bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Sclerosis: Levels of Interleukin‐10‐Secreting Blood Mononuclear Cells are Low in Untreated Patients but Augmented During Interferon‐β‐1b Treatment
Authors: Özenci  , Kouwenhoven  , Huang  , Xiao  , Kivisäkk  , Fredrikson  , Link
Published: March 2003

The cytokine interleukin (IL)‐10 has immune response down‐regulatory properties, which include suppression of the synthesis of pro‐inflammatory cytokines such as interferon (IFN)‐γ and of major histocompatibility complex (MHC) class II expression on monocytes. To further elucidate the involvement of IL‐10 in multiple sclerosis (MS), an enzyme‐linked immunospot assay was adopted to enumerate IL‐10‐secreting mononuclear cells (MNC) in peripheral blood. IFN‐γ secreting MNC were detected in parallel. Levels of IL‐10‐secreting cells were lower in patients with MS compared with other neurological diseases (OND) and healthy subjects. This difference was seen only in patients with untreated MS, and not in those undergoing treatment with IFN‐β‐1b. No differences were observed when subgrouping the patients with MS regarding clinical phase (exacerbation, remission, secondary progression), duration of MS or disability status. Levels of IFN‐γ‐secreting blood MNC did not differ in patients with MS, irrespective of treatment with IFN‐β‐1b, compared with OND and healthy subjects. Patients with MS, but not the two groups of controls, had elevated numbers of IL‐10‐ and IFN‐γ‐secreting cells upon stimulation with MBP compared with culture in the absence of antigen. The data suggest that IL‐10 is decreased in MS and that treatment resulting in its up‐regulation beneficially influence the disease.
A Human Whole‐Blood Assay for Analysis of T‐Cell Function by Quantification of Cytokine mRNA
Authors: HÄRTEL  , BEIN  , KIRCHNER  , KLÜTER
Published: March 2003

A whole blood assay was developed for T‐lymphocyte analysis which allows the quantification of induced cytokine mRNA expression. We applied a novel kinetic reverse transcription polymerase chain reaction method which directly measures product accumulation using Taqman technology. Quantitative results were obtained by using β‐actin and cytokine standard curves generated from synthetic external standards. Since quantification relies on threshold cycles for fluorescence detection (Ct), this technique proved to be accurate over a dynamic range of at least five orders of magnitude. To evaluate the method a study was undertaken to find optimal conditions for whole‐blood stimulation with soluble anti‐CD3 monoclonal antibodies in the presence of a costimulatory signal mediated by anti‐CD28 monoclonal antibody. Therefore, whole blood was taken from healthy individuals (n = 10) and aliquots for mRNA measurement were withdrawn after 0, 4, 8 and 24 h of stimulation. Optimal assay conditions were reached with 1 : 10 diluted heparinized whole blood and after stimulation with equimolar amounts of anti‐CD3 and anti‐CD28 monoclonal antibodies (1 μg/ml). Interferon‐γ and tumour necrosis factor‐α proved to be early response cytokines with peak expression at 4 h. In contrast, interleukin (IL)‐2, IL‐4 and IL‐10 required 8 h of stimulation. This novel whole‐blood assay is potentially useful for monitoring T‐cell‐specific immune functions in a variety of clinical settings. Using whole blood obviates the need for T‐cell purification and may therefore closely approximate the state of responsiveness of circulating T cells in vivo.
Cytotoxic T‐Lymphocyte Clones, Established by Stimulation with the HLA‐A2 Binding p5365–73 Wild Type Peptide Loaded on Dendritic Cells In Vitro, Specifically Recognize and Lyse HLA‐A2 Tumour Cells Overexpressing the p53 Protein
Authors: Barfoed  , Petersen  , Kirkin  , Thor Straten  , Claesson  , Zeuthen
Published: March 2003

Mutations in the tumour suppressor gene p53 are among the most frequent genetic alterations in human malignancies, often associated with an accumulation of the p53 protein in the cytoplasm. We have generated a number of cytotoxic T lymphocyte (CTL) clones that specifically recognize the HLA‐A*0201 p53 wild type peptide RMPEAAPPV [65–73], designated R9V, by the in vitro stimulation of CD8 enriched peripheral blood lymphocytes from a healthy HLA‐A*0201 donor using peptide loaded autologous dendritic cells. A total of 22 CTL clones were generated from the same bulk culture and demonstrated to carry identical T‐cell receptors. The CTL clone, 2D9, was shown to specifically lyse the HLA‐A*0201+ squamous carcinoma cell line SCC9 and the breast cancer cell line MDA‐MB‐468. Our data demonstrate that human peripheral blood lymphocytes from normal healthy individuals comprise T cells capable of recognizing p53 derived wild type (self) peptides. Furthermore, the capacity of R9V specific T cell clones to exert HLA restricted cytotoxicity, argues that the R9V peptide is naturally presented on certain cancer cells. This supports the view that p53 derived wild type peptides might serve as candidate target antigens for the immunotherapeutic treatment of cancer.
Neutralization of Disease Associated Autoantibodies by An Immunoglobulin M‐ and Immunoglobulin A‐Enriched Human Intravenous Immunoglobulin Preparation
Authors: Bar‐Dayan  , Bar‐Dayan  , Bonnin  , Bloch  , Schweitzer  , Ravid  , Kazatchkine  , Kaveri
Published: March 2003

