bluuebunny/crossref_metadata_2025 huggingface dataset:
Three New Cross‐Reacting Idiotopes as Markers for the Products of Two Distinct Human VH3 Genes Expressed in the Early Repertoire
Authors: SULEYMAN S. , THOMPSON K. M. , FØRRE Ø. , SIOUD M. , RANDEN I. , MAGEED R. A. , NATVIG J. B.
Published: July 2006

This article describes characterization of three new cross‐reacting idiotopes, as recognized by mouse MoAbs, on human antibodies utilizing VH3 genes that are expressed in the early repertoire. Two of the mouse MoAbs (3H7 and 3H1) were raised against a human MoAb utilizing the DP47 (VH26) VH3 gene, whilst the third (7B4) was raised against a DP46 (GLSJ2) gene product. Evidence for the anti‐idiotypic specificity of the mouse MoAbs was provided by their reactivity with the immunizing IgM, but not with Fcα, and by their specific inhibition of the binding between each immunizing antibody and its antigen. The three anti‐idiotypic MoAbs were shown to be VH‐specific reagents by the independence of their reactivity upon the L‐chain type, or the untigenic specificity of the human MoAbs tested. Specificity of each mouse MoAb for VH3 gene‐products was demonstrated by its sole cross‐reactivity with VH3 proteins. Each anti‐Id had a different reactivity pattern with a panel of MoAbs utilizing different VH3 genes. By relating the VH sequences of the tested VH3 proteins to their germline counterparts, 3H7 and 3H1 appeared to be specific for DP47‐encoded proteins, although 3H1 had weak cross‐reactivities with a few other VH3 gene‐products. 7B4 appeared to be specific for antibodies utilizing DP46‐related genes. Both 3H7 and 3H1 were also completely different to B6 and D12, two previously described MoAbs that also recognize VH3 proteins. Although 7B4 was similar to B6 and D12 in its binding to DP46‐related gene products, B6 and D12 additionally recognized non DP46‐related proteins and were thus different to 7B4.
Accessory Signalling by B7‐1 for T Cell Activation Induced by Anti‐CD2: Evidence for IL‐2‐Independent CTL Generation and CsA‐Rcsistant Cytokine Production
Authors: VAN GOOL S. W. , KASRAN A. , WALLAYS G. , DE BOERJ M. , CEUPPENS J. L.
Published: July 2006

Resting T cells can be activated by selected pairs of anti‐CD2 MoAb. Activation is dependent on the presence of accessory cells, which can be replaced by either anti‐CD28, or by the combination of IL‐1β and IL‐6. The present study was undertaken to investigate accessory signalling by B7‐1, the natural ligandof CD28, in this pathway of T cell activation. 3T6 mouse fibrobiasts were transfected with human B7‐1 and used as accessory cells in cultures of purified resting human T cells. In the presence of a stimulating pair of anti‐CD2 MoAb, T cell proliferation, production of cytokines (IL‐2, IL‐4, IL‐10, GM‐CSF, IFN‐α and TNF‐α), and generation of cytotoxic T lymphocytes were all supported by B7‐l(+) 3T6 cells but not by control 3T6 cells. Blocking studies with anti‐IL‐2 + anti‐IL‐2R MoAb revealed both IL‐2‐dependent and IL‐2‐independent CTL generation after B7‐1 ‐mediated costimulation. Moreover, a partial or complete resistance to inhibition with CsA was observed for IL‐2 production and CTL generation respectively in the presence of the costimulatory signal derived from B7‐1 ‐ CD28 interaction. Anti‐CD2 MoAb with B7‐1 costimulation could directly induce proliferation, IL‐2 production and generation of CTL activity in highly purified CD8+ T cells without the heip of CD4+ T cells. We conclude that CD28 ligation with the natural ligand B7‐1 provides a strong accessory signal for CD4 and CD8 cell activation through CD2.
Intraembryonic Haemopoietic Cells and Early T Cell Development
Authors: LAMPISUO M. , KARVINEN J. , ARSTILA T. PETTERI , LASSILA O.
Published: July 2006

T cell precursors in the chick embryo have been localized into the intraembryonic mesenchyme (IEM) and into the para‐aortic region before the first wave of the thymic colonization on embryonic day (ED) 6, 5–8. The cell surface markers of avian prethymic stem cells are not known. It is also not known whether these precursor cells are already committed to the T cell lineage before their thymic colonization.

In 7‐day‐old chick embryos Ov+ cells were found in the para‐aortic region. Also the endothelial cells of the embryonic dorsal aorta were positively stained. Ov antigen might represent a very primitive marker for precursor cells having the potentiality to differentiate both to haemopoietic and endothelial cells. Scattered CD45+ cells were observed in the same para‐aortic area as in many haemopoietic areas in the loose embryonic mesenchymal tissues. CD8α (MoAb 3–298) expressing haemopoielic cells were detected before thymic colonization on ED6. In flow cytometric analysis of IEM precursors Ov, CD45 and CD8α expressing cells seemed to form distinct subsets suggesting heterogeneity of these haemopoietic cells.
Interferon‐α Production and Tissue Localization of Interferon‐α/β Producing Cells after Intradermal Administration of Aujeszky's Disease Virus–Infected Cells in Pigs
Authors: ARTURSSON K. , LINDERSSON M. , VARELA N. , SCHEYNIUS A. , ALM G. V.
Published: July 2006

