) to MHC—I followed by cross‐linking with secondary rabbit anti mouse Ig or streptavidin. Cross linking of MHC—I molecules on NK cells resulted in a clear inhibition of the NK activity against the target cells K562, Molt‐4 and U937. The inhibitory effect was selective for MHC—I and was not seen with MoAb to MHC—II or CD56 molecules. The inhibition was not mediated via Fc receptors since F(ab)2 fragments of the MHC—I MoAb W6/32 were as effective as the intact antibody. The best inhibition of NK activity was obtained using biotin‐labelled F(ab)2 fragments of W6/32 and streptavidin as a cross‐linker, where up to 70 % reduction in NK cell activity was observed. Antibody dependent cellular cytotoxicity (ADCC) was also inhibited by cross‐linking MHC—I molecules on the effector cells.

The results show that antibody mediated cross‐linking of MHC—I proteins on NK cells can inhibit their killing capacity. This indicates that MHC—I molecules on NK cells can be involved in the regulation of NK cytotoxicity, perhaps by transmitting inhibitory signals into the NK cell.
Comparison of the Number of IL–4 and IFN–7 Secreting Cells in Response to the Malaria Vaccine Candidate Antigen Pf155/RESA in Two Groups of Naturally Primed Individuals Living in a Malaria Endemic Area in Burkina Faso
Authors: ELGHAZALI G. , ESPOSITO F. , TROYE‐BLOMBERG M.
Published: July 2006

The enzyme‐linked immunospot (ELISPOT) assay was used to enumerate the number of IFN‐γ and IL–4 producing cells after in vitro stimulation with a highly purified recombinant malaria vaccine candidate antigen (r‐Pf155/RESA) or synthetic peptides corresponding to its major T‐cell epitopes. Two groups of naturally primed individuals living in rural areas of Burkina Faso were studied. The donors comprised one group of healthy (non‐parasitemic) mainly adult people and one parasitemic mainly younger people. IL–4 producing cells were detected in response to PHA but no such cells were detected in response to the malarial antigens. The most frequent IFN–7 responses were seen with r‐Pf155/RESA. Thus, after stimulation with this antigen 52% of the donors responded positively in the ELISPOT assay, while only 17% responded to the synthetic peptides, suggesting that the r‐Pf155/RESA contained T‐cell epitopes not covered by the peptides used in this study. The number of IFN‐γ producing cells in response to the malarial antigens did not differ between the two groups. However, IFN‐γ levels found in sera from the parasitemic individuals were significantly higher than in those from healthy donors. This latter finding and the lack of differences seen in the number of IFN‐γ producing spots in the two groups indicate that IFN‐γ producing cells may have sequestered to other organs in the parasitemic group.
Homology Between the MPB70 and MPB83 Proteins of Mycobacterium bovis BCG
Authors: HARBOR M. , NAGAI S. , WIKER H. G. , SLETTEN K. , HAGA S.
Published: July 2006

Isolation of MPB83 from Mycobacterium bovis BCG Tokyo culture fluid is described. MPB70 and MPB83 have similar molecular mass as judged by SDS‐PAGE but differ in isoelectric points. Peptides isolated after CNBr cleavage of MPB83 revealed extensive homology as well as distinct differences from corresponding parts of the amino acid sequence deduced from the mpb70 gene cloned by Terasaka et al. Antibodies produced by immunization with MPB70 and MPB83 had distinctly different fine specificity revealing cross‐reactivity between the proteins. These findings indicate that two distinct, homologous genes code for these proteins. Sensitization with live BCG Tokyo also induced T cell responses to MPB83 with development of delayed type hypersensitivity in guinea pigs.
The Somatic Hypermutation Activity of a Follicular Lymphoma Links to Large Insertions and Deletions of Immunoglobulin Genes
Authors: WU H. Y. , KAARTINEN M.
Published: July 2006

A biopsy specimen from a patient with follicular lymphoma was divided into two fragments. DNA was extracted from one fragment and a 1.2 kb region of the functional heavy chain (IgH) gene was amplified, cloned and sequenced (eight clones). From the other fragment a cell line (HF‐1) was started. The IgH gene region was amplified from the cell line, and sequenced without cloning. The nine sequences obtained could be arranged into a genealogical tree where the individual sequences differed from the deduced ancestor by 16–29 single nucleotide changes, some also by an insertion and/or a deletion. It is apparent that the sequence alterations were caused by somatic mutations during the growth of the lymphoma.

