bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokine expression by high‐density human lymphocytes
Authors: SEWELL W. A. , BERGER M. F. , SKIPSEY L. J. , COOLEY M. A. , WARREN H. S.
Published: November 2003

T lymphocytes spend much of the time as small non‐cycling cells. To determine the pattern of cytokine expression in such resting cells, they were purified from human peripheral blood mononuclear cells (PBMC) on the basis of high buoyant density. The cells were stimulated and cytokine mRNA expression was assessed by reverse transcription–polymerase chain reaction (RT‐PCR). Expression of interleukin‐2 (IL‐2), IL‐3 and interferon‐γ (IFN‐γ) was similar in high‐density lymphocytes and in unfractionated PBMC. In contrast, the high‐density lymphocytes expressed less IL‐4 than PBMC, and little or no IL‐5. Because a substantial minority of the high‐density lymphocytes was CD45RO+, the presence of this marker was not an indicator of the ability to express IL‐4 and IL‐5. In the high‐density lymphocytes, IFN‐γ expression was confined to the CD45RO+ fraction, whereas IL‐2 was expressed by both CD45RO+ and CD45RO− subsets. To assess whether high‐density lymphocytes could give rise to cells with a broader range of inducible cytokine expression, they were activated and then restimulated between 10 and 22 days of culture. Cells derived from both the CD45RO+ and CD45RO− fractions of high‐density lymphocytes expressed IL‐5 after restimulation. Thus the high‐density lymphocyte population has the potential to acquire a broader range of inducible cytokine expression.
Venom immunotherapy modulates interleukin‐4 and interferon‐γ messenger RNA expression of peripheral T lymphocytes
Authors: AKOUM H. , TSICOPOULOS A. , VORNG H. , WALLAERT B. , DESSAINT J. P. , JOSEPH M. , HAMID Q. , TONNEL A. B.
Published: November 2003

The mechanism by which specific immunotherapy exerts its beneficial effect remains unclear. In order to evaluate the influence of venom immunotherapy on the T‐cell cytokine pattern of allergic reactions, we studied interleukin‐4 (IL‐4) and interferon‐γ (IFN‐γ) mRNA expression of peripheral T lymphocytes from 12 patients undergoing rush venom desensitization, before treatment at Day 0 (D0), at Day 15 (D15) and Day 90 (D90) after treatment, and from seven controls. Antigen‐specific T‐cell proliferation was also determined. Cytokine mRNA expression was evaluated using in situ hybridization, 24 hr after culture of peripheral T cells with medium, venom, or an unrelated allergen. Allergen‐induced T‐cell proliferation decreased at D15 and D90 of rush immunotherapy (P ≤ 0.02). In venom‐stimulated cultures of the patient group, there was a decrease in IL‐4 mRNA‐positive cells at D15 and D90 (P ≤ 0.001). Before desensitization, IFN‐γ mRNA expression was lower in patients than in controls and did not increase after in vitro allergen stimulation. In contrast, after immunotherapy, spontaneous IFN‐γ mRNA expression increased, but only at D90 (P ≤ 0.001). The cytokine pattern observed at D90 after immunotherapy was similar to that observed in control subjects. In conclusion, venom immunotherapy induced an altered cytokine mRNA pattern in allergen‐stimulated T cells which was dissociated from the early changes of allergen‐induced T‐cell responsiveness.
Expression of functional molecules by human CD3− decidual granular leucocyte clones
Authors: GUDELJ L. , DENIZ G. , RUKAVINA D. , JOHNSON P. M. , CHRISTMAS S. E.
Published: November 2003