Intradermal administration of glutaraldehyde–fixed Aujeszky's disease virus (ADV) infected autologous or allogeneic cells resulted in the induction of an interferon(IFN)–α/β response in pigs. Using a sensitive dissociation–enhanced lanthanide fluoroimmunoassay (DELFIA), IFN–α/β was detected in blood at 8 and 24 h after injection of ADV–infected cells. In parallel, by means of in situ hybridization, IFN–α/β mRNA containing cells were demonstrated in regional lymph nodes. Occasional IFN–α/β mRNA positive cells were also seen in injected dermal areas, but not in contralateral lymph nodes, spleen, bone marrow, blood or liver. The ability of leucocytes in whole blood cultures to produce IFN–α/β upon stimulation by ADV was markedly diminished 3–7 days after intradermal injection of ADV–infected cells. In contrast, cultures of purified peripheral blood mononuclear cells (PBMC) had intact IFN–α/β responses. Further, serum from ADV–injected pigs inhibited the in vitro ADV–induced IFN‐α/β responses in PBMC from control pigs, most likely due to the demonstrated presence of anti–ADV antibodies. We suggest that the IFN‐α/β producing cells in lymph nodes may participate in the development of antiviral immunity and could be equivalent to Natural IFN–α/β producing (NIP) cells.
A Polyclonal T Cell Repertoire of V‐Alpha and V‐Beta T Cell Receptor Gene Families in Intrathyroidal T Lymphocytes of Graves' Disease Patients
Authors: CASO‐PELÁEZ E. , McGREGOR A. M. , BANGA J. P.
Published: July 2006

We have examined for the presence or absence of T cell receptor V‐alpha (VA) and V‐beta (VB) gene expression in infiltrating T lymphocytes (ITL) isolated from Graves' thyroid glands in comparison to paired peripheral blood lymphocyte (PBL) samples using a qualitative based polymerase chain reaction (PCR) assay. Sequence specific oligonucleotides for VA and VB T cell receptor gene (TCR) families that had previously been validated in other studies, were used for the PCR analysis, followed by Southern blot hybridization with a labelled, internal C‐region primer. A total of seven Graves' disease patients who had been treated with carbimazole were studied. T ceil receptor VA and VB gene usage was examined in freshly isolated, unstimulated ITLs from five patients. A widespread usage of VA and VB gene families with 12 to 18 families being used was apparent. Use of oiigo‐dT or C‐region priming of the mRNA prior to reverse transcription of the mRNA did not have any significant affect on the results nor did the use of whole Graves' thyroidmRNA as the starting material (n = 2) or perfusion of one gland with saline to remove as much of the contaminating blood from the gland. Our results contrast with those of Davies and colleagues who have previously shown a restricted repertoire of VA gene families m ITLs in comparison to autologous PBLs, and are much more in line with other recent reports indicating a diverse VA repertoire of the infiltrating T cells in Graves' thyroid glands derived from patients treated with anti‐thyroid drugs.
Monosialoganglioside GM3 Induces CD4 Internalization in Human Peripheral Blood T Lymphocytes
Authors: SORICE M. , PAVAN A. , MISASI R. , SANSOLINI T. , GAROFALO T. , LENTI L. , PONTIERI G. M. , FRATI L. , TORRISI M. R.
Published: July 2006

Gangliosides modulate the expression of CD4 molecules on the cell surface of T lymphocytes. We report here that treatment of human peripheral blood lymphocytes with exogenous monosialoganglioside GM3 induces a rapid down‐modulation of the CD4 molecules on the plasma membrane of CD4+ T lymphocytes, as assessed by cytofluorimetric analysis and quantitative immunoelectron microscopy. The CD4 down‐modulation was gang Hoside‐dose dependent and was already evident after 5 min of treatment, reaching the maximum after 20 min. The expression of other surface antigens was not affected by GM3 treatment. The immunoelectron microscopic analysis showed that, following GM3 addition, gold labelled CD4 molecules were rapidly redistributed on the cell surface, clustered and internalized via endocytic pits and vesicles. These results indicate that CD4 down‐modulation induced by GM3 occurs through an endocytic mechanism. A persistent low level of CD4 expression on the cell surface up to 24 h after GM3 treatment, compared with a stable expression of either CD4 in untreated cells and CD3 in GM3‐treated cells, suggests intracellular degradation of the internalized CD4 molecules.
Abnormal Splenic and Thymic IL‐4 and TNF‐α Expression in MRL‐lpr/lpr Mice
Authors: TSAI C.‐Y. , WU T.‐H. , HUANG S.‐F. , SUN K.‐H. , HSIEH S.‐C. , HAN S.‐H. , YU H.‐S. , YU C.‐L.
Published: July 2006

The MRL‐lpr/lpr and MRL‐++ mice were studied for the expression of cytokines in the spleen, lymph node., thymus, kidney and brain through the reverse transcription‐polymerase chain reaction (RT‐PCR). The frequencies of IL‐4 and TNF‐a expression in the thymus and spleen were significantly higher in MRL‐lpr/lpr mice than in MRL‐++ mice from the age of 17 to 32 weeks. More importantly, IL‐4 transcript was demonstrated in the early rather than in the terminal stage of the lupus disease. At the 20th week, MRL‐lpr/lpr mice with active disease exhibited higher concentrations of IL‐1α, IL‐6 and TNF‐a in serum than MRL‐++ mice. Interestingly, in MRL‐lpr/lpr but not MRL‐++ mice, the IL‐6 concentration in culture supernatants of the thymic cells was significantly higher than that of the splenic or lymph node cells. On the other hand, IL‐6 and IL‐l/? were expressed in the brain and kidney of MRL‐lpr/lpr mice but not of MRL‐++ mice. Cultured MRL‐lpr/lpr mesangial cells could also express IL‐6 but to a lesser extent. These results suggest that the abnormal splenic and thymic IL‐4 and TNF‐α expression may predispose the development of autoimmune reactions. The expression of IL‐1ß and IL‐6 in the brain and kidney may be implicated in the damage of these two organs in MRL‐lpr/lpr mice.
Cytokine Gene Expression During Infeetion of Miee Lacking CD4 and/or CD8 with Trypanosoma cruzi
Authors: ROTTENBERG M. E. , SPORRONG L. , PERSSON I. , WIGZELL H. , ÖRN A.
Published: July 2006