Immunoglobulin preparations enriched with IgM and IgA are used in the therapy of severe bacterial infections and for the treatment of acute graft‐versus‐host disease, but not as yet, in the treatment of autoimmune diseases. We investigated the potential of an IgM‐ and IgA‐enriched immunoglobulin preparation to neutralize activity autoantibodies from patients with autoimmune diseases. We demonstrate that Pentaglobin® was at least as effective as intravenous immunoglobulin (Sandoglobulin®) in inhibiting autoantibody activity. Each of the immunoglobulin isotypes present in Pentaglobin® may be responsible for the inhibitory effect. Pentaglobin® immobilized on an affinity matrix retained the disease associated autoantibodies and interacted with F(ab′)2 fragments of IgG autoantibodies. Suppression of autoantibody activity is dependent, at least in part, on idiotypic interactions. The present findings provide a rationale for considering these preparations for the immunomodulation of autoimmune disease.
Chemokine Expression during Acute Rejection of Rat Kidneys
Authors: Grau  , Gemsa  , Steiniger  , Garn
Published: March 2003

During acute rejection of fully allogeneic rat renal allografts, few neutrophil granulocytes are detected, whereas an abundant infiltrate of macrophages and T lymphocytes becomes apparent. The mechanisms leading to this specific pattern of infiltration are not understood. We performed a sequential daily Northern blot analysis of the mRNA expression of the CC‐chemokines MCP‐1, MIP‐1α and RANTES and of the CXC‐chemokines GRO/KC and MIP‐2 in rat renal isografts (LEW → LEW, n = 1 per day) and allografts during acute rejection (DA → LEW, n = 3 per day). MCP‐1 gene expression strongly increased on days 3–4 after allotransplantation and returned to control levels on day 6. The expression of MIP‐1α and RANTES continuously rose until day 3–4 and remained stable thereafter. Isografts displayed minor changes in CC‐chemokine expression. In contrast to CC‐chemokines, GRO/KC was expressed in low amounts during rejection and MIP‐2 mRNA remained undetectable. In conclusion, the expression of the CC‐chemokines MCP‐1, MIP‐1 and RANTES was clearly upregulated during rejection, whereas the mRNA of the CXC‐chemokines MIP‐2 and GRO/KC was not detected at all or remained at low levels. This pattern of chemokine gene expression is in good accordance with the predominant mononuclear leukocyte infiltrate in allografts.
Evidence that Human Immunoglobulin M Rheumatoid Factors Can Be Derived from the Natural Autoantibody Pool and Undergo an Antigen Driven Immune Response in which Somatically Mutated Rheumatoid Factors Have Lower Affinities for Immunoglobulin G Fc than Their Germline Counterparts
Authors: Carayannopoulos  , Potter  , Li  , Natvig  , Capra
Published: March 2003

The question of whether immunoglobulin (Ig)M rheumatoid factors (RF) arise as the result of an abnormal expansion of already existing clones producing natural autoantibodies or emerge as new clones that are somatically mutated owing to an antigen driven immune response has never been conclusively answered. In this study, an inhibition ELISA was utilized to measure the affinities of recombinant antibodies using VH segments reverted back to their closest germline counterparts (germline revertants). In all cases, the somatically mutated parental RFs had a decreased affinity for immunoglobulin (Ig)G Fc compared to the germline revertant, indicating that the antibodies in the germline configuration had the higher affinities. This demonstrates that somatic mutation is not a prerequisite to generate disease associated antibodies. The presence of mutations in the parental IgM RFS suggests that these cells had been involved in a germinal centre reaction. As the germinal centre is the conventional site of the acquisition of mutations during an antigen driven response, these data suggest a role for germinal centres in the generation of the antibody diversity in addition to the selection of higher affinity antibodies. Assuming that only antigen selected cells survive deletion, these data support the hypothesis that IgM RFS can be derived from the natural autoantibody repertoire and result from an antigen driven response. Mechanisms controlling the survival of B cells based on the affinity/avidity of the immunoglobulin receptor are shown to be functional in patients with rheumatoid arthritis.
Fine Specificity of Anti‐Fibrillin‐1 Autoantibodies in Primary Pulmonary Hypertension Syndrome
Authors: Morse  , Antohi  , Kasturi  , Saito  , Fotino  , Humbert  , Simonneau  , Basst  , Bona
Published: March 2003

Autoantibodies to fibrillin‐1 (Fbn‐1) have been found in systemic sclerosis (SSc), calcinosis, Raynaud's esophagael dysmotility, sclerodectyly, and telaengectasia (CREST) and mixed connective tissue disease (MCTD) diseases. The purpose of this study was to determine whether patients with primary pulmonary hypertension (PPH) and appetite‐suppressant‐associated PPH have anti‐Fbn‐1 autoantibodies. In addition we assessed the human leucocyte antigen (HLA) class II alleles (DRB1, 3, 4, 5 and DQB1) in these patients in order to determine whether the response is genetically restricted. The frequency of anti‐Fbn‐1 autoantibodies in patient groups was compared with that of a control group of 88 healthy patients, and HLA was correlated similarly with a group of 51 healthy subjects. Anti‐Fbn‐1 autoantibodies were found at high frequency in PPH: in 70 of 75 adults with PPH (93%), in 28 of 33 children with PPH (84.8) and in 12 of 18 (67%) patients with appetite‐suppressant‐associated PPH. Utilization of two Fbn‐1 fusion proteins allowed us to determine the dominant determinant region, recognized by anti‐Fbn‐1 autoantibodies, which may be located on the N‐terminal fragment of the Fbn‐1 protein. No significant immunogenetic correlations were found when the PPH patient groups were compared with normal controls. This novel category of autoantibodies is found in diseases characterized by endothelial and extracellular matrix protein alterations and fibrosis.
Intestinal Intraepithelial Lymphocytes Contain a CD3− CD7+ Subset Expressing Natural Killer Markers and a Singular Pattern of Adhesion Molecules
Authors: Eiras  , Leon  , Camarero  , Lombardia  , Roldan  , Bootello  , Roy G.
Published: March 2003