The comparison of the sequences with two published (allelic) germline sequences of the human JH region showed ∼20% non‐homology. The differences included five additional multinucleotide insertion/deletion changes, the longest of them a 101‐nucleotide insertion. Two long insertions were homologous to the adjacent germline sequences. We propose that most of the changes observed, including long deletions and insertions, represent or are linked to somatic hypermutation events of the Ig gene type. Although in a few cases large deletions and insertions (>2 bp) have been found in mutated immunoglobulin genes, our results, for the first time, firmly link these deletions/insertions to somatic hypermutations; their frequency was found to be 2.2% of the observed mutational events in the non‐translated gene regions. HF‐1 is the first follicular lymphoma line successfully established from a lymphoma known to have hypermutated its Ig genes during the malignant growth. It is a candidate cell line to be studied for its ability to generate mutations of B cell type in cell cultures.
Characterization of TAP‐Independent and Brefeldin A‐Resistant Presentation of Sendai Virus Antigen to CD8+ Cytotoxic T Lymphocytes
Authors: ZHOU X. , LIU T. , FRANKSSON L. , LEDERER E. , LJUNGGREN H.‐G. , JONDAL M.
Published: July 2006

H‐2Kb‐transfected T2 cells, which lack both TAP1/2 and LMP2/7 genes, are able to efficiently process and present Sendai virus Antigen to Kb‐restricted Sendai virus‐specific CTL. This presentation is not inhibited by Brefeldin A (BFA). Here we extend our analysis of this novel antigen presentation pathway. We show that presentation of Sendai virus antigen was not due to sensitization of T2Kb cells by peptides in the virus preparation or peptides released from virus infected cells. Also, the ability to present Sendai virus in a BFA resistant fashion was specific for cells of the T2 lineage. Re‐expression of TAP1/2 genes in T2Kb cells did not alter the capability to present antigen in a BFA resistant fashion, i. e. the presence of a functional TAP transporter complex did not relocate (all) peptides to the classical pathway for antigen processing and presentation. We found that co‐infection of T2Kb cells with either Sendai virus plus influenza virus or Sendai virus plus VSV did not relocate presentation of influenza or VSV antigen to the TAP independent BFA resistant antigen presentation pathway. Peptide elution experiments and studies with peptide‐specific CTL firmly demonstrated that the antigen presented by T2Kb cells after infection with Sendai virus was the natural Sendai virus epitope NP324‐332. The same epitope, when expressed as a minigene in vaccinia virus, could be presented also by T2Kb cells but this presentation could be blocked by BFA. Thus, the TAP independent BFA resistant presentation of antigen seem cell (T2 lineage) and virus (Sendai virus) specific, but not epitope specific. The ability of T2Kb cells to present Sendai virus antigen in a TAP independent BFA resistant fashion was only partially blocked by lysosomal inhibitors such as methylamine, ammonium chloride and chloroquine. These findings demonstrate that TAP1/2‐independent and BFA‐resistant class I processing is only expressed in certain cell types, in parallel with classical MHC class I processing, and that Sendai virus selectively can enter this pathway. Hypothetical models for the TAP‐independent class I processing are discussed.
Specific and Long‐Lasting Protection from Collagen‐Induced Arthritis and Oil‐Induced Arthritis in DA Rats by Administration of Immunogens
Authors: LORENTZEN J. C. , ERLANDSSON H. , MÜSSENER Å. , MATTSSON L. , KLEINAU S. , NYMAN U. , KLARESKOG L.
Published: July 2006

DA rats develop transient arthritis after subcutaneous immunization with adjuvant‐oil, while chronic arthritis and collagen autoreactivity ensues when collagen is added to the oil. We show here that DA rats can be protected from oil‐induced arthritis (OIA) and rat collagen‐induced arthritis (rCIA) by addition of antigen to these arthritogenic inocula.

We have investigated this remarkable phenomenon and demonstrate that both foreign and self antigens can be protective, apparently provided they are immunogenic; hence HSP‐65kDa, ovalbumin, rat myelin basic protein, rat IgG and bovine albumin are effective while rat albumin is not. This protection is long‐lasting and disease‐specific because rats protected from rCIA resist a later attempt to induce arthritis, but not experimental autoimmune encephalomyelitis (EAE). Protection from rCIA depends neither on the blocking of humoral autoreactivity to collagen nor on a change in the isotype profile of anti‐collagen antibodies. We demonstrate that immunogens can also be protective when injected intraperitoneally only a few days before onset of arthritis. Our results indicate that protection is mediated through bystander immune reactions towards the co‐immunized antigen and that the arthritogenicity of a given provocation, be it adjuvants, microbes or autoantigens, may be a complex net result of arthritogenic and contra‐arthritogenic immune reactions.
Female Preponderance for Development of Arthritis in Rats is Influenced by both Sex Chromosomes and Sex Steroids
Authors: HOLMDAHL R.
Published: July 2006