The expression of lymphokine genes during infection of virulent (Tulahuén) or mild (CA‐I) strains of T. cruzi was studied in mice lacking CD4 and/or CD8 molecules. The increased susceptibility of CD4− and CD4−CD8− mice to infection with CA‐I or Tuiahuen was parallelled by diminished IFN‐γmRNA levels. Nitric oxide release and inducible nitric oxide synthase mRNA accumulation by cells from Tulahuen infected CD4− mice was also diminished. CD8− (but not CD4−CD8− mice) showed an increased IL‐4 and IL‐10mRNA accumulation upon infection with both strains of T. cruzi. A Th2‐like’ response (higher IL‐4 and IL‐10mRNA to IFN‐γmRNA ratio), was also observed when cells from non‐infected CD8‐ mice were stimulated with T cell mitogens.
Increased mRNA Expression of IL‐10 in Mononuclear Cells in Multiple Sclerosis and Optic Neuritis
Authors: NAVIKAS V. , LINK J. , PALASIK W. , SÖDERSTRÖM M. , FREDRIKSON S. , OLSSON T. , LINK H.
Published: July 2006

The inflammatory nature of multiple sclerosis (MS) implicates the participation of immunoregulatory cytokines, including the Th2 related IL‐10. We describe the use of in situ hybridization with cDNA oligonucieotide probes to detect and enumerate mononuclear cells (MNC) expressing mRNA for IL‐10 which is known to down‐regulate Thl cell related cytokines such as interferon‐γ. Expression of IL‐10 was studied in blood MNC of MS and blood and cerebrospinal fluid (CSF) MNC of optic neuritis (ON) patients without culture and after culture in the presence of myelin basic protein (MBP), the control antigen acetylcholine receptor (AChR), and without antigen. Numbers of IL‐10mRNA expressing MNC were elevated in the MS patients' blood both when enumerated without culture and after culture with MBP. Control patients with myasthenia gravis had elevated numbers of AChR‐reactive IL‐10mRNA expressing cells, while numbers of MBP‐reactive IL‐10 positive ceils did not difl'er from numbers registered in cells without antigen. Patients with ON, in many instances representing early MS, had IL‐10 positive blood MNC that were elevated to the same extent as in clinically deflnite MS, and further increased in the CSF. ON patients examined within 1 month after onset had lower numbers of MBP induced IL‐10mRNA expressing blood MNC compared with patients examined later suggesting that IL‐10 is related to the degree of inflammation and outcome in ON.
Composition and Immunoreactivity of the A60 Complex and Other Cell Fractions from Mycobacterium bovis BCG
Authors: COCITO C. , VANLINDEN F.
Published: July 2006

Surface static cultures of Mycobacterium bovisBCG contained cells embedded in an extracellular matrix, whose mechanical removal yielded free cells that were pressure disrupted and fractionated into cytoplasm and walls. Cell envelopes were either mechanically disrupted or extracted with detergents. Intracellular and extracellular fractions were analysed for proteins, polysaccharides, and antigen 60 (A60), a major complex immunodominant in tuberculosis. A60 was present in extracellular matrix, cytoplasm and walls: it represented a substantial portion of the proteins and polysaccharides of these fractions. While the protein/polysaccharide ratio varied according to the origin of A60 preparations, the electrophoretic patterns of A60 proteins (which accounted for the immunogenicity of the complex) remained unchanged. Western blots pointed to the proteins present within the 29–45 kDa range as the A60 components endowed with the highest immunogenicity level. Since the most heavily stained protein bands in SDS‐PAGE patterns were located outside the region best recognized by antisera, a striking discordance was found between concentration and immunogenicity patterns of A60 proteins. The electrophoretic patterns of A60‐ and non‐A60‐proteins from cytoplasm were also different. A60 complexes in dot blots and some electrophoresed A60 proteins reacted with monoclonal antibodies directed against lipoarabinomannan (LAM), a highly immunogenic polymer of cell envelope. This contaminating compound was removed from A60 with organic solvents and detergents. SDS‐PAGE and Western blot patterns of proteins from delipidated A60 were similar to those of native A60 proteins.
Aberrant Patterns of Immunoglobulin Levels in Wiskott‐Aldrich Syndrome
Authors: INOUE R. , KONDO N. , KUWABARA N. , ORII T.
Published: July 2006

We have investigated IgM deficiency in Wiskott‐Aldrich syndrome patients. From the assessment of T and B cell functions in pokeweed mitogen‐induced immunoglobulin (Ig) production. IgM deficiency was chiefly thought to result from B cell dysfunction. The percentages of surface IgM‐bearing cells were decreased in peripheral blood mononuclear cells (PBMCs) and the number of IgM‐secreting cells was also decreased. Lymphoblast cell lines (LCLs) from the patients have produced IgG and IgA, but never IgM. Moreover the expression of the Cμ transcript from the patients' PBMCs and their LCLs were decreased, whereas the Cμ gene was well expressed. No germ‐line polymorphism existed between the patients and the controls in the Cμ region, and no mutation was detected in the μs C‐terminal and the M exon by nucleotide sequencing. These suggest that the Ig heavy chain (IGHC) isotype switch may be abnormally accelerated in the patients' B cells. While the methylation patterns of the human Ig enhancer gene region were the same between the patient and the control, the methylation patterns of the Iγ1 region showed less methylation in the patient than in the control, which may cause low IgM expression and high expression levels of other classes located downstream of the IGHC gene.
Human Anti‐Ro Autoantibodies Bind Peptides Accessible to the Surface of the Native Ro Autoantigen
Authors: HUANG S. C. , YU H. , SCOFIELD R. H. , HARLEY J. B.
Published: July 2006