Intestinal intraepithelial lymphocytes (i‐IEL) represent one of the largest, non‐organized lymphoid population in the body. They are located outside the epithelial basement membrane among the mucosal epithelial cells. We, and previously other groups, have reported the presence of a CD7+CD3−IEL subset in the epithelium of human small intestine. This subset is drastically reduced in coeliac disease (CD) patients. In the present work we accomplish a better phenotypic characterization of this CD3−IEL subset and demonstrate the expression of typical natural killer (NK) cell markers. Most, if not all, CD3−CD7+ cells express NKPR1 (CD161)[98% ± 2] and CD122[92% ± 6]. In addition, a variable percentage express CD2[55% ± 16], CD94[24% ± 18], CD56[44% ± 21] and CD16[12% ± 4], however, no CD57 expression was observed. Moreover, these cells contain perforin granules[75% ± 5], supporting a potential cytolytic ability. Regarding adhesion molecules, CD18 and CD44 expression is absent, which is consistent with a limited capacity of migration. Altogether, these data suggest the presence of intraepithelial NK cells in human intestinal epithelium, a compartment where cytotoxic effectors have not been clearly defined.
Hypersusceptibility of A/J Mice to Tuberculosis is in Part Due to a Deficiency of the Fifth Complement Component (C5)
Authors: Jagannath C. , Hoffmann H. , Sepulveda E. , Actor J. K. , Wetsel R. A. , Hunter R. L.
Published: March 2003

Mycobacterium tuberculosis (MTB) causes tuberculosis in man, which occurs as an acute, chronic or dormant disease reactivating over several years. The mechanisms of persistence and reactivation are not well understood and there is a need for animal models. Moderate‐dose, aerosol infection killed A/J mice earlier than partially resistant C57Bl/6 mice, whereas a low‐dose, aerosol‐induced chronic infection exacerbated earlier in A/J mice. A/J mice lethally infected with MTB but drug cured of disease underwent reactivation of tuberculosis at least 100 days before similarly infected C57Bl/6 mice. Because A/J mice were C5 deficient, congenic B10 mice sufficient and deficient for C5 were infected intravenously with MTB to define the role of C5. C5‐deficient mice again showed enhanced growth of MTB in the lungs. MTB‐infected macrophages from C5‐deficient mice showed enhanced growth of MTB coinciding with a reduced secretion of both cytokines (TNF‐α, IL‐1β, IL‐6, IL‐12) and chemokines (KC, MIP‐2 and MIP‐1α) in A/J and TNF‐α and chemokines in C5‐deficient mice. Because C5‐deficient macrophages could be activated from extraneous C5 and TNF‐α we suggest that both play a role in the macrophage‐mediated killing as well as containment mechanisms in tuberculosis.
Detection of a Soluble Form of the Complement Membrane Attack Complex Inhibitor CD59 in Plasma after Acute Myocardial Infarction
Authors: Väkevä A. , Lehto T. , Takala A. , Meri S.
Published: March 2003

Activation of the complement system has been documented in both experimental and clinical studies of acute myocardial infarction (AMI). Our earlier immunohistochemical studies have shown that the deposition of the membrane attack complex (MAC) of complement is associated with the loss of protectin (CD59), a glycosyl‐phosphatidylinositol (GPI)‐anchored sarcolemmal regulator of MAC, from the human and rat infarcted myocardium. In this study we detected, using an enzyme immunoassay (EIA), CD59 in the plasma of AMI patients at a concentration of 23.0 ± 8.4 ng/ml (mean ± SD; n = 17) at 4 h and 27.3 ± 11.8 ng/ml (n = 24) at 24 h after AMI. Both values were significantly higher than in healthy controls (7.8 ± 6.4 ng/ml; n = 20; P < 0.001). The amount of CD59 correlated with the level of soluble terminal complement complexes (SC5b‐9; r = 0.84; P < 0.01) in the plasmas of AMI patients. Our results suggest that myocardial damage leads to release of CD59 from the sarcolemmal cell membranes during AMI.
The Vδ1 T Cell Receptor Repertoire in Human Nasal Mucosa
Authors: Hirata N. , Takeuchi K. , Majima Y. , Sakakura Y.
Published: March 2003

The γ/δ T cells play important roles as the first line of defence in mucosal immunity. Vδ1 bearing T cells comprise the major population of γ/δ T cells in the human nasal mucosa. Recently, it was reported that certain receptors are predominant at given anatomical locations. The purpose of this study was to elucidate the γ/δ T‐cell receptor repertoire in human nasal mucosa. Nasal epithelia were scraped and peripheral blood were obtained from three healthy volunteers. We performed reverse‐transcription polymerase chain reaction (RT‐PCR) analysis and characterized the junctional sequences of Vδ1 T‐cell receptor (TCR) transcripts. Vγ and Vδ gene usage in intraepithelial lymphocytes (IELs) in right and left nasal mucosa were almost the same, but they were different from those in peripheral blood lymphocytes (PBLs). The VDJ sequence of nasal Vδ1 T‐cell receptor transcripts showed diversity and were different for each individual. Vδ1 in the IELs were rearranged to Jδ1 and Vδ1 in the PBLs were rearranged not only to Jδ1 but also to Jδ3. Vδ1 sequences in the IELs in the nasal mucosa showed no overlap with those in the PBLs. These results indicate that γ/δ T cells in the nose are organ specific lymphocytes.
Regulation of the IL‐10 Production by Human T Cells
Authors: Rafiq K. , Charitidou L. , Bullens D. M. A. , Kasran A. , Lorré K. , Ceuppens J. L. , Van Gool S. W.
Published: March 2003