Cell surface and cytoplasmic antigen expression by 35 CD3− decidual granular leucocyte (DGL) clones, derived from human endometrial tissue in the first trimester of pregnancy, has been compared with both that of fresh CD3− decidual leucocytes and that of CD3− peripheral blood natural killer (PBNK) cell clones (n = 12). The majority of DGL clones retained the antigenic phenotype of fresh cells, although CD103 (HML‐1) was expressed on 50% of DGL clones but only 17% of fresh DGL. Both cytoplasmic CD3ζ and CD3ε chains were detected in > 90% of DGL clones in the absence of cell surface CD3. Cytoplasmic CD3ζ was present in almost all fresh CD3− DGL, whereas CD3ε was not. Most DGL clones did not express surface Fcγ receptors I‐III (CD64, ‐32 and ‐16, respectively) and complement receptors (CR) types 1 and 2 (CD35 and 21, respectively), but 43% expressed CR3 (CD11b/18); in contrast, all PBNK clones were CR3+. The NK cell‐associated molecules Kp43 (CD94) and the p58 molecule recognized by the HP3E4 monoclonal antibody were both present on a higher proportion of CD3− PBNK (91% and 50%, respectively) than DGL clones (31% and 14%, respectively), despite expression of CD94 by > 90% of fresh CD56+ decidual leucocytes. Five of 35 CD3− DGL clones expressed cytoplasmic CD3ζ in the absence of expression of CD2, CD16 or the p58 molecule recognized by HP3E4. These variations between CD3− DGL and PBNK cell clones in expression of functional molecules may be related to previously reported differences in major histocompatibility complex‐non‐restricted cytotoxic activities between these two cell types.
How partial C7 deficiency with chronic and recurrent bacterial infections can mimic total C7 deficiency: temporary restoration of host C7 levels following plasma transfusion
Authors: WÜRZNER R. , PLATONOV A. E. , BELOBORODOV V. B. , PEREVERZEV A. I. , VERSHININA I. V. , FERNIE B. A. , HOBART M. J. , LACHMANN P. J. , ORREN A.
Published: November 2003

An apparently completely complement C7‐deficient patient with refractory otitis media and two episodes of meningococcal disease was given therapeutic plasma transfusions in 1992 and 1994. Following these transfusions unexpected changes were found in C7 levels. Immediately after transfusion the serum C7 levels failed to rise to the expected levels but then rose to 5–10% of the normal mean during the next 5 days and remained at that level for more than 2 weeks before eventually returning to zero. The patient's DNA genotyped C7 M, and therefore C7 N donor plasma was selected for the second transfusion to allow identification of the source of the C7 circulating post‐transfusion. This C7 phenotyped C7 M, demonstrating it to be of recipient origin. Therefore, the apparently completely C7‐deficient patient was able to secrete some C7. By a combination of DNA typing and isoelectric focusing of the C7 appearing after transfusion, it was demonstrated that the patient was heterozygous for combined subtotal C6/C7 deficiency (inherited from his father) and a different, so far uncharacterized, subtotal C7 deficiency (inherited from his mother). The low amount of C7 secreted appeared to be constantly consumed, probably by generation of C5b6 as a result of his chronic infection. He had been shown to have circulating C5b6 most of the time, and thus only when sufficient exogenous C7 was given to consume the free C5b6 did his own C7 appear in circulation.
Rat bone marrow‐derived mast cells co‐cultured with 3T3 fibroblasts in the absence of T‐cell derived cytokines require stem cell factor for their survival and maintain their mucosal mast cell‐like phenotype
Authors: MACDONALD A. J. , THORNTON E. M. , NEWLANDS G. F. J. , GALLI S. J. , MOQBEL R. , MILLER H. R. P.
Published: November 2003

When cultured without fibroblasts, rat bone marrow‐derived mast cells (BMMC) contain abundant rat mast cell proteinase type II (RMCP‐II), and exhibit survival and proliferation when maintained in mesenteric lymph node conditioned medium (CM). When BMMC were co‐cultured with 3T3 fibroblasts in the absence of CM, BMMC numbers increased for 7 days and the BMMC survived for up to 23 days. There was a gradual loss of stored RMCP‐II in BMMC that were co‐cultured with 3T3 cells, but the fibroblast microenvironment did not induce a detectable increase in the low levels of the connective tissue mast cell (CTMC)‐associated proteinase, RMCP‐I, in the BMMC. Nor did 3T3 cell co‐culture induce significant heparin synthesis in BMMC as judged by the cells' reactivity with the fluorescent heparin‐binding dye, berberine sulphate. These results suggest that rat BMMC, unlike murine BMMC, do not have the potential to develop multiple CTMC‐like characteristics upon co‐culture with 3T3 cells. However, when BMMC and fibroblast co‐cultures were treated with an antibody to recombinant rat stem cell factor (rrSCF), mast cell survival was completely abrogated. This result suggests that endogenous, fibroblast‐derived SCF is essential for the maintenance of rat BMMC viability in the absence of CM. On the other hand, prior treatment of the fibroblasts with the anti‐rrSCF antibody did not affect the adherence of BMMC to the monolayer, implying that (an)other molecule(s) is(are) involved in the attachment process. The demonstration that rat BMMC survival on fibroblasts in vitro is dependent upon SCF may indicate an important mechanism by which tissue mucosal cells can be maintained in vivo in the absence of T‐cell derived factors.
Staphylococcal enterotoxin B‐specific adhesion of murine splenic T cells to a human endothelial cell line
Authors: KITA M. , EGUCHI K. , KAWABE Y. , TSUKADA T. , MIGITA K. , KAWAKAMI A. , MATSUOKA N. , NAGATAKI S.
Published: November 2003