The relationship between fine specificity of linear epitopes and conformational determinants has been explored in a naturally arising human autoimmune response. In particular, the hypothesis tested is that the linear epitopes of the human Ro autoantigen are components of its conformational epitopes. Twenty groups among the 531 overlapping octapeptides 60kDa Ro are variably bound by anti‐Ro precipitin positive lupus sera whose reactivity was easily distinguished from sera of normal controls and of anti‐Ro precipitin negative lupus patients. The specific activities of anti‐peptide antibodies and of anti‐native Ro autoantibodies are similarly increased after affinity enrichment using native human Ro as ligand. Moreover, affinity‐enriched anti‐native Ro autoantibodies bind virtually the same 20 groups of epitopes recognized by whole anti‐Ro positive sera. Two peptides (residues 274–290 and 480–494) from the defined 60 kDa Ro octapeptide epitopes have been prepared and used as ligands for affinity purification of peptide specific autoantibodies. The binding of both whole IgG and affinity‐enriched peptide specific autoantibodies is inhibited by native Ro autoantigen. Thus, none of the available data can be construed to support the existence of cryptic linear epitopes in this system. Indeed, the data are only consistent with the conclusion that all of the anti‐Ro octapeptide autoantibodies are part of the population of anti‐native Ro autoantibodies in this naturally arising autoimmune response.
Interleukin‐4 Gene Expression in Mercury‐Induced Autoimmunity
Authors: GILLESPIE K. M. , QASIM F. J. , TIBBATTS L. M. , THIRU S. , OLIVEIRA D. B. G. , MATHIESON P. W.
Published: July 2006

Mercuric chloride (HgCl2) induces autoitnmunity in Brown Norway (BN) rats, with necrotizing vasculitis in the gut. Circumstantial evidence implicates the Th2 subset of CD4+ T lymphocytes, which produces IL‐4. We developed a quantitative polymerase chain reaction (PCR) technique to quantify IL‐4 gene expression. A phagemid containing rat IL‐4 cDNA was modified to act as the template for a synthetic RNA construct; a known amount of synthetic RNA was added to total RNA from spleen and caecum of BN rats at various times after HgCl2, followed by reverse transcriptase PCR. IL‐4 gene expression increased markedly in spleen and caecum after HgCl2. Splenic levels peaked by 10 days at approximately five‐times baseline, then returned towards normal as the autoimmune response was spontaneously regulated. Caecal IL‐4 expression peaked at 48 h, at which time we observed a previously unreported early phase of tissue injury, with necrotizing vasculitis qualitatively similar to that reported previously in the later phases of the model. These data support a key role for IL‐4 in this experimental model of autoimmunity. The quantitative PCR technique can be modified for analysis of other cytokines, allowing further investigation of the role of T ceil subsets in this model.
Cloning and Sequencing of a Unique Antigen MPT70 from Mycobacterium tuberculosis H37Rv and Expression in BCG Using E. coli‐Mycobacteria Shuttle Vector
Authors: MATSUMOTO S. , MATSUO T. , OHARA N. , HOTOKEZAKA H. , NAITO M. , MINAMI J. , YAMADA T.
Published: July 2006

MPB70 is known to be an immunogenic mycobacterial protein secreted in large amounts from Mycobacterium bovis BCG (BCG) Tokyo. The analogous gene for MPT70 was cloned from Mycobacterium tuberculosis H37Rv which produces this protein in only a small amount. The gene encoding 193 amino acid residues inciuding 30 amino acids for the signal peptide. the promoter‐like sequence, and the ribosome‐binding site, was completely identical to that of BCG Tokyo. Computer analysis revealed that the carboxy‐terminal half of MPT70 was homologous to amino acid sequences of fasciclin 1, osteoblast‐specific factor 2 (OSF‐2), and human transforming growth factor‐beta induced gene product (βIG‐H3). Escherichia coli (E. coli) ‐mycobacteria shuttle vectors containing mpt70 or mpb70 genes 0.7kbp upstream of the 5′ end of them were able to be expressed in BCG Pasteur which is a MPB70 low‐producer, but the extent of the expression was not that of a high‐producer.
Dendritic Leucocytes Pulsed with Monoclonal Antibody‐Hapten Conjugates Elicit Vigorous Primary Humoral ResponsesIn Vivo
Authors: MJAALAND S. , FOSSUM S.
Published: July 2006

Purified dendritic leucocytes (DL) were pulsed brieflyin vitrowith haptenated monoclonal antibodies (MoAb) to MHC class II and immediately injected i. v. into syngeneic recipients. Strong anti‐hapten humoral responses were observed even though only a few picomoles of specific MoAb‐hapten conjugates were injected with the DL. In contrast, DL pulsed with control conjugates, i. e. haptenated non‐binding MoAbs, gave only weak responses. DL thus, can take up, process and present protein antigens even after brief exposurein vitro, and their immunogenicity is enhanced by pulsing with antigen conjugated to specific MoAbs. Furthermore, in the presence of fetal calf serum (FCS), but not normal rat serum, the control MoAb W6/32 (against human MHC class I) bound to DL. The vigorous primary humoral response achieved following this pulsing indicates that it is the binding and the corresponding increased uptake that enhances the immunogenicity of the DL.
Differential Effects of Interleukin‐2 and Interleukin‐7 on the Induction of CD4 and CD8 Expression by Double‐Negative Human Thymocytes*
Authors: HE W. , KABELITZ D.
Published: July 2006

Expression of ceil surface CD4 (in the absence of CD3/T cell receptor) characterizes an early stage of intrathymic T cell development. Here, we investigated the appearance of CD4 and CD8 expression on highly purified CD4−8− double‐negative human thymocytes in response to interleukin (IL)‐7 and IL‐2. While IL‐7 preferentially promoted the appearance of CD4 single‐positive and CD4+8+ double‐positive thymocytes, I1‐2 primarily induced CD8 single‐positive thymocytes. A significant fraction of CD4 single‐positive cells generated from double‐negative thymocytes via IL‐7 lacked cell surface CD3 expression. In contrast, the majority of CD8 single‐positive cells generated from double‐negative thymocytes via IL‐2 coexpressed CD3. We conclude that IL‐7 and IL‐2 exert differential effects on the differentiation of early human T cell progenitors.
Predominant V‐Region Gene Configurations in the Human Antibody Response to Haemophilus influenzae Capsule Polysaccharide
Authors: PINCHUK G. V. , NOTTENBURG C. , MILNER E. C. B.
Published: July 2006