Interleukin (IL)‐10, an immunomodulatory cytokine predominantly produced by monocytes/macrophages and T cells, inhibits several functions of dendritic cells (DC), monocytes and T cells including their cytokine production, but it stimulates B cell immunoglobulin (Ig) production and cytotoxic T lymphocyte (CTL) generation. A precise knowledge of the mechanisms that control the IL‐10 production is therefore highly important for understanding the immunoregulation. The IL‐10 production was studied in cultures of freshly isolated human T cells. A rise in intracellular calcium as well as the common γ‐chain containing cytokine receptor triggering or CD28 triggering were found to be important signals for IL‐10 induction. CD80, CD58, rIL‐12 and rIFN‐α all had efficacious and independent costimulatory activities on the IL‐10 production, while PGE2 was inhibitory. Dependence on autocrine IL‐2 signalling was shown by the effects of anti‐IL‐2 and anti‐IL‐2R monoclonal antibodies (MoAb), but the IL‐10 production proceeded partly IL‐2‐independent when CD80 provided costimulation. Sensitivity to inhibition by CsA was not removed by CD80 or CD58 costimulation and/or by addition of rIL‐12 or rIFN‐α, pointing to the absolute requirement for calcineurin activity. These data reveal important differences in the regulatory pathways between IL‐10 (a cytokine‐inhibitory interleukin) and IL‐2 (a cytokine‐inducing interleukin), which can potentially be exploited therapeutically. The fact that CsA blocks the production of IL‐10, which itself has important immunosuppressive properties, should be taken into account in defining immunosuppressive treatment schedules which include the use of CsA.
Autoantibody to the Leucine Zipper Region of 52 kDa Ro/SSA Binds Native 60 kDa Ro/SSA: Identification of a Tertiary Epitope with Components from 60 kDa Ro/SSA and 52 kDa Ro/SSA
Authors: Kurien B. T. , Chambers T. L. , Thomas P. Y. , Frank M. B. , Scofield R. H.
Published: March 2003

Anti‐Ro (or SSA) is found in the sera of patients with autoimmune rheumatic illnesses. All patients with anti‐Ro defined by precipitation bind a 60 000 Da antigen (60 kDa Ro), whereas some patients also bind a 52 000 Da molecule (52 kDa Ro). In general, antibody binding is directed against native 60 kDa Ro and denatured 52 kDa Ro. The mechanism by which anti‐52 kDa Ro arises in the setting of anti‐60 kDa Ro is unknown. Conflicting data exist as to the existence of a physical interaction between the two proteins in cells and as to cross‐reacting antibodies. Antibodies were affinity purified from a peptide within the leucine zipper region of 52 kDa Ro. These purified antibodies binding the 197–207 peptide from 52 kDa Ro (anti‐52LZ) bound native 60 kDa Ro as well as denatured 52 kDa Ro. In addition, anti‐52LZ also bound up to four regions from the sequence of 60 kDa Ro and a single conformational epitope of 60 kDa Ro. Thus, these primary sites represent components of the tertiary epitope. We hypothesized that if this was the case, these peptides making up a tertiary epitope would show molecular interaction. In fact, peptides from 60 kDa Ro have a molecular interaction with the 52 kDa Ro peptide as well as full‐length 52 kDa Ro when assessed by surface plasmon resonance. The leucine‐zipper region peptide from 52 kDa Ro bound three of the four peptides from 60 kDa Ro. These data suggest that these two molecular species, 60 and 52 kDa Ro, form a conformational epitope. This relationship may explain why anti‐52 kDa Ro is found in association with anti‐60 kDa Ro.
Administration of BiMAb‐Retargeted T Cells in a Rat Hepatic Metastases Colon Tumour Model Results in T‐Cell Tumour Infiltration Independent of the Route of Administration
Authors: Koelemij R. , Hagenaars M. , Ensink N. G. , Eggermont A. M. M. , Van De Velde C. J. H. , Fleuren G. J. , Kuppen P. J. K.
Published: March 2003

In this study, cultured T cells, pre‐incubated with the bispecific monoclonal antibody (BiMAb) R73IgG1 × CC52IgG1 were adoptively transferred, via systemic and regional routes, to rats bearing day 10 hepatic metastases of the CC531 adenocarcinoma of the colon to investigate the role of the route of administration in tumour infiltration by these BiMAb‐retargeted effector cells. The BiMAb, directed against the T‐cell receptor and the tumour‐associated antigen CC52, were used to crosslink CC531 tumour cells and T cells to induce tumour cell lysis. Retargeted T cells were administered via the jugular vein, hepatic artery or the portal vein. The number of BiMAb‐retargeted T cells that reached the liver tumours was independent of the route of administration. There was also no difference between the number of T cells that reached the portal tracts, central veins of parenchyma of the liver, after loco‐regional or systemic administration. These findings are in contrast to the interleukin (IL)‐2 activated NK (A‐NK) cells biodistribution studies earlier performed in the same animal model in our laboratory. Compared with A‐NK cells, a lower number of BiMAb‐retargeted T cells reached the tumours, irrespective of their route of administration while for A‐NK cells, there was an advantage of administration via the hepatic artery.
Connectivity Patterns in Tuberculosis and Leprosy Patients are Indistinguishable from that of Healthy Donors
Authors: Tovar‐Rivera T. , Sánchez‐Colón S. , Padierna‐Olivos L. , Massó‐Rojas F. , Estrada‐Parra S. , Mondragón‐González R. , Jiménez‐Martínez M. C. , Sánchez‐García F. J.
Published: March 2003

Connectivity, the self‐defined interactions between antigen‐recognising molecules in a network system can in part be assessed by measuring the reactivity of a given serum against an ordered set of immunoglobulin (Ig)G F(ab′)2 fractions, separated by means of isoelectric focusing so that, the serum reactivity against the whole set of fractions defines a characteristic pattern of connectivity. Deviations from the normal condition (healthy donors) have so far been documented for two autoimmune diseases: systemic lupus erythematosus (SLE) and pemphigus vulgaris, as well as for human immundeficiency virus (HIV)‐1 infection. We tested here if bacterial infections lead to alterations in connectivity. In addition, we wanted to test if two antigenically related bacteria would produce similar or otherwise distinctive connectivity patterns. Connectivity analysis was applied on the sera from tuberculosis and leprosy patients and the sera from healthy donors were used as control.