Autoimmune arthritis was induced after a single injection of the non‐immunogenic adjuvant (avridine) or with autologous rat type II collagen. Females of two different rat strains, DA and LEW, were found to be more susceptible than males. To investigate further the mechanisms behind the female preponderance, we selected the avridine induced arthritis model. This is known to be a chronic jointspecific disease which is T‐cell dependent and associated with MHC genes and, therefore, is an appropriate model for rheumatoid arthritis. To address the possibility of sex chromosome involvement, reciprocal F1 hybrids were produced. Female (DAxLEW)F1 rats were found to be more prone to arthritis than their male counterparts. This difference could be explained, at least partly, by the influence of sex chromosomes since reciprocal (LEWXDA)F1 rats showed no sex linkage. However, the sex linkage was more pronounced in normal rats when compared to castrated (DAxLEW)F1, rats indicating a role for sex hormones in conjunction with the sex chromosome‐linked effect. Both oestrogen and testosterone had a suppressive effect on the development of arthritis. The findings presented here suggest the presence of a sex chromosome gene, which mediates its function only in the presence of sex hormones and is associated with a female preponderance for development of arthritis.
No Evidence for TCR Vβ Repertoire Changes Influencing Disease Protection in E‐Transgenic NOD Mice
Authors: SPETZ A. L. , BRENDEN N. , PILSTRÖM B. , BÖHME J.
Published: July 2006

In order to study whether positive selection of T cells plays any role in the MHC‐dependent protection from diabetes in the non‐obese‐diabetic (NOD) mouse, the T cell Vβ repertoire has been studied in NOD mice and in NOD mice either transgenic for the wildtype MHC class II Eα gene, or for ΔY, a promotor‐mutagenized Eα gene with a restricted tissue expression. The Eα transgenic line is protected from both insulitis and diabetes. The ΔY transgenic line is neither protected from insulitis nor from diabetes, although it can perform both positive and negative E‐mediated selection in the thymus. The Vβ repertoire was studied in the pancreatic lymph nodes as these drain the area which is the target for the autoimmune attack. We see no evidence for Ea TCR Vβ repertoire differing from both nontransgenic NOD mice and ΔY mice despite its striking difference in susceptibility to autoimmunity. We conclude that none of the differences in the TCR Vβ repertoire of Eα‐transgenic NOD mice hitherto observed are likely to explain the protective effect of E molecule expression in NOD mice.
Monozygotic Rheumatoid Arthritis Twin Pairs Express Similar Levels of Conserved Immunoglobulin V Gene in Polyclonal Rheumatoid Factors Irrespective of Disease Status
Authors: VENCOVSKY J. , MAGEED R. A. , OLLIER W. E. , MAINI R. N.
Published: July 2006

The degree of polyclonal RF heterogeneity was assessed in diseased and non‐diseased twins with rheumatoid arthritis (RA). The distribution of variable region determinants encoded by a set of immunoglobulin germline, or minimally mutated germline, genes within IgM RF, IgG RF and IgA RF isotypes was determined by ELISA using specific mouse monoclonal antibodies (MoAb) in fractionated plasma from 12 members of six monozygotic twin pairs with RA, The results reveal that at least 40% (range ∼ 18–87%) of IgM RF are encoded by a small set of ∼ 10 genes from the VH1, 3 and 4 families. Furthermore, a significant proportion of IgG RF and IgA RF (∼ 30%) are also encoded by these same genes. Comparison with RF‐negative fractions of immunoglobulins showed that the examined variable region determinants were overrepresented in the RF fractions.

The level of expression of the variable region determinants in RF were generally similar within twins but different between unrelated twin pairs irrespective of disease status. The variability of VH gene usage between unrelated individuals suggests that the level of expression and regulation of the variable region determinants may be genetically regulated or influenced by common environmental factors.
TNF Inhibitors are Produced Spontaneously by Rheumatoid and Osteoarthritic Synovial Joint Cell Cultures: Evidence of Feedback Control of TNF Action
Authors: BRENNAN F. M. , GIBBONS D. L. , COPE A. P. , KATSIKIS P. , MAINI R. N. , FELDMANN M.
Published: July 2006

We have proposed the hypothesis that tumour necrosis factor α (TNF‐α) has a pivotal role in the pathogenesis of rheumatoid arthritis, based on in vitro observations that in RA synovial joint cell cultures removal of TNF‐α, inhibited other potentially pathogenic cytokines such as the equally pro‐inflammatory cytokine interleukin 1 (IL‐I) and the macrophage activating factor, GM‐CSF. Here we describe that in both rheumatoid (RA) and osteoarthritic (OA) synovial cultures there is a homeostatic mechanism to regulate the activities of TNF‐α. This concept is based on several observations. First in these synovial joint cell cultures the substantial discrepancy between the levels of bioactive and immunoreactive TNF‐α indicates the presence of an inhibitor. Second, TNF binding proteins are produced spontaneously, which are the soluble variants of surface p75 and p55 TNF receptor. The amount of soluble TNF receptors (sTNF‐R) produced varied between cultures; p75 sTNF‐R was more abundant than p55 sTNF‐R (as detected by ELISA), and both were produced at higher levels by RA synovial joint cells in culture, compared to OA cultures. These TNF binding proteins act as endogenous inhibitors of TNF‐a, since blocking their activity in synovial joint cell culture supernatants with MoAb to p55 and p75 sTNF‐R enhanced their cytotoxic activity in the TNF bioassay. The regulation of production of these sTNF‐R in synovial joint cell cultures is important as the balance between TNF‐a and sTNF‐R production may determine the outcome of the inflammatory process.
Characterization of Chicken CD8‐Specific Monoclonal Antibodies Recognizing Novel Epitopes
Authors: LUHTALA M. , KOSKINEN R. , TOIVANEN P. , VAINIO O.
Published: July 2006