The presence of a putative autoantigen of autoimmune disorder in a target organ may cause accumulation of specific T cells in the inflammatory region. One of the mechanisms of such accumulation involves the migration of specific‐circulating T cells through the endothelial cells into the target lesion. The presence of only a few specific T cells responsive to a putative autoantigen has hampered the investigation of specific migration of circulating T cells to the target organ. We used a superantigen to investigate specific T‐cell adhesion to endothelial cells, because it stimulates a large proportion of T cells with particular Vβ elements and adhesion of T cells to the endothelium is a vital step in the migration process. Adhesion of murine T cells to the human endothelial cell line, EA.hy926, was specifically increased in the presence of staphylococcal enterotoxin B (SEB). The increase was interferon‐γ (IFN‐γ)‐dependent, and consisted mainly of CD4+ T cells. Vβ8.1,2+ T cells preferentially adhered to endothelial cells in the presence of SEB compared with Vβ6+ T cells. Pretreatment of endothelial cells with SEB increased the adherence of Vβ8.1,2+ T cells, while anti‐human leucocyte antigen (HLA)‐DR and ‐DQ antibodies inhibited the increased adherence of Vβ8.1,2+ T cells. Our results demonstrate that increased T‐cell adhesion to endothelial cells is SEB specific, and that the specificity is dependent on major histocompatibility complex (MHC) class II molecules expressed on endothelial cells and on the recognition of the SEB–MHC class II complex by Vβ8.1,2+ T cells.
HL‐60 myeloid leukaemia cells acquire immunostimulatory capability upon treatment with retinoic acid: analysis of the responding population and mechanism of cytotoxic lymphocyte activation
Authors: GEARY S. M. , ASHMAN L. K.
Published: November 2003

HL‐60 myeloid leukaemia cells are ineffective as stimulators of allogeneic lymphocytes in mixed leucocyte culture (MLC). These cells can be induced to differentiate along the monocytic or granulocytic pathways with or without acquisition of major histocompatibility complex (MHC) class II antigen by various agents. Surprisingly, treatment of HL‐60 cells with 10 nMall‐trans retinoic acid (RA) for 7 days (HL‐60‐R7) resulted in a marked increase in MLC stimulation although the cells lacked detectable MHC class II antigen expression at the initiation of the MLC. In contrast, treatment with interferon‐γ (IFN‐γ), with or without RA, induced MHC class II antigen expression but failed to enhance MLC stimulation. Lymphocytes responding to HL‐60‐R7 were predominantly CD8+ and/or CD16+ and displayed enhanced cytolytic capacity for HL‐60 and HL‐60‐R7 cells as well as natural killer (NK)‐sensitive K562 cells. Nevertheless, monoclonal antibodies (mAb) to MHC class II antigens substantially inhibited the MLC and some CD4+ lymphocytes in the responding population were required, although this requirement could be replaced by the addition of interleukin‐2 (IL‐2). HL‐60‐R7 (and HL‐60) cells were shown to acquire detectable MHC class II antigen expression during the first 3 days of the MLC. Thus a low level of activation by MHC class II+ stimulator cells appears to be required for the response. Analysis of the role of cytokines with costimulatory activity for T cells and/or NK cells indicated that tumour necrosis factor‐α (TNF‐α) was important in the proliferative response, while interleukins‐1, −6 and −12 and stem cell factor did not seem to be involved. Cell interaction molecules lymphocyte function‐associated antigen‐1 (LFA‐1) (CD11a), intracellular adhesion molecule‐1 (ICAM‐1) (CD54), ICAM‐3 (CD50) and B7.2 (CD86) were up‐regulated on HL‐60‐R7. Blocking mAb to LFA‐1 and B7.2 potently inhibited the proliferative response indicating a key role for these molecules in the enhanced immunostimulation by HL‐60‐R7 cells. The results may have implications for the mechanism of the therapeutic effect of RA in acute promyelocytic leukaemia and may also provide valuable information in regard to the immunogenicity of tumour cells in general.
Regulation by non‐major histocompatibility complex genes of the allo‐4‐hydroxy‐phenylpyruvate dioxygenase (F liver protein) response
Authors: BRUNNER M. C. , MITCHISON N. A.
Published: November 2003