No statistically significant differences between the three groups studied were found. These results have implications for theories that set the origin of autoimmune diseases in microbial infections. To the best of our knowledge, this is the first attempt to analyze the connectivity status in bacterial infections.
Structural Analysis of TCRα and β Chains from Human T‐Cell Clones Specific for Small Nuclear Ribonucleoprotein Polypeptides Sm‐D, Sm‐B and U1–70 kDa: TCR Complementarity Determining Region 3 Usage Appears Highly Conserved
Authors: Talken B. L. , Bailey C. W. , Reardon S. L. , Caldwell C. W. , Hoffman R. W.
Published: March 2003

Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are systemic autoimmune diseases that are characterized by the presence of autoantibodies reactive with U small nuclear RNP (snRNP) autoantigens. Both B and T cells are important in the pathogenesis of the disease, and T‐ and B‐cell immunity against snRNP polypeptides have been shown to be linked in vivo. Currently, several alternative hypotheses for the pathogenesis of these diseases have been proposed. These include loss of tolerance, modified self‐antigens, molecular mimicry and nondirected immune activation. To help distinguish between the various models of disease pathogenesis, we have characterized the T‐cell receptor (TCR) CDR3 from a large panel of well‐characterized human T‐cell clones and lines specific for individual snRNP polypeptides. The results presented here reveal highly restricted TCR usage across patients by the snRNP‐reactive T cells based on the deduced amino acid sequence of the CDR3 loop. These data support the hypothesis that T‐cell responses against self antigens in SLE and MCTD are antigen driven and that there are a limited number of T‐cell epitopes present on the snRNP autoantigens.
Analysis of HCV‐Ig Isotype Complexes by a Novel Immuno‐Capture RT–PCR Method
Authors: Peng X. X. , Zhu H. , Zhang J. Y. , Wang S. Y.
Published: March 2003

HCV can be present in the circulating blood either as a free virus or as a virion‐immunoglobulin (Ig) complex. All isotypes of Igs may form the virus complexes, but it remains unclear what specific role of each Ig plays in the clearance of HCV. In the present study, we have combined immuno‐capture and RT–PCR, and developed a quick double‐specificity method for detecting and distinguishing different HCV‐Ig complexes. We compared our new method, the immuno‐capture RT–PCR (iRT–PCR), with the conventional RT–PCR (cRT–PCR) for the sensitivity of detecting HCV in 35 clinically diagnosed patients with HCV infection. The results showed that 31 patients were detected to be positive by using iRT–PCR, whereas 16 patients were positive with the use of cRT–PCR. HCV‐IgM, HCV‐IgG, HCV‐IgA could separately be detected by iRT–PCR and their positive rates were 66.7%, 51.0%, 62.7%, respectively. HCV bound to antibody was a common feature of hepatitis C (HC) and 86.3% of patients were positive at least by one of the HCV‐Ig tests. The patterns of HCV RNA constituents varied according to disease categories. In summary, iRT–PCR is a valuable method for analysis of the composition of the immune complexes, which may provide new and valuable insights into HCV pathogenesis.
Unresponsiveness of Specific T Cells to IL‐12 is Associated with Active Cutaneous Leishmaniasis Owing to Leishmania guyanensis
Authors: Bourreau E. , Prévot G. , Pradinaud R. , Launois P.
Published: March 2003

The production of interleukin (IL)‐13 and unresponsiveness to IL‐12 in T cells were analyzed in patients with active localized cutaneous leishmaniasis (untreated or not responsive to treatment) and in patients who had been treated successfully for the disease. More IL‐13 was produced by specific T cells in response to Leishmania guyanensis (L. guyanensis) antigens in active compared to in inactive leishmaniasis. Furthermore, unresponsiveness of specific T cells to IL‐12 was detected only in patients with active leishmaniasis, i.e. in patients with detectable parasites such as untreated patients and patients unresponsive to treatment. These results support that IL‐12 unresponsiveness of Leishmania‐specific T cells is responsible for the persistence of infection.
CD8+ T Cells Induce Medullary Thymic Epithelium and CD4+CD8+CD25+ TCRβ− Thymocytes in SCID Mice
Authors: Ekholm Pettersson F. , Schneider M. K. J. , Andersson J. , Grönvik K.‐O.
Published: March 2003

During T‐cell development the transition in the thymus of CD4−CD8− double negative (DN) progenitor T cells into CD4+CD8+ double positive (DP) cells is dependent on the expression of a T‐cell receptor (TCR)‐β‐chain protein. In this study purified peripheral CD4+ and CD8+ T lymphocytes from the C.B‐17 strain of mice were adoptively transferred into syngeneic, neonatal SCID mice, where donor cells resided at constant numbers in thymus from 2 weeks until 10 weeks post cell transfer. In the recipient thymus the CD8+ donor cells outnumbered the CD4+ cells by a factor of three to five and both subsets contained a large fraction of activated cells. During the late phase of treatment, CD8+ T cells induced high numbers of DP thymocytes in the SCID mice, a process accompanied by the maturation of medullary epithelial cells. Such thymic development in the SCID mouse was inhibited by coresiding CD4+ donor T cells. These results indicate a regulatory role by mature peripheral T cells on medullary epithelial growth and thymocyte development in the treated SCID mice.
Regulation of Surface and Intracellular Expression of CTLA‐4 on Human Peripheral T Cells
Authors: Wang X.‐B. , Zheng C.‐Y. , Giscombe R. , Lefvert A. K.
Published: March 2003