The antibody response to Haemophilus influenzae type b polysaccharide (Hib PS) is known to be encoded by a few V‐region genes. We have obtained four human monoclonal Hib PS antibodies from four healthy adult subjects immunized with diphtheria toxin‐conjugated Hib PS vaccine. The VH gene segments that encode for these antibodies belong to the VH3 gene family, of which two are related to the V3‐23 gene and two to the VH3b subfamily. Both hybridomas that express a V3‐23‐related gene use short D‐segments (3 bp), the JH6 gene segment and a Vk gene derived from the A2 germline gene. The two hybridomas that express VH3b genes use D‐segments of conventional length (24–33 bp), the JH4 gene segment and a non‐A2 Vk gene. Comparison of our sequences with those reported by others suggests that the above patterns of V‐region gene segment association exemplify two V‐region gene configurations that are predominant in the Hib PS antibody response. The first configuration is reminiscent of antibodies produced by B‐l B cells while the second is more characteristic of antibodies produced by conventional B cells. The possibility that these two configurations, in fact, represent the products of two different B cell lineages remains to be elucidated.
A Human Melanoma Cell Line, Recognized by Both HLA Class I and Class II Restricted T Cells, is Capable of Initiating both Primary and Secondary Immune Responses
Authors: OLSEN A. C. , POSSUM B. , KIRKIN A. F. , ZEUTHEN J. , GAUDERNACK G.
Published: July 2006

We have characterized a melanoma cell line, FM3, established from a metastasis of a 75 year old female patient (HLA–A2, HLA–DQ7) with malignant melanoma. This cell line expresses both HLA class I and class II antigens, as well as several important accessory molecules at high levels. FM3 cells were shown to function as a stimulator of both allogeneic as well as autologous mixed lymphocyte tumour cell culture (MLTC). From these autologous MLTC we were able to generate cytotoxic T cell clones indicating that FM3 is capable of processing and presenting endogenous antigens.

We have used this cell line in a model system to investigate whether these cells were able to initiate and support an immune response with specificity for selected peptide antigens. The FM3 cell line was capable of presenting a HLA–DQ7 restricted ras derived peptide (5–21, 13Gly–Asp) to a previously established T cell clone, RM70. The ability of FM3 to function as an antigen presenting cell (APC) was comparable to that of an autologous Epstein Barr virus (EBV) transformed B cell line. The CD4+ T cell clone RM70 showed a peptide–specific anti–proliferative effect on FM3 cells. This growth inhibition was not due to cytotoxicity as measured in a standard 4h chromium release assay. The FM3 cell line also presented a HLA–A2 restricted nonapeptide derived from the influenza matrix protein. M 1 (58–66) to a CD8+ T cell line specific for this peptide. This resulted in an effective killing of the melanoma cells.

Together, these data suggest that some melanomas may initiate an immune response by presenting their own specific antigens in an immunogenic context, and subsequently serve as targets for T cells of both the CD4+ and CD8+ phenotype.
Limitations of the Severe Combined Immunodeficiency (SCID) Mouse Model for Study of Human B‐Cell Responses
Authors: SOMASUNDARAM R. , JACOB L. , ADACHI K. , CLASS R. , SCHECK S. , MARUYAMA H. , HERLYN D.
Published: July 2006

Mice lacking functional T and B lymphocytes offer an in vivo animal model for the study of human immune functions. We have attempted to optimize the reconstitution of severe combined immunodeficiency (SCID) mice with human peripheral blood lymphocytes (PBL) using radiation, anti‐asialo GM1 antibody or cyclophosphamide (Cy) treatment of the mice and in vitro stimulation of human PBL with interleukin (IL)‐2 prior to their transfer to the mice. Total human IgG and tetanus‐toxoid (TT)‐specific human IgG responses of the mice were used as parameters of successful reconstitution. Treatment of the mice with anti‐asialo GM1 antibody significantly enhanced total human IgG levels, but not TT‐specific antibody responses, whereas irradiation or Cy treatment of the mice had no effect on human antibody production. In vitro treatment of human PBL with IL‐2 prior to engraftment significantly decreased total human IgG responses of human PBL‐grafted SCID mice. The immune responses of individual mice within a group were highly variable, which constitutes a major disadvantage of this model.
Subclass Distribution of IgA and IgG Antibodies Against Clq in Patients with Rheumatic Diseases
Authors: COREMANS I. E. M. , DAHA M. R. , VAN DER VOORT E. A. M. , SIEGERT C. E. H. , BREEDVELD F. C.
Published: July 2006

To obtain insight into the immunoregulatory mechanisms in patients with different rheumatic diseases, the occurrence and the subclass distribution of IgA and IgG antibodies against Clq (anti‐ClqAb) was determined. In patients with systemic lupus erythaematosus (SLE) the highest frequency of increased serum levels of IgG anti‐ClqAb were found, whereas IgA anti‐ClqAb were predominantly present in patients with ankylosing spondylitis (AS) and patients with rheumatoid arthritis complicated by vasculitis (RV). In all the IgA anti‐ClqAb positive AS and RV patients the antibody reactivity involved the IgAl subclass while the IgA2 subclass was found in 47% of the patients. Further characterization of the IgA anti‐Clq binding activity in sera of AS patients revealed that both subclasses of IgA anti‐ClqAb were predominantly polymeric; the binding of both IgA subclasses with solid phase CI q was inhibitable by aggregated fluid phase Clq; we found no delectable interference of rheumatoid factor in the test system for the measurement of IgA anti‐ClqAb. In patients with SLE the IgG anti‐ClqAb reactivity was mainly of the IgG2 and IgG3 subclass, whereas in the same patients the IgG anti‐tetanus toxoid response was not restricted to these subclasses.