Although rapid progress is being made in the quantitative genetics of multifactorial disease, no response to a simple antigen has yet been subjected to full genomic analysis. The well‐characterized antigen allo‐HPPD (4‐hydroxy‐phenylpyruvate dioxygenase, previously known as F liver antigen) is a good candidate for such treatment. Old and new data bearing on this possibility are here assembled. In respect of antibody production and an early burst of interleukin‐4 (IL‐4) transcription, introduction of the non‐major histocompatibility complex (MHC) background from A/J strain mice into F1 hybrids with C57BL10 strains up‐regulates the response. These findings can be aligned with previous quantitative genetics carried out on airway hyper‐responsiveness in related strains, and to a lesser extent with the genetics of autoimmune diabetes in the mouse. Taken together, the findings suggest that regulation of the pro‐inflammatory cytokines are largely responsible for the variation. Additional data indicate that these non‐MHC genes are are to a variable extent (depending on the response parameter) epistatic to the down‐regulatory MHC allele H‐2Ab
Prostaglandin E2 and monoclonal antibody to lymphocyte function‐associated antigen‐1 differentially inhibit migration of T lymphocytes across microvascular retinal endothelial cells in rat
Authors: MESRI M. , LIVERSIDGE J. , FORRESTER J. V.
Published: November 2003

Lymphocyte‐transendothelial cell migration is a complex event, and although much is known about the receptor–ligand interactions involved, little is understood about the intracellular events which accompany these interactions, or their regulation by inflammatory mediators. In this study we have shown that activation of T lymphocytes increased the proportion of cells migrating across monolayers of cultured retinal microvascular endothelial cells by both lymphocyte function‐associated antigen‐1 (LFA‐1)‐dependent and LFA‐1‐independent mechanisms. In preliminary experiments, it was found that activation of T cells with mitogens such as concanavalin (Con A) increased significantly T‐cell migration across the endothelial monolayers. In contrast, activation of the endothelial monolayer with interferon‐γ (IFN‐γ) (96 hr) had no effect on transendothelial migration. Investigation of adhesion molecule requirements for transendothelial migration indicated that LFA‐1 was necessary (P < 0.02) but that intracellular adhesion molecule‐1 did not appear to be involved. Investigation of the role of prostaglandins in transendothelial migration revealed that, while prostaglandin E2 (PGE2) did not affect adhesion molecule expression on endothelial cells or T cells, treatment of either cell significantly blocked transendothelial migration (P < 0.05). Pretreatment with PGE2 combined with LFA‐1 blockade had an additive effect on inhibition of T‐cell transendothelial migration, indicating that two independent mechanisms were operative. PGE2 also had a direct inhibitory effect on T‐cell adhesion to the endothelium. These results highlight the importance of considering non‐adhesion receptor‐mediated mechanisms, perhaps involving cytoskeletal and/or motility, in the migration of T cells across endothelial monolayers.
Mechanisms by which HLA‐class II molecules protect human B lymphoid tumour cells against NK‐ and LAK‐mediated cytolysis
Authors: LOBO P. I. , CHANG M. Y. , MELLINS E.
Published: November 2003