CDS is a heterodimeric cell surface glycoprotein expressed primarily on thymocytes and a subpopulation of mature T lymphocytes. It binds to the invariant part of the major histocompatibility complex class I molecule and participates in antigen recognition by the major histocompatibility complex class I restricted T cells. As in mammalian species, the majority of chicken thymocytes express both CD4 and CD8, whereas peripheral T cells are either CD4‐ or CD8‐positive. We have created a panel of mouse monoclonal antibodies detecting different cell surface epitopes on chicken CD8. The antibodies precipitate a 32–34 kDa dimeric protein from surface labelled thymocytes under reducing conditions. The identical N‐deglycosylation pattern confirms that these MoAb precipitate the same heterodimeric molecule from chicken thymocyte lysates. Binding of 11–38 and 11–39 MoAb to peripheral blood T cells is totally inhibited by 11–39 and previously characterized CT8 and EP72 MoAb, further confirming their CD8 specificity. CD8α‐chain specificity of MoAb 11–39, 11–38, 11–30 and 11–13 is conclusively proven by staining COS‐cells transfected with a plasmid containing CD8α cDNA. However, MoAb 11–13, 11–30 and 11–38 do not compete with MoAb 11–39 in binding to CD8. These results demonstrate recognition of different epitopes by these MoAb. Monoclonal antibodies detecting novel epitopes on chicken CD8 provide a valuable tool for further studies on T cell development.
Lamina Propria T Cell Subsets in the Small and Large Intestine of Euthymic and Athymic Mice
Authors: BOLL G , REIMANN J.
Published: July 2006

We investigated lamina propria T cells from the small intestine (jejunum/ileum) and the large intestine (colon) of euthymic (BALB/c, C. B‐17, C57BL/6) and athymic (C57BL/6 nu/nu; BNX bg/bg nu/nu xid/ xid) mice. CD3+ T cells represented about 40% of the lamina propria lymphocytes (LPL) from the small or the large intestine of euthymic mice, and 20–30% of the LPL populations from the small or large intestine of athymic mice. In the lamina propria T cell population of the small intestine, 85% were of the αβ lineage in euthymic mice, but only 40% were of the αβ lineage in athymic mice. T cells of the αβ lineage were thus more frequent than T cells of the αβ lineage in the intestinal lamina propria T cells of extrathymic origin. CD4+ T cells represented 40% of the lamina propria T cells in the small as well as in the large intestine of euthymic mice, and 20–30% of the T cells in the lamina propria of the nude mouse gut. In euthymic mice, 40% of the T cells in the small intestine lamina propria, and 30% of the T cells in the colonic lamina propria were CD8+, In intestinal lamina propria T cell populations of athymic mice, the CD8+ T cell population was expanded. Most (60–70%) CD8+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice expressed the homodimeric CD8α+β− form of the CD8 coreceptor. A fraction of 15–20% of all CD3+ T cells in the lamina propria of the small and the large intestine of euthymic and athymic mice were ‘double negative’ CD4− CD8−. A large fraction of the TCRαβ+ T cells in the colonic lamina propria (but not in the small intestine lamina propria) of euthymic mice expressed the CD2 and the CD28 costimulator molecules, the adhesion molecule LECAM‐1 (CD62 L), and could be activated in vitro by CD3 ligation. These data reveal a considerable heterogeneity in the surface phenotype and the functional phenotype of murine lamina propria T cells.
Augmented Lung Adenocarcinoma Cytotoxicity by the Combination of a Genetically Modified Anti‐Lewis Y Antibody and Antibodies to Complement Regulatory Proteins
Authors: AZUMA A. , YAMANO Y. , YOSHIMURA A. , HIBINO T. , NISHIDA T. , YAGITA H. , OKUMURA K. , SEYA T. , KANNAGI R. , SHIBUYA M. , KUDOH S.
Published: July 2006

Complement‐dependent cytotoxicity (CDC) mediated by a chimeric anti‐Lewis Y monoclonal antibody (cH18A; human IgGl) was investigated in this study. Human lung adenocarcinoma cell lines (PC7, PC9, and PC 14) were used as the target cells. PC7 and PC9 cells, expressed Lewis Y antigen and were lysed by cH18A as effectively as by the parent mouse anti‐Lewis Y antibodies (mH18A) in a concentration‐dependent manner. PC14 cells did not express Lewis Y antigen and were not lysed by either cH18A or mH18A. cH18A mediated CDC activity against PC7 and PC9 cells was enhanced by the combined use of monoclonal antibodies directed against CD46(MCP), CD55(DAF), and CD59. These molecules are complement‐regulatory proteins which protect host cells from CDC. PC7 and PC9 cells, showed high levels of surface expression of these proteins, PC7 cells were more susceptible to cH ISA‐mediated CDC than PC9 cells. Use of multiple blocking antibodies to the complement‐regulatory proteins produced more enhancement of cH18A‐mediated CDC than a single antibody. Moreover, expression of CD55 and CD59 by PC7 and PC9 cells was decreased after treatment with Pl‐PLC, resulting in increased susceptibility to cHISA‐mediated CDC. Although the reason is unknown, PC7 cells became more susceptible to CDC than PC9 cells after PI‐PLC treatment even in the absence of cH18A. These data suggest that chimeric monoclonal antibodies can be used to induce CDC against lung adenocarcinoma, and that such CDC is potentiated by a variety of antibodies blocking compliment‐regulatory proteins on the tumour cell surface.
Mechanisms by which the I‐ABM12 Mutation Influences Susceptibility to Experimental Myasthenia Gravis: a Study in Homozygous and Heterozygous Mice
Authors: KARACHUNSKI P. I. , OSTLIE N. , BELLONE M. , INFANTE A. J. , CONTI‐FINE B. M.
Published: July 2006