The predominance of IgG2 and IgG3 subclass of anti‐ClqAb in sera of SLE patients, suggests a skewing of the anti‐ClqAb response. The observation that the IgA anti‐ClqAb of both subclasses is predominantly polymeric in nature and the notion that polymeric IgA is associated with activation of inflammation cascades, suggests that IgA anti‐ClqAb may contribute to tissue damage.
First Evidence for the Existence of Multiple Isoforms of Bovine Serum Amyloid‐A (apoSAA)
Authors: ALSEMGEEST S. P. M. , HORADAGODA A. , HULSKAMP‐KOC C. K. , TOOTEND P. C. J. , KIM H. , NIEWOLD TH. A. , GRUYS E.
Published: July 2006

Bovine serum amyloid‐A (SAA) was purified from acute‐phase high density iipoprotein (HDL) by affinity chromatography and subsequent gel filtration chromatography. The identity of the isolated protein was checked by Western blotting following SDS‐urea‐PAGE using antisera raised against the purified protein fraction (SAA) and Amyloid A (AA). The antiserum raised against the purified SAA stained Congo red positive regions in the kidney of an AA‐amyloidotic cow and reacted on Western blot with an AA‐related protein of approximately 14kDa. Moreover, it immunostained two to three bands, of approximately 14kDa, present in serum from diseased cows, proportionally to the serum SAA concentration as measured by ELISA.

Isoelectric focusing of the purified bovine SAA fraction revealed three major (pI 5.5, 6.0, 6.4) and three minor (pi 4.8, 5.0, 7.3) isoforms and two‐dimensional SDS‐urea‐PAGE confirmed the identity of the major isoforms. Isoelectric focusing of SAA isolated from sera, obtained from cows affected with different diseases, showed a variable ratio of the isoforms. In SAA isolated from serum obtained from acow suffering from spontaneous AA‐amyloidosis only one isoform (pI 4.8) was detectable.

It is concluded that the results give first evidence for the existence of multiple isoforms of bovine SAA, occurring in different plasma concentration ratios during different diseases.
Origin and Immunoregulation of Autoantibodies Against Intestinal Alkaline Phosphatase
Authors: KOLBUS N. , BEUCHE W. , SCHLAF G. , NÜSSLEIN H. G. , FELGENHAUER K. , MÁDER M.
Published: July 2006

In the sera of patients with acute bactetial infections specific autoantibodies (sIAPa) of the imtnuno‐globulin class G (IgG) were found which bind to intestinal alkahne phosphatase (IAP) through the Fab portion. This was demonstrated using immunoaffinity (IA) isolation of sIAPa from patients' sera (particularly bacterial meningitis and ventriculitis) digestion with pepsin, purification of F(ab')2 fragments on protein A and subsequently binding on IAP coupled to CNBr (cyanogen bromide)‐activated Sepharose. Immunoblots using specific anti‐Fc and anti‐Fab antibodies showed that the bulk of F(ab')2 fragments had bound. Additionally, binding of native IAP to the F(ab')2 fragments was observed after separation of F(ab')2 fragments using isoelectric focusing (IEF), blotting onto nitro cellulose and incubation with IAP. Moreover, we have demonstrated the occurrence of natural anti‐IAP autoantibodies (nIAPa) which were isolated from sera of healthy individuals using IA chromatography. Investigation of isotype distribution revealed that IgG but not IgM or IgA were predominant even among nIAPa. The nIAPa fraction exhibited lower binding efficiencies on IEF blots than the sIAPa fraction, however, in contrast to sIAPa, cross‐reactions with other autoantigens were observed for nIAPa. NIAPa and sIAPa did not show subclass restriction. As revealed by IEF the spectrotypes of sIAPa were found to be patient‐specific, poly‐ to oligoclonal and stable during longer periods.
Placental Thrombosis and Fetal Loss After Passive Transfer of Mouse Lupus Monoclonal or Human Polyclonal Anti‐Cardiolipin Antibodies in Pregnant Naive BALB/c Mice
Authors: PIONA A. , LA ROSA L. , TINCANI A. , FADEN D. , MAGRO G. , GRASSO S. , NICOLETTI F. , BALESTRIERI G. , MERONI P. L.
Published: July 2006

In the present study we evaluated the effect of passive transfer of a mouse monoclonal (CAM) or a human polyclonal anti‐cardiolipin IgG on pregnancy outcome in BALB/c mice. The mice were immunized through the tail vein immediately after mating with 10 μg of monoclonal or polyclonal anti‐cardiolipin antibodies. Two other groups of mice were given a mouse irrelevant monoclonal antibody or normal human polyclonal IgG respectively, at the same dose. In mice immunized with monoclonal or polyclonal anti‐cardiolipin antibody we observed a significant increase in the number of fetal resorptions and a significant reduction of the mean weights of the embryos and the placentas. In mice immunized with CAM we also found a significant decrease in the number of healthy pups, while mice infused with human aCL antibody expressed a significant reduction in the fecundity rate. The histological examination showed widespread thrombosis and necrosis in the placentas derived from the mice immunized with the anti‐cardiolipin antibodies. The model supports a possible direct pathogenetic effect of anti‐phospholipid antibodies in recurrent fetal loss and points out that thrombotic events at placental level can be instrumental in the pathogenesis of the obstetric complications.
Characterization of the Gene Encoding the MPB51, One of the Major Secreted Protein Antigens of Mycobacterium bovis BCG, and Identification of the Secreted Protein Closely Related to the Fibronectin Binding 85 Complex
Authors: OHARA N. , KITAURA H. , HOTOKEZAKA H. , NISHIYAMA T. , WADA N. , MATSUMOTO S. , MATSUO T. , NAITO M. , YAMADA T.
Published: July 2006