We have previously shown that mutant B lymphoblastoid cell lines, totally deficient in expression of human leucocyte antigen (HLA)‐class II molecules, but with normal HLA‐class I expression, develop enhanced susceptibility to natural killer (NK) and lymphokine‐activated killer (LAK) cell lysis. The current investigations were aimed at examining the role of HLA‐DR and native peptides occupying the antigen‐presenting grooves of HLA‐class II molecules in protecting mutants of the same B‐lymphoid lines against LAK‐mediated lysis. No augmentation in LAK lysis was observed despite using two mutant B‐cell lines (9.22.3 and 3.1.0) that lacked HLA‐DR. Both these lines expressed HLA‐DP and HLA‐DQ. However, when using other B‐cell lines with point mutations in certain regions of the HLA‐DR α‐chain (78, 80 and 96) significantly increased their susceptibility to LAK lysis despite normal expression of HLA‐DR and the other class I and II molecules. Of particular interest was the finding that absence of native peptides in antigen‐presenting grooves of all the HLA‐class II molecules did not render the mutant B cell (9.5.3) susceptible to LAK lysis. These observations support the concept that there are different NK or LAK clones. Certain LAK clones recognize ‘self’ major histocompatibility complex (MHC) antigens (but not the native peptides in their antigen‐presenting grooves). Presence of ‘self’ MHC antigens inhibits such clones. Conversely, other NK or LAK clones recognize ‘non‐self’ in the context of MHC antigens. Hence, point mutations at certain specific sites on the MHC molecules or foreign peptides in the antigen‐presenting grooves enhances the susceptibility of these cells to LAK clones recognizing ‘non‐self’.
Afferent lymph veiled cells stimulate proliferative responses in allogeneic CD4+ and CD8+ T cells but not γδ TCR+ T cells
Authors: HOWARD C. J. , SOPP P. , BROWNLIE J. , PARSONS K. R. , KWONG L.‐S. , COLLINS R. A.
Published: November 2003

Dendritic cells were identified in afferent lymph derived by lymphatic cannulation of cattle, stained with monoclonal antibody (mAb) to the bovine workshop cluster 6 (WC6) antigen, which is highly expressed on bovine afferent lymph veiled cells, and sorted with a fluorescence‐activated cell sorter. These cells expressed major histocompatibility complex (MHC) class I and II and CD1b but not CD14. They bound human and murine CTLA4–immunoglobulin (CTLA4–Ig) fusion proteins indicating expression of CD80 and or CD86. Dendritic cells induced proliferative responses in allogeneic CD4+ and CD8+ cells sorted from blood but did not induce responses in purified allogeneic WC1+, γ/δ T cells, which are CD2−, CD4−, CD8− and are the major γδ T‐cell population in cattle blood, even when interleukin‐2 (IL‐2) was added to cultures. A WC1−, CD2+γδ T‐cell receptor (TCR)+ population predominates in cattle spleens and proliferation of a T‐cell line with this phenotype was not induced by allogeneic dendritic cells, with or without added IL‐2. The observations imply that the ligand for the γδ TCR expressed on the two populations is not present on allogeneic dendritic cells or that the costimulatory molecules expressed on dendritic cells that render them highly effective at stimulating MHC class I‐ and class II‐restricted CD8+ and CD4+ T cells are not recognized by the WC1+ or WC1−γ/δ T cells. Expression of CD28 by the four cell types was assessed by reverse transcriptase–polymerase chain reaction (RT‐PCR). Purified CD4+ and CD8+ cells both produced CD28 transcripts but neither purified WC1+ cells nor the WC1−γδ TCR+ cell line did so. The findings indicate that CD80 and or CD86 are involved in the stimulation of CD4+ and CD8+αβ TCR+ T cells but not in the stimulation of either of the two γδ TCR+ populations.
A defect in inducible β‐galactosidase of B lymphocytes in the osteopetrotic (mi/mi) mouse
Authors: YAMAMOTO N. , NARAPARAJU V. R.
Published: November 2003

Macrophages were activated by administration of an inflammatory lipid metabolite, lysophosphatidylcholine (lyso‐Pc), to wild type mice but not murine (microphthalmic) osteopetrotic (mi/mi) mutant mice. In vitro treatment of wild type mouse peritoneal cells with lyso‐Pc efficiently activated macrophages whereas lyso‐Pc‐treatment of mi mutant mouse peritoneal cells resulted in no activation of macrophages. Generation of macrophage activating factor requires a precursor protein, serum vitamin D binding protein (DBP), and participation of lyso‐Pc‐inducible β‐galactosidase of B lymphocytes. Lyso‐Pc‐inducible β‐galactosidase of B lymphocytes was found to be defective in mi mutant mice.
Chemotaxis of human B lymphocytes to anti‐IgD
Authors: KOMAI‐KOMA M. , WILKINSON P. C.
Published: November 2003