The I‐Abm12 mutation in C57B1/6 (B6) mice yields the B6. C‐H‐2bm12 (bm12) strain, which is resistant to Experimental Myasthenia Gravis (EMG) induced by immunization with Torpedo acetylcholine receptor (TAChR), while the parental B6 strain is highly susceptible to EMG. CD4+ cells from bm12 mice immunized with TAChR do not recognize three sequence regions of the TAChR Q subunit which dominate the CD4+ cell sensitization in B6 mice. We immunized with TAChR bm12, B6 and (bm12B6)Fl mice. B6 and F1 mice developed EMG with comparable frequency. Their CD4+ cells recognized the same TAChR α subunit peptide sequences (Tα150–169, Tα181–200 and Tα360–378). CD4+ cells from TAChR‐sensitized Fl mice were challenged with TAChR and α subunit epitope peptides, using F1, B6 or bml 2 APC. B6 and F1 APC presented all these Ag efficiently, while bm 12 APC presented TAChR and peptide Tα150–169 poorly and erratically. Anti‐TAChR and anti‐α subunit epitope CD4+ lines propagated from Fl and B6 mice had similar TcR Vβ usage. All lines but those specific for the sequence Tα150–169 had unrestricted Vβ usage. Anti‐Tα150–169 lines from both B6 and Fl mice had a strong preferential usage of Vβ6. Anti‐Tα150–169 lines from Fl mice had also a slightly higher Vβ14 usage. B6, bm12 and Fl mice developed similar anti‐TAChR Ab titres, and had Ab bound to muscle AChR in comparable amounts. Therefore EMG resistance of bm12 mice must be due to a subtle shift in the anti‐AChR Ab repertoire, and absence of special Ab able to cause destruction and/or dysfunction of muscle AChR. This is probably related to the absence of CD4+ cells sensitized to epitopes within the sequence Tα 150–160, consequent to the inability of the I‐Abm12 molecule to present this sequence.
Induction of Tolerance to Experimental Anti‐Phospholipid Syndrome (APS) by Syngeneic Bone Marrow Cell Transplantation
Authors: BLANK M. , TOMER Y. , SLAVIN S. , SHOENFELD Y.
Published: July 2006

Previously, we have shown the ability to induce experimental autoimmune conditions (e. g. SLE, APLS, Wegener's granulomatosis) following active immunization with the pathogenic autoantibody emulsified in adjuvant. The mice first develop anti‐autoantibodies (Ab2: anti‐id) and eventually generate anti‐anti‐autoantibodies (Ab3: anti‐anti‐id) which carry the same antigen binding characteristic as the autoantibody (Ab1), The appearance of the specific autoantibodies in mice sera was associated with the emergence of the compatible laboratory and with the clinical findings characteristic to the respective autoimmune disease. The effects of syngeneic bone marrow transplantation (BMT) on experimental anti‐phospholipid syndrome (APS) were investigated. BALB/c mice were immunized with anti‐cardiolipin monoclonal antibody (MoAb) named CAM and developed elevated serum titres of anti‐phospholipid Abs accompanied by prolonged activated partial thromboplastin time, thrombocytopenia and a high percentage of fetal resorptions. These mice were then lethally irradiated and transfused with bone marrow (BM) cells (T cell depleted) from syngeneic naive mice. The titres of antiphospholipid antibodies were reduced in the recipients. The decrease in titre of autoantibodies was found to be related to depletion of antibody forming cells in vivo, associated with reduced proliferative response of lymph node cells to anti‐cardiolipin MoAbs. The recipients showed improvement in clinical parameters following syngeneic BMT. The same recipients developed specific unresponsiveness to a second challenge with the anticardiolipin MoAb (CAM), but developed experimental systemic lupus erythe‐matosus upon immunization with a monoclonal anti‐DNA antibody. We conclude that acute myeloablative immunosuppression combined with syngeneic bone marrow transplantation may induce a state of tolerance to the pathogenic autoantibodies in mice with experimental APLS. Our results suggest that a similar approach may be useful in treating life‐threatening autoimmune syndromes (e. g. catastrophic APLS) in clinical practice.
Two Nuclear Dot‐Associated Proteins, PML and SplOO, are Often Co‐Autoimmunogenic in Patients with Primary Biliary Cirrhosis
Authors: STERNSDORF T. , GULDNER H. H. , SZOSTECKI C. , GRÖTZINGER T. , WILL H.
Published: July 2006