The secreted protein MPB51 is one of the major proteins in the culture filtrate of Mycobacterium bovis BCG (BCG) and is a protein immunologically cross‐reacting with the fibronectin binding 85 complex secreted by this bacterium. The gene encoding MPB51 (mpb51) was cloned, sequenced, and expressed in Escherichia coll. The mpb51 gene was mapped downstream of the gene for 85A component with 179 bp spaces. The mpb51 gene encoded 299 amino acids, including 33 amino acids for the signal peptide, followed by 266 amino acids for the mature protein with a molecular mass of 27807.37 Da. This is the first complete sequence of MPB51. MPB51 showed 37–43% homology to the components of 85 complex. Two‐dimensional electrophoresis of culture fluids of BCG and Western blotting indicated the existence of the other novel protein(s) which strongly cross‐reacted with the α antigen (85B) and MPB51.
Inhibition of Complement‐Mediated Red Cell Lysis by Immunoglobulins is Dependent on the IG Isotype and its Cl Binding Properties
Authors: MOLLNES T. E. , HOGÅSEN K. , HOAAS B. F. , MICHAELSEN T. E. , GARRED P. , HARBOE M.
Published: July 2006

We have investigated the effect on complement activation of human irnmunoglohulins (Ig) using several therapeutic Ig preparations including two for intravenous use (IVIG), and various purified myeloma proteins. Ig inhibited lysis in a dose‐dependent manner in the classical pathway assay whereas no alternative pathway inhibition was observed. The Fc part of the molecule was responsible for all the inhibitory effect. Purified IgG3 myeloma proteins were potent inhibitors whereas IgGl inhibited to a lesser extent and lgG2 and IgG4 did not inhibit at all. Inhibition was obtained both when Ig was added to the solution and when it was coated onto a solid matrix. Analysis of the soluble and solid phase Ig after incubation revealed binding of Clq and activated C4 and C3 to the isotypes which inhibited lysis. Using selectively depleted sera and reconstitution with their respective purified components, efficient inhibition of lysis was seen when Ig was added prior to serum (C1), some inhibition was seen at the C4 level, whereas no effect was seen when Ig was added at the C9 level. We conclude that the complementmodulatory effect of Fg in vitro is isotype specific and dependent mainly on competitive C1 binding by the Ig molecule in the absence of antigen.
Interleukin‐10 Directly Inhibits the Interleukin‐6 Production in T‐Cells
Authors: HEMPEL L. , KÖRHOLZ D. , BÖNIG H. , SCHNEIDER M. , KLEIN‐VEHNE A. , PACKEISEN J. , MAUZ‐KÖRHOLZ C. , BURDACH S.
Published: July 2006

IL‐6 is a potent regulator of T‐cell activation, proliferation and differentiation. Since IL‐10 inhibits cytokine production by T cells, the effect of IL‐10 on IL‐6 production by T cells was investigated. IL‐6 production by purified monocytes or T cells was detected from cell‐free culture supernatants by ELISA after stimulation of the cells with LPS or an anti‐CD3 monoclonal antibody for 3 days. Although the main source of IL‐6 are LPS activated monocytes (29.6 × lOng/ml), T cells secreted sufficiently high levels of IL‐6 (790 × 200pg/ml) to stimulate the high affinity IL‐6 receptor. IL‐10 decreased anti‐CD3 induced IL‐6 mRNA expression by up to 80%. In addition, IL‐10 significantly inhibited IL‐6 release from T‐cells. Highly purified, anti‐CD3 activated T‐cells secreted 600 × 150pg/ml IL‐6 compared to 21 × 2pg/ml IL‐6 following addition of IL‐10 (10ng/ml; P <0.001). FACS analysis revealed a monocyte contamination of the T‐cell preparations of less than 0.5%. In addition, no IL‐1 production was detectable. Thus, in our experiments the effect of TL‐10 on IL‐6 production was independent of the presence of monocytes. Finally, inhibition of IL‐6 production was not reversed by IL‐2 (100U/ml). In conclusion, IL‐10 suppressed the synthesis of IL‐6 by T‐cells via a monocyte‐and IL‐2‐independeni mechanism. These results may help to understand the complex regulation of T‐cell mediated cytokine production by IL‐10.
The Identification of Germinal Centres and Follicular Dendritic Cell Networks in Rheumatoid Synovial Tissue
Authors: RANDEN I. , MELLBYE O. J. , FØRRE Ø. , NATVIG J. B.
Published: July 2006

We document here the identification of germinal centres with dark and light zones, a follicular dendritic cell network and clonal expansion in the synovium of rheumatoid arthritis patients. Synovial tissue from 24 patients suffering from rheumatoid arthritis or the polyarticular form of juvenile rheumatoid arthritis were screened for the presence of lymphoid follicles. The synovial tissues of 14 patients contained follicles and four of these had germinal centres and a follicular dendritic cell network. There was a statistically significant association between follicles in the synovium and the presence of rheumatoid factor autoantibodies in the patients' serum indicating a link between local germinal centre formation and the presence of pathological rheumatoid factors. Nucleotide sequencing of monoclonal rheumatoid factors from one of the patients' synovial tissue which contained germinal centres clearly supports the possibility that these rheumatoid factors have gone through a germinal centre reaction. While rheumatoid factors from healthy immunized donors are regulated through a tolerization mechanism which selects against replacement mutations and does not allow affinity maturation, synovial rheumatoid factors seem to lack this tolerization mechanism. The formation of germinal centres where B cells affinity mature and expand at the central site of disease in rheumatoid arthritis may explain why rheumatoid factors in rheumatoid arthritis develop into auto‐aggressive antibodies.
Vβ‐Specific Activation of T Cells by the HIV Glycoprotein gp 160
Authors: AKOLKAR P. N. , CHIRMULE N. , GULWANI‐AKOLKAR B. , PAHWA S. , KALYANARAMAN V. S. , PERGOLIZZ R. , MACPHAIL S. , SILVER J.
Published: July 2006