Cytotoxic T‐lymphocyte‐associated antigen (CTLA‐4) is an important downregulator of T‐cell activation. In order to analyze the expression and regulation of CTLA‐4 on human peripheral T cells, CTLA‐4 mRNA and protein expression were determined using analysis by reverse transcription–polymerase chain reaction (RT–PCR) and FACs, respectively. Intracellular CTLA‐4 was constitutively expressed in unstimulated CD4+ and CD8+ T cells. Interleukin (IL)‐2 induced a dose‐dependent increase of both intracellular and surface expression of CTLA‐4 (CD152). Most of the CD4+ and CD8+ cells expressing CTLA‐4 also expressed CD25. Interferon (IFN)‐γ induced the upregulation of CTLA‐4 expression via antigen‐presenting cells (APC) activation. The CTLA‐4delTM mRNA (550 bp) had a shorter half‐life than the full length CTLA‐4 mRNA and the expression was downregulated upon activation of the cells by treatment with IL‐2. Given an inhibitory role of CTLA‐4 and CD4+ CD25+ T cells in immune responses, the present findings suggest that IL‐2‐induced immunosuppression may result from its stimulatory effect of the CTLA‐4 expression.
Naive CD4+ T Cells Can be Sensitized with IL‐7
Authors: Vakkila J. , Äystö S. , Saarinen‐Pihkala U. M. , Sariola H.
Published: March 2003

We addressed the question whether it is possible to lower the threshold for naive T cells to respond to antigens. Purified adult and cord blood derived CD4+ CD45RA+ naive T cells were incubated in the presence of various cytokines for two days (‘primed T cells’), after which the cytokines were removed by extensive washing. Primed and unprimed cells were activated with solid phase‐coupled anti‐CD3 and soluble anti‐CD28 monoclonal antibodies (MoAb). Naive T cells, primed with interleukin(IL)‐7 proliferated more vigorously than unprimed cells. Primed cells required 6 h for antigenic stimulation, whereas unprimed cells required 20 h. The priming also shifted the threshold of naive T cells in order to stimulate the antigen concentration to a lower level. The addition of IL‐10 almost completely abrogated the enhancing effect of IL‐7 on naive T cells. Other cytokines (IL‐1, IL‐2, IL‐6, IL‐12, interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α had less effect on the cell proliferation. However, priming of naive T cells with IL‐7 had no impact on the proliferation to allogeneic immature or mature dendritic cells (DC). We conclude that the antigen‐independent activation of naive T cells with IL‐7 prior to antigen stimulation sensitizes cells, and may be of help in trying to stimulate immune responses against weak antigens. This approach, however, does not enhance proliferative responses stimulated by DC.
Stronger Proliferative Response to Membrane Versus Cell‐Wall Streptococcal Proteins by Peripheral Blood T Cells in Chronic Plaque Psoriasis
Authors: Baker B. S. , Brown D. , Fischetti V. A. , Ovigne J.‐M. , Porter W. , Powles A. , Fry L.
Published: March 2003

Proliferative responses of peripheral blood mononuclear cells (PBMC) to group A streptococcal (GAS) antigens have been studied in 24 patients with psoriasis and 15 disease controls. Extracts of cell wall (including M protein) from types M4 and M12 GAS, recombinant M6 protein, and both cell‐wall and cell‐membrane extracts from type M6 (M6+) GAS and its corresponding M gene deletion mutant (M6‐) were tested. PBMC from psoriatic patients proliferated more strongly to cell‐wall extracts from M12 versus M4 (P = 0.0348), and to M6+ versus M6‐ (P = 0.0019) GAS with, in most cases, moderate proliferation to recombinant M6 protein. The psoriatic response to M12 cell wall was significantly increased compared to the controls (P = 0.0032). In psoriatics, M6+ membrane extracts induced a markedly greater proliferation than those of cell wall (P = 0.0002); responses to M6+ (P = 0.0039) and M6‐ (P = 0.0114) membrane extracts were higher than those of the control PBMC. Both groups showed a decreased response to the M6‐ versus M6+ membrane extracts (P = 0.0030; P= 0.0181, respectively). This study has demonstrated that patients with psoriasis have a heightened circulating T‐cell response to cell wall M protein and to non‐M proteins present on the cell wall and membrane of GAS.
Megakaryothrombopoiesis During Ex Vivo Expansion of Human Cord Blood CD34+ Cells Using Thrombopoietin
Authors: Woo S.‐Y. , Kie J.‐H. , Ryu K.‐H. , Moon H.‐S. , Chung W.‐S. , Hwang D.‐H. , Kim S.‐K. , Han T.‐H. , Hahn M.‐J. , Chong Y.‐H. , Park H.‐K. , Seoh J.‐Y.
Published: March 2003

Thrombopoietin (TPO) is one of the most promising stimulants for ex vivo expansion of haematopoietic stem cells. Previously, we have found that TPO induces a characteristic pattern of apoptosis during ex vivo expansion of human cord blood (CB) CD34+ cells and that the TPO‐induced apoptotic cells belong to megakaryocyte (MK) lineage. In this study, we have examined the maturation of MK and platelet production in association with the TPO‐induced apoptosis. CD34+ cells, purified from human CB, were expanded in serum‐free conditions stimulated with TPO. Apoptosis was confirmed by terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate‐biotin nick end labelling (TUNEL) assay and electron microscopy (EM). Simultaneous measurement of DNA content and immunophenotyping revealed that the cells with higher DNA content (>8 N) constituted less than 5% of the CD41+ fractions until day 14, implying premature apoptosis of MKs before full polyploidization. Nevertheless, EM observation showed not only platelet territories but also newly produced platelets in which granules and microfilaments could be identified. Furthermore, flow cytometry demonstrated that the platelet fraction expressed P‐selectin and an activation motif on GPIIb/IIIa recognized by monoclonal antibody PAC‐1 upon stimulation with adenosine diphosphate (ADP). In addition, periodic acid‐Schiff (PAS)‐positive materials and nonspecific esterase activities could be demonstrated. Therefore, it is suggested that platelet production and the accompanying processes, rather than apoptosis only, be hastened during the ex vivo expansion of CB CD34+ cells when using TPO.
A Novel Lacrimal Gland Autoantigen in the NOD Mouse Model of Sjögren's Syndrome
Authors: Esch T. R. , Poveromo J. D. , Aikins M. C. , Levanos V. A.
Published: March 2003