The nucleoproteins Sp100 and PML, the first an autoantigen predominant in patients with primary biliary cirrhosis (PBC) and the second a transformation and cell growth suppressing protein aberrantly expressed in promyelocytic leukaemia ceils, were recently shown to colocalize in dot‐like nuclear domains. Here we analysed whether PML, like Sp100, is also an autoantigen in patients with PBC and other autoimmune diseases, and whether both proteins interact directly. Testing sera from autoimmune patients using an immunoprecipitation assay with radiolabelled PML and an immunofluorescence assay based on a cell hne overexpressing PML, autoantibodies (Aabs) against PML were found in the majority of anti‐Sp100 Aab positive patients. Only very few patients with PBC or other autoimmune diseases contained anti‐PML or anti‐Sp100 Aabs exclusively. In contrast to Sp100, immunoreactivity of recombinant PML in immunoblots was only weak and was directed to one region. This suggests that anti‐PML Aabs recognize fewer and preferentially conformation‐dependent epitopes. In an immuno precipitation assay using in vitro synthesized Sp 100 and PML proteins and Abs to recombinant proteins, no direct interaction was observed. Taken together, these data indicate that Aabs against PML are as highly prevalent and specific for patients with PBC as those against Sp100. The colocalization of these autoantigens and the frequent co‐occurence of the corresponding Aabs might reflect an association of both proteins mediated by one or several other proteins.
Systemic and Secretory Humoral Immunity in the Normal Human Vaginal Tract
Authors: HOCINI H. , BARRA A. , BELEC L. , ISCAKI S. , PREUD'HOMME J.‐L. , PILLOT J. , BOUVET J.‐P.
Published: July 2006

The molecular status of Abs in the vaginal fluid is reconsidered as a basis for immunization strategies for women's vaccination against HIV. Analysis of separated immunogiobulins (Igs) shows a large proportion of uncleaved IgG, whereas the low amount of IgA includes SIgA, monomers and fragments. SIgM is at a very low level, while free SC molecules are abundant. In addition to the already documented local synthesis, vaginal IgG contains serum‐derived tetanus antitoxins. The IgG could reach the lumen by diffusion, and/or be transported by an Fc receptor‐associated mechanism as suggested by the subclass imbalance in favour of the IgGl isotype. Vaginal SIgA contains very low levels of antibodies to the cell‐wall carbohydrates from a dental caries‐associated streptococcus confirming the participation of the secretory immune system. In addition, the low percentage of IgA2 suggests that a proportion of vaginal SIgA can also derive from actively transported serum polymers. In agreement with our previous studies showing induction of vaginal tetanus antitoxins by intramuscular immunization, these results are in favour of classical parenteral vaccinations to induce protection of the human vagina.
Anti CD5 Sustains the Proliferative Response of IgM‐Activated Human CD5+B Cells
Authors: JAMIN C. , DUEYMES M. , LYDYARD P. M. , YOUINOU P.
Published: July 2006

The CD5 molecule is expressed by most T cells but it is present on a minor B cell subset. Whilst several studies have provided information on the physiological role of T cell CD5, the functional role of CD5 on B lymphocytes remains unclear. To address this question, tonsillar CD5+ B cells were sorted by dual‐colour fluorescence and FACS. Sorted cells were stimulated with polyclonal anti‐IgM antibodies (Ab), and monoclonal (MoAb) F(ab')2 fragments of anti‐CD5. Proliferative responses were evaluated by enumeration of Ki‐67 positive cells using quantitative flow cytometry. Co‐stimulation with anti‐CD5 MoAb for 3 days did not affect the anti‐IgM and IL2‐induced proliferation of CD5+ B cells. This was seen under conditions where the anti‐CD5 was soluble, adsorbed to the microwells or cross‐linked by anti‐mouse antibodies. Fewer CD25+ cells were detected, however, in the presence of anti‐CD5. In contrast, the proliferative response of CD5+ B cells prestimulated for 3 days with IL‐2 and anti‐IgM, was sustained in a further 3‐day culture period when anti‐CD5 was added. It is concluded that CD5 occupancy might provide an additional signal to activated CD5+ B cells favouring their proliferation and differentiation into autoantibody secreting cells.
Chemical Denaturation of Ovalbumin Abrogates the Induction of Oral Tolerance of Specific IgG Antibody and DTH Responses in Mice
Authors: PENG H.‐J. , CHANG Z.‐N. , HAN S.‐H. , WON M.‐H. , HUANG B.‐T.
Published: July 2006