The resting population of small surface IgM+ and surface IgD+ B cells from the human tonsil can be preactivated by overnight culture in interleukin‐4 (IL‐4) to show locomotor responses to anti‐IgM and anti‐IgD at between 10 ng and 1 μg/ml. Because this locomotion is activated through the antigen receptor and may simulate a response to antigen, we set out to establish whether this was a chemotactic response using a checkerboard filter assay with a range of concentrations and concentration gradients of anti‐IgD. At high concentrations (100 ng/ml to 1 μg/ml), a chemokinetic response, but no chemotaxis, to anti‐IgD was seen. However, in concentration gradients set up at lower concentrations (0–50 ng/ml) a chemotactic response was demonstrable. During the period of culture in anti‐IgD at 1 μg/ml, a progressive loss of surface IgD from the cells was seen, but there was no loss at 10 ng/ml. This receptor loss from the cell surface may account for the lack of chemotactic effect of the anti‐IgD at higher concentrations.
Induction of intercellular adhesion molecule‐1 in human nasal epithelial cells during respiratory syncytial virus infection
Authors: MATSUZAKI Z. , OKAMOTO Y. , SARASHINA N. , ITO E. , TOGAWA K. , SAITO I.
Published: November 2003

The effects of infection with respiratory syncytial virus (RSV) on expression of intercellular adhesion molecule (ICAM)‐1 was determined in vitro in nasal epithelial cell cultures. Functional consequences of changes in ICAM‐1 expression were assessed by measuring adhesion of a human leukaemic T‐cell line to RSV‐infected epithelial cells. Also, adhesion of phytohaemagglutinin‐activated tonsillar lymphocytes (TL) to RSV‐infected epithelial cells caused a significant increase in interleukin (IL)‐4 or IL‐5 production. Release of these cytokines was adhesion dependent as non‐adherent TL produced significantly less IL‐4 or IL‐5. However, no significant difference was observed for IL‐2 or interferon‐γ (IFN‐γ) production. These observations suggest that RSV‐infected epithelial cells may induce T‐helper type‐2 (Th2)‐like cytokines by mucosal lymphocytes during mucosal infection in vivo
Contribution of extrathymic γδ T cells to the expression of heat‐shock protein and to protective immunity in mice infected with Toxoplasma gondii
Authors: HISAEDA H. , SAKAI T. , NAGASAWA H. , ISHIKAWA H. , YASUTOMO K. , MAEKAWA Y. , HIMENO K.
Published: November 2003

We demonstrated that γδ T cells contribute to protective immunity against Toxoplasma gondii by inducing the expression of a 65 000 MW heat‐shock protein (hsp 65) in host macrophages. Here we examined the role of extrathymic and intrathymic γδ T cells in protective immunity and hsp 65 expression in mice infected with T. gondii. Intrathymic γδ T cells were obtained from severe combined immunodeficiency (SCID) mice grafted with syngeneic fetal thymus (TG‐SCID), in which only T cells derived from the donor thymus developed, whereas extrathymic γδ T cells were obtained from nude mice that lack thymus. Extrathymic γδ T cells from T. gondii‐infected nude mice differed from intrathymic γδ T cells of infected TG‐SCID mice, in terms of Thy1.2 expression and V‐region gene usage of T‐cell receptor (TCR) γδ. Extrathymic γδ T cells expressed extremely high levels of Thy1.2, and had Vγ7 repertoire but lacked Vγ5,6 and Vδ1,5. On the other hand, intrathymic γδ T cells express intermediate and low levels of Thy1.2. These cells possessed Vγ5,6 and Vδ1,5 but failed to rearrange the Vγ7 gene. Peritoneal macrophages from infected nude mice contained hsp 65, whereas this protein was scarcely expressed in those of infected TG‐SCID mice. Transfer of extrathymic, but not of intrathymic γδ T cells to SCID mice enabled their macrophages to express hsp 65. Athymic nude mice were significantly resistant to the infection compared with SCID mice which lack γδ T as well as αβ T cells. The resistance was dependent upon extrathymic γδ T cells, since nude mice depleted of γδ T cells using a corresponding monoclonal antibody became extremely susceptible. These results indicated that extrathymic rather than intrathymic γδ T cells play some crucial roles in protection against T. gondii and in hsp 65 expression.
Treatment of established adjuvant arthritis in rats with monoclonal antibody to CD18 and very late activation antigen‐4 integrins suppresses neutrophil and T‐lymphocyte migration to the joints and improves clinical disease
Authors: ISSEKUTZ A. C. , AYER L. , MIYASAKA M. , ISSEKUTZ T. B.
Published: November 2003