Recent research has demonstrated a crucial role for autoantibodies in the pathogenesis of Sjögren's syndrome (SS)‐like disease in the non‐obese diabetic (NOD) mouse, but it remains to be determined which antibody species among all those present are directly related to the various aspects of pathology. To identify autoantigens in the NOD mouse system, we have taken the approach of using immunoglobulin (Ig)G purified from sera of NOD mice exhibiting SS‐like symptoms to screen cDNA expression libraries derived from exocrine gland mRNA. Here we report the identification of a novel autoantigen, designated LGP10, expressed in lacrimal and submandibular glands. Autoantibodies to this protein are prominent in the sera of NOD mice starting at 11–12 weeks of age, but not in control nonautoimmune mice. LGP10 has no known function, but bears similarities to various other proteins produced by epithelia. Interestingly, all of these similar proteins have been linked to immunosuppression and/or steroid binding, both processes that could have a significant impact on pathological features of SS.
Decreased Urokinase Receptor Expression on Granulocytes in HIV‐Infected Patients
Authors: Storgaard M. , Obel N. , Black F. T. , Møller B. K.
Published: March 2003

Pericellular proteolysis initiated by receptor‐bound urokinase‐type plasminogen activator (uPA) is considered important for directed migration of granulocytes to inflammatory sites. Using flow cytometry and whole‐cell binding of radiolabelled‐uPA, we found a high level of uPA‐receptor (uPAR) expression in granulocytes (3.9 × 104 ± 0.9 × 104 sites/cell). Modulation of uPAR expression was assessed in the presence of chemoattractant gradients. Our findings demonstrate that interleukin (IL)‐8, leukotriene B4(LTB4) and formyl‐methionyl‐leucyl‐phenylalanine (f MLP) caused a dose‐dependent upregulation of uPAR on granulocytes in healthy controls. Modulation of uPAR expression is known to regulate chemotactic response. As determined by flow cytometry, uPAR expression by granulocytes from human immunodeficiency virus (HIV)‐infected patients was distinctly lower than that of healthy control cells (P < 0.001). However, upregulation of uPAR in response to chemoattractants was similar to that observed in healthy controls. In HIV‐infected patients, the uPAR expression on granulocytes correlated (P < 0.001, n = 10) with the number of CD4+ blood cells. In contrast, the expression of IL‐8 receptor, CD11b, CD18 and CD62 was not significantly altered in HIV‐patients compared with healthy controls.
CD80 Expression is Induced on Activated B Cells Following Stimulation by CD86
Authors: Sahoo N. C. , Rao K. V. S. , Natarajan K.
Published: March 2003

We examined the effects of CD86 ligation on surface densities of CD80 on activated B cells. For this, splenic B cells where CD86 levels had been enhanced upon anti‐immunoglobulin M (IgM) treatment were employed. Incubation of such CD86‐expressing B cells with anti‐CD86 monoclonal antibody (MoAb) resulted in a marked upregulation in cell surface levels of CD80. Such an enhancement in surface levels was also accompanied by a marked increase in CD80 mRNA, suggesting that the effects of CD86‐triggering are exerted at the level of CD80 message. Furthermore, coculture of CD86‐upregulated B cells with CD28‐expressing fibroblasts also led to the enhancement of CD80 levels, which was not obtained upon incubation with cells expressing cytotoxic T‐lymphocyte‐associated molecule (CTLA)‐4‐Ig. Finally, we also demonstrate that CD86‐induced CD80 is fully competent to function as a costimulatory molecule, as revealed by its ability to enhance cytokine secretion by allogeneic T cells. Thus, these results reveal the existence of another pathway that regulates the expression of CD80, at least on activated B cells, which could have important implications in directing the course of an immune response.
Rat Complement Factor H: Molecular Cloning, Sequencing and Quantification with a Newly Established ELISA
Authors: Demberg T. , Pollok‐Kopp B. , Gerke D. , Götze O. , Schlaf G.
Published: March 2003

Factor H (FH) is the predominant soluble regulatory protein of the complement system. With a concentration of 300–600 µg/ml in human plasma it acts as a cofactor for the FI‐mediated cleavage of the component C3b to iC3b. Furthermore, it competes with factor B for binding to C3b and C3(H2O) and promotes the dissociation of the C3bBb complex (i.e. it has decay accelerating activity). FH is a monomer of about 155 kDa which comprises 20 short consensus repeats (SCR), each of which is composed of nearly 60 amino acid residues. For the screening of a rat liver cDNA library, we used two hybridization probes which had been produced by polymerase chain reaction (PCR). The probes were generated using degenerated primers which corresponded to conserved parts of the human and the murine factor H nucleotide sequences. The entire rat sequence spanned 4240 nucleotides with an open reading frame of 3708 nucleotides. These were preceded by 23 nucleotides of the 5′ untranslated region, followed by a stop codon and a 3′ untranslated region of 478 nucleotides including the polyadenylation‐signal up to the beginning of the poly A tail. Comparison of the rat cDNA‐derived coding sequence revealed identities of 74% to the human and 87% to the mouse FH nucleotide sequence. The translation product of rat FH mRNA was 1236 aa in length (leader sequence included) with an identity of 63% to the human and 81.5% to the murine protein. The degree of glycosylation of rat FH‐Mr is about 9.5%. To quantitate FH in rat serum and supernatants of primary cultures of rat hepatocytes (HC), a reliable and sensitive sandwich‐enzyme‐linked immunosorbent assay (ELISA) was established. The concentration of FH in rat serum was calculated to be 238 µg ± 21 µg/ml (mean ± SD). Its concentration in the culture supernatants of HC was upregulated about three‐fold by interferon (IFN)‐γ (100 U/ml).
Analysis of HCV‐Immunoglobulin Isotype Complexes Provide New Insights into Antibody Response to HCV
Authors: Peng X. X. , Wang S. Y. , Zhang J. Y. , Zhu H.
Published: March 2003