We have examined the effects of ingestion of chemically denatured ovalbumin (OVA) in mice. Both 8 M urea‐denatured OVA (UD‐OVA) and carboxymethylated UD‐OVA (CM‐OVA) were purified by gel filtration. Specific IgG antibody and systemic delayed‐type hypersensitivity (DTH) responses to OVA were not suppressed by CM‐OVA fed prior to or after immunization with OVA in complete Freund's adjuvant (CFA). When CM‐OVA was used instead of OVA for immunization, serum IgG and DTH responses to CM‐OVA were orally tolerized by OVA, but not by UD‐OVA or CM‐OVA. Studies of antigen uptake in mice using sandwich ELISA tests showed that OVA, but not CM‐OVA, was absorbed after antigen ingestion. In vitro studies further demonstrated that CM‐OVA was digested much more rapidly than OVA. Moreover, studies using bovine serum albumin (BSA) demonstrated that both IgG and DTH responses to BSA were orally tolerant to BSA, but not to denatured BSA. Finally, studies using human gamma‐globulin (HGG), a well‐known tolerogen, also found that the IgG antibody response to HGG was not orally tolerized by denatured HGG. These results suggest that complete denaturation of globular proteins may affect their processing and absorption in the gut and thus abrogates oral tolerance induction.
Cytokines and Adherence Molecules Involved in Spontaneous Dendritic Cell‐Lymphocyte Clustering in Skin Afferent Lymph
Authors: GALKOWSKA H. , WOJEWODZKA U. , OLSZEWSKI W. L.
Published: July 2006

The skin afferent lymph dendritic cell (DC) spontaneously forms clusters with autologous T cells. The role of adhesion molecules and cytokines in this process was investigated. Analysis of the expression of adhesion receptors on the canine peripheral lymph DC revealed the presence of CD54, CD58, CD18 as well as CD49d and CD49e molecules and cell surface fibronectin. The CD54 and CD58 molecules were found to play a key role in the ‘spontaneous’ lymph cell clustering. Antibody against fibronectin, a substrate for CD49d and CD49e receptors, reduced DC‐lymphocyte binding. Analysis of the effect of cytokines revealed that the pro‐inflammatory IL1β rather than ILlα, and TNFα may be responsible for the enhanced lymph cell in vitro clustering. The IL6 had no such augmenting effect. The enhancing effect of endogenous ILlβ present in lymph was reduced by the ILlβ neutralizing antibody. The effect of exogenously added ILlβ was also limited by the IL1 receptor antagonist. The ILlRa alone had no effect on cell binding, even when used in the high doses. Neutralizing of ILlRa in lymph with the specific antibody brought about augmented cluster formation. The enhancing properties of TNFα on cell binding were reduced by the TNFα neutralizing antibody. The IL10 significantly limited lymph DC cluster formation with T cells. In conclusion, these data demonstrate that the present in lymph IL1β and TNFα may be responsible for the observed in vitro enhanced cluster formation of lymph DC with autologous T lymphocytes. Cell binding can be reduced by ILlRa and by IL10. It provides insight into the potential clinical use of these inhibitors.
Stimulation of Human Peripheral Lymphocytes via CD3 and Soluble Antigen Abrogates Specific Antibody Production by Reducing Memory B Cell Numbers
Authors: INGVARSSON S. , LAGERKVIST A. C. SIMONSSON , CARLSSON R. , BORREBAECK C. A. K.
Published: July 2006

Human B cells are polyclonally activated in vitro by T cells stimulated with immobilized anti‐CD3 monoclonal antibodies. We have analysed the effect of CD3 ligation on the production of antigen‐specific antibodies, using peripheral blood lymphocytes from tetanus toxoid vaccinated blood donors. High levels of antigen‐specific antibodies were obtained after stimulation with anti‐CD3 antibodies for 7 days. Addition of a soluble recall antigen did not affect the total amount of Ig produced, but dramatically decreased the antigen‐specific response. The addition of IL‐2, IL‐4, anti‐CD40 or anti‐CD28 antibodies or the removal of antigen did not restore the B cell response. Analysis using limiting dilution of B cells showed that the frequency of antigen‐specific memory B cells decreased significantly in cultures stimulated with antigen. The antigen‐specific B cell response could be completely restored only if the soluble antigen was cross‐linked on the surface of the B cells. These results suggested that peripheral memory B cells were eliminated or anergized in the presence of soluble antigen.
Macrophage Response to Microbial Pathogens: Modulation of the Expression of Adhesion, CD14, and MHC Class II Molecules by Viruses, Bacteria, Protozoa and Fungi
Authors: KELLER R. , KEIST R. , JOLLER P.
Published: July 2006