Studies by several groups have suggested that HIV infection in vivo results in a Vβ‐specific alteration of the TCR repertoire and that this might play a role in the pathogenesis of AIDS. However, there is very little agreement as to which Vβ segments are affected. In order to circumvent the confounding factors present in vivo we have examined the abilities of both a crude protein extract of HIV and purified gp 160 to alter the Vβ repertoire of normal T cells in vitro. We find that both a crude extract of HIV as well as gp 160 specifically activate T cells expressing a common set of Vβ segments (Vβ3, 12, 14, 15, and sometimes Vβ17 and 20) in individuals of disparate HLA type. This set of Vβ segments is remarkably similar to those recognized by staphlococcal enterotoxin B and supports the hypothesis that bacterial superantigens produced by opportunistically acquired micro‐organisms could have an exacerbating effect in AIDS.
T Lymphocytes Bearing the γδ T Cell Receptor are Susceptible to Steroid‐Induced Programmed Cell Death
Authors: SPINOZZI F. , AGEA E. , BISTONI O. , TRAVETTI A. , MIGLIORATI G. , MORACA R. , NICOLETTI I. , RICCARDI C. , PAOLETTI F. P. , VACCARO R. , BERTOTTO A.
Published: July 2006

The mechanisms by which glucocorticoids suppress immune responses have not yet been clearly defined. In steroid‐sensitive pathological conditions, an increase in γδ T cells can occur in certain untreated systemic autoimmune disorders and seems to be a persistent feature in most cases of systemic lupus erythematosus (SLE). Our previously published data demonstrated that immunosuppressive therapy normalized this expanded SLE T cell subset in parallel with clinical remission of the symptoms. To establish how corticosteroid treatment determines the disappearance of peripheral blood γδ T lymphocytes, circulating αβ and γδ T lymphocytes from seven SLE subjects with active disease and seven healthy individuals were cultured in the presence or absence of 10‐7M Dexamethasone (DEX). Cell suspensions were then analysed for DNA fragmentation, characteristic of apoptotic cell death, by a new cytofluorimetric method. Conventional agarose‐gel electrophoresis on the same T cell populations was carried out for comparison. Regular follow‐ups for 6 months revealed in vivo steroid treatment determined a dramatic fall in SLE blood γδ T cells, and in vitro experiments seem to indicate that DEX‐triggered apoptotic signals are confined to the double negative (CD4‐ CD8‐) γδ T cell subpopulation which disappears after in vivo immunosuppressive therapy. Clinical and pathological remission of some autoimmune diseases is often obtained by corticosteroids. Our results offer new insights on the mechanisms through these hormones exert their potent inhibitory activities on immune system cells postulated to play a role in the generation of autoimmune responses.
Cytokine Production and Serum Proteins in Depression
Authors: SEIDEL A. , AROLT V. , HUNSTIGER M. , RINK L. , BEHNISCH A. , KIRCHNER H.
Published: July 2006

One, as yet unemployed, approach to investigating immunology in depression is the assessment of the cytokine production by leucocytes, which would allow the determination of immune response under standardized conditions. Thus we measured the production of mitogen‐induced cytokines (IL‐1β, IL‐2, IL‐6, IL‐10, interferon‐7) and sIL‐2R in a whole blood assay, and serum protein levels such as C‐reactive protein (CRP), haptoglobin (Hp) and α2‐macroglobulin (α2 M) in a longitudinal 6‐week study in an attempt to assess leucocyte function during and after acute clinical stage of depression in 39 patients. Shortly after admission to hospital we found higher levels of all measured cytokines in the patients. Serum protein levels were significantly higher in the patients than in controls, and decreased over the study period. Whereas slightly elevated monokine levels in patients tended to reach control values, lymphokines showed a significant decrease over the 6 weeks as compared to baseline. These results suggest that the increase in immune activity seen at the beginning of the study may be followed by a suppressed cell‐mediated immune function.
Expression of the Human Mucosal Lymphocyte Antigen, HML‐1, by T Cells Activated with Mitogen or Specific Antigen In Vitro
Authors: BREW R. , WEST D. C. , BURTHEM J. , CHRISTMAS S. E.
Published: July 2006

Expression of the human mucosal lymphocyte antigen, HML‐1 (CD103), recently identified as a novel äEβ7 integrin, was studied on peripheral blood lymphocytes activated with mitogen or specific antigen. HML‐1 was up‐regulated on PHA activated T‐lymphoblasts cultured in 100IU/ml interleukin‐2 (IL‐2), reaching a peak of >50% positive cells at day 7, and expression was maintained at this level throughout the 28‐day culture period. Following a transient decrease in the percentage of L‐selectin cells, expression of this molecule was maintained on most PHA T‐lymphoblasts. Cells activated by purified protein derivative of M. tuberculosis (PPD) or in mixed lymphocyte culture also up‐regulated and maintained HML‐1 expression for 14 days. In contrast, in all cases the percentage of CD25+ cells rose initially but subsequently declined over the same time periods. When freshly isolated cells from tonsil, spleen, mesenteric lymph node and lung were analysed, only lung contained significant numbers (39 × 6%) of HML‐1+ cells. In both freshly isolated and activated cell populations the great majority of HML‐1+ cells co‐expressed CD8 although some HML‐1+CD8− cells were also present. Production of TGF‐β1 peaked early during T‐lymphoblast and MLR cultures and was not related to induction of HML‐1 expression. Immunoprecipitation studies showed that the HML‐1 molecule expressed on 10‐day PHA T‐lymphoblasts was indistinguishable from that found on intestinal intraepithelial lymphocytes and that no α4β7 integrin was expressed by these cells. Although HML‐1 expression is essentially restricted to mucosal leucocytes in vivo, these experiments show that it is readily induced and maintained along with co‐expression of L‐selectin following CD8+ T‐lymphocyte activation in vitro.