Hepatitis C virus (HCV) is known for its ability to establish persistent infection and cause chronic hepatitis in most infected individuals. The antibody response to HCV in HCV‐circulating immune complexes (CIC) is unknown. In the present study, we have characterized distinct changes in patterns of HCV‐immunoglobulin (Ig) constituents with disease category, viral mutation and clinical markers. The number of samples positive for single HCV‐Ig, HCV‐IgG and HCV‐IgA, HCV‐IgM and HCV‐IgA, HCV‐IgM and HCV‐IgG, HCV‐IgM, HCV‐IgG and HCV‐IgA in 47 samples tested were 8 (17%), 1 (2.1%), 9 (19.1%), 4 (8.5%) and 17 (36.2%), respectively. The occurrence of HCV‐IgM and HCV‐IgA in combination of two isotypes of HCV‐Ig became predominant. These results show that defective IgG in HCV‐CIC may contribute to long‐term viremia. Further analysis indicated that the frequency of HCV RNA/IgA‐CIC in the abnormal aspartic aminotransferase (AST) group was significantly higher than that of the normal AST group, and HCV RNA/IgA‐CIC frequency in the abnormal alanine aminotransferase (ALT) group was slightly higher than that in the normal ALT group. IgA complexes may reflect the damage degree of liver function during the course of HCV infection. We also found that there were more mutations in supernatant than in other constituents from single‐strand conformation polymorphism (SSCP) analysis. Our results suggest that Ig‐complexed virions and free virions may have different biological consequences, with the latter being elusive to immunological elimination. The findings in this study may provide some new insights into antibody response to HCV.
Identification of Rat Quantitative Trait Loci that Regulate LPS‐Induced Pro‐Inflammatory Cytokine Responses
Authors: XU H.‐W. , WALLSTRÖM E. , BECANOVIC K. , DAHLMAN I. , LORENTZEN J. C.
Published: March 2003

Bacterial lipopolysaccharides (LPSs) trigger innate immune effector functions, such as the production of pro‐inflammatory cytokines. Here we utilized major histocompatibility complex (MHC)‐congenic rats to dissect the genetic basis of strain‐dependent variations of LPS‐induced tumour necrosis factor‐α (TNF‐α) levels in a whole blood in vitro assay. PVG.1AV1 background was associated with a high response, ACI background with a medium response, and LEW.1AV1 and DA backgrounds were associated with low responses. To determine the location of regulating non‐MHC genes, a genome‐wide linkage analysis with 236 microsatellite markers was performed on 186 F2 progeny of high TNF‐α responder PVG.1AV1 and MHC identical but low TNF‐α responder LEW.1AV1 rats. A region on rat chromosome 1 displayed linkage to LPS‐induced TNF‐α responses (P = 3.3 × 10−5). In addition, a locus on chromosome 2 was linked to responses of both interleukin‐6 (IL‐6) (P = 2.3 × 10−5) and TNF‐α (possible linkage, P = 8 × 10−3). Both chromosome regions have been linked to inflammatory diseases in rats, and so have the homologous regions in mice and humans. We therefore suggest that continued genetic dissection of the described in vitro phenotypes will give clues to both normal physiological regulation of LPS‐induced TNF‐α production and disease pathways.
Specific Acquired Resistance in Mice Immunized with Killed Mycobacteria
Authors: Agger E. M. , Weldingh K. , Olsen A. W. , Rosenkrands I. , Andersen P.
Published: March 2003

Past attempts to raise resistance against Mycobacterium tuberculosis using various preparations of killed mycobacteria have questioned the specificity of the generated immune response. In the present study, we have focused on the protective efficacy of experimental vaccines based on killed mycobacteria. We demonstrate that killed mycobacteria can confer high levels of protection, which can be adoptively transferred to recipient T‐cell‐deficient mice. Moreover, protective antigens can be found in the cell wall, membrane and cytosol of the mycobacterial cell, and hence emphasize the importance of searching for protective antigens in various compartments of the mycobacterial cell.
Clonal Analysis of a Human Antimouse Antibody (HAMA) Response
Authors: Thorpe S. J. , Turner C. , Heath A. , Feavers I. , Vatn I. , Natvig J. B. , Thompson K. M.
Published: March 2003

Circulating human antimouse antibodies (HAMAs) directed to mouse immunoglobulin G (IgG) are clinically significant, compromising mouse antibody therapy and imaging, and interfering in immunological assays. To investigate the HAMA response, 20 stable cell lines secreting human monoclonal antibodies reactive with mouse IgG were established from a donor with a history of exposure to mice. Their subclass and domain specificities were established by solid‐phase binding, indirect haemagglutination assays and immunoblotting, using Igs of known subclass and Ig fragments. The heavy‐chain variable region gene usage was determined for 12 HAMAs.

Eight HAMAs were IgM, 11 HAMAs were IgG4 and one HAMA was IgG1, indicating an IgG4‐dominated response. All of the IgG HAMAs reacted with epitopes present on the Fc portion; one was subclass‐specific, nine were subclass‐restricted and two were pan‐IgG‐reactive. Measurement of their affinities gave dissociation constants typically in the nanomolar range. Seven and five HAMAs were derived from variable heavy‐chain 3 (VH3) and VH1 gene segments, respectively. The IgG HAMAs used different VH segments to the IgM HAMAs. JH regions were coded by JH4 in eight HAMAs. DH segment usage appeared to be restricted in the IgM HAMAs. Two IgG HAMAs were clonally related. These monoclonal HAMAs are potentially useful as reagents for detecting mouse IgG and as reference reagents for the investigation of the HAMA response in patients undergoing mouse monoclonal antibody therapy and for the investigation of the influence of HAMAs on immunodiagnostic tests.