The ability of inactivated viruses, bacteria, protozoa and fungi to modulate the expression of CD14, CD49d, CD49f, CD11a (LFA‐1), CD61, and CD54 (ICAM‐1) molecules in unprimed bone marrow‐derived mononuclear phagocytes (BMMø) was investigated by means of flow cytometry. Incubation with bacterial agents resulted in the large majority of experimental situations in enhanced expression of these macrophage surface molecules. In contrast, viruses and fungi down‐regulated the expression of several adhesion receptors, especially integrins. Amplification of MHC class II expression triggered in macrophages by interferon γ was clearly inhibited by viruses, bacteria, protozoa and fungi. The findings explain earlier results showing that, under the same experimental conditions, bacterial agents are, for the most part, potent stimulators of secretory and cell‐mediated macrophage activities while viruses, protozoa and fungi are poor in this respect.
Stimulation of the Same B‐Cell Population by Thymus‐Independent Dextran and by Thymus‐Dependent Oligosaccharide‐Carrier
Authors: WILLERS J. , WEILER E. , KOLB C.
Published: July 2006

The immune response of adult BALB/c mice against the bacterial antigen dextran B13558 (Dex) is well characterized as thymus independent (TI type 2) and Igha linked. The antisera consist of mainly IgM/ antibodies directed against the λ(1 → 3) glucosidic linkage. This study describes the immune reponse against the α(1 → 3) linkage in the thymus dependent form (TD), i. e. tetra‐ or heptasaccharides (N4 or N7) of glucose as hapten coupled to chicken serum albumin (CSA) as carrier. Whereas athymic BALB/c‐nu/nu mice did not respond to the TD antigens N4‐CSA and N7‐CSA, euthymic BALB/c showed high anti‐Dex antibody titres of IgM and, after 2° immunization, a class switch to IgG (mainly IgGl) isotypes with λ light chains. The hapten N4 inhibited Dex‐binding of M104E or of antisera from Dex or N4‐CSA or N7‐CSA immunized mice at 1.7–10 × 104M. The idiotype composition of these antibodies resembled those after Dex immunization. We conclude that the same Dex‐specific precursor B cells have been stimulated by either form of antigen. The ontogenic development of a Dex‐specific response could not be accelerated by the aid of T cells, even of adult origin. It seems, therefore, that the maturation of antigen specific B cells is the limiting step in ontogeny.
Multiple Ligand Interactions for Bacterial Immunoglobulin‐Binding Proteins on Human and Murine Cells of the Hematopoetic Lineage
Authors: AXCRONA K. , BJÖRCK L. , LEANDERSON T.
Published: July 2006

A group of bacterial Ig‐binding surface proteins were studied: protein H and Ml are from Streptococcus pyogenes and interact with IgG, protein L is expressed by Peptostreptococcus magnus and shows affinity for Ig light chains, whereas protein LG is a chimeric construction combining the binding properties of protein L with the IgG‐binding activity of protein G from group C and G streptococci. Proteins L and H coupled to Sepharose were mitogenic for human peripheral blood lymphocytes (PBL) and mouse splenic B cells, but not when added in soluble form. Differentiation to Ig secretion was induced by protein H‐Sepharose in mouse splenic B cells but not in human PBLs. In FACS analysis FITC‐labelled protein H stained virtually all CD19+ cells in human peripheral blood as well as a majority of the CD3+ population. Protein L bound the majority of the CD19+ population, but also a fraction of the CD19−/CD3 population. Protein Ml was not mitogenic but stained the entire CD19+ population and 70% of the CD3+ population. Identical staining patterns were observed with mouse splenocytes using B220 and T‐cell receptor as lineage markers. The chimeric protein LG was a potent mitogen for mouse splenic B cells when added either coupled to Sepharose or in soluble form. In addition, protein LG induced differentiation to Ig secretion of the responding mouse splenic B cells. In FACS analysis, protein LG stained the entire CD19+ and the majority of the CD19−/CD3 lymphocyte population as well as all B220+ mouse splenocytes and a fraction of the splenic T cells. These data indicate that the bacterial proteins studied interact with surface structures of several leucocyte populations and can hence interfere with the immune system at multiple levels.
Impact of MHC Class I Gene on Resistance to Murine AIDS
Authors: MAKINO M. , MURPHY D. B. , MELVOLD R. W. , HARTLEY J. W. , MORSE H. C.
Published: July 2006

Development of murine AIDS in mice following infection with LP‐BM5 murine leukaemia virus (MuLV) is highly strain dependent, with strain differences determind by genes within and outside H‐2. Among H‐2 genes, the Dd gene is the most closely associated with resistance to LP‐BM5 MuLV infection. However, the Dd‐mediated resistance is highly influenced by outside H‐2 genes, i. e. A lineage strains are more resistant than mice strains of B6/B10 lineage. In this study, the mice having BALB background were analysed and, similarly to A lineage mice, only Dd gene products were found to be required to provide resistance to LP‐BM5 MuLV infection. Furthermore, BALB/c Kh mice bearing both Dd and Ld genes clearly showed obviously higher resistance than BALB/c‐H‐2dm2 mice solely having the Dd gene. In addition, in the long‐term observation of the effect of the Dd gene on B6/B10 background mice, D8 mice having the Dd gene as a transgene and expressing a high level Dd gene product showed higher resistance than naturally recombinant B10. A(18R) mice. These results suggest that the MAIDS resistance associated with the D end loci is dependent on the level of expression of an MHC class I gene.