The migration of leucocytes from blood into the joint is a key feature of human and experimental arthritis. Adhesion molecules on leucocytes and vascular endothelium are important in this process and may be therapeutic targets for intervention in arthritis. We investigated whether monoclonal antibody treatment to block the α4 integrin, very late activation antigen‐4 (VLA‐4), and β2 integrins (CD11/CD18) administered to rats during the preclinical (day 5) or clinical phase (day 10+) would modify disease. When treatment was initiated 5 days after induction of disease, development of arthritis was significantly reduced by either anti‐α4 (TA‐2) or anti‐β2 (WT.3) monoclonal antibodies (mAb) and the combination of both mAb was even more effective (clinical scores: control 11.4; anti‐α4 6.6; anti‐β2 6.8; anti‐α4 + anti‐β2 3.9). When treatment was delayed until arthritis was apparent (day 10), the anti‐α4 + anti‐β2 mAb combination still significantly diminished the arthritis score on day 14 (control 13; anti‐α4 + anti‐β2 7.9). Treatment with anti‐α4 + anti‐β2 mAb decreased the migration to the joints of blood polymorphonuclear leucocytes (PMNL) by 66–79% and of spleen T lymphocytes by 56–75%, depending on the joint. In contrast, PMNL migration was abolished (>98%) and T‐cell migration markedly (87%) inhibited to dermal inflammatory reactions in the same animals. These findings demonstrate: that blocking mAb to α4 and β2 integrins can reduce the severity of adjuvant arthritis, even after joint inflammation has developed; that this treatment can markedly inhibit PMNL and T‐lymphocyte migration to the joints; and that yet to be defined mechanisms distinct from α4 (CD49d) and β2 (CD11/CD18) integrins, also contribute to leucocyte migration to inflamed joints. Identifying these additional adhesion mechanisms may be required to control joint inflammation further.
Isotype and antibody specificity of spontaneously formed immunoglobulins in pig fetuses and germ‐free piglets: production by CD5− B cells
Authors: CUKROWSKA B. , ŠINKORA J. , ŘEHÁKOVÁ Z. , ŠINKORA M. , ŠPLÍCHAL I. , TUČKOVÁ L. , AVRAMEAS S. , SAALMÜLLER A. , BAROT‐CIORBARU R. , TLASKALOVÁ‐HOGENOVÁ H.
Published: November 2003

Pig fetuses, colostrum‐deprived newborns and germ‐free (GF) piglets, animals in which B‐cell development is not influenced by maternal regulatory factors, were employed to study the occurrence and specificity of natural antibodies (NAb). Serum immunoglobulins of all isotypes were found in 44‐day‐old fetuses (the gestation period in pigs lasts 114 days) and their level, with predominating IgM, was increased during fetal ontogeny. In sera of fetuses at the end of embryonic life as well as of newborns and older GF piglets, antibody activity against autoantigens (thyroglobulin, hormones, ssDNA), phylogenetically conserved proteins (myosin), haptens (trinitrophenyl; TNP) and bacterial components (Escherichia coli O86, tetanic anatoxin) was detected by enzyme‐linked immunosorbent assay. The antigen‐binding activity of IgM NAb increased after isolation of the serum immunoglobulins on a Staphylococcus Protein A (SPA)–Sepharose column. IgM reactivity similar to that detected in serum was found in supernatants from polyclonally stimulated cultures of spleen of 8‐ and 12‐day‐old GF piglets. Pig fetal liver IgM+ B cells, which were able to produce IgM after polyclonal stimulation, did not express the CD5 molecule. Our results indicate that pig preimmune repertoire is comparable to that described in humans and mice, although in contrast to these species pig B‐1 cells do not express CD5.
The Nramp1 antimicrobial resistance gene segregates independently of resistance to virulent Mycobacterium tuberculosis
Authors: M EDINA E. , ROGERSON B. J. , NORTH R. J.
Published: November 2003

Nramp1 is a recently cloned gene that is involved in resistance of mice to infection with certain microbial pathogens, including the attenuated bacillus Calmette–Guérin (BCG) strain of Mycobacterium bovis, as well as certain other mycobacteria. With a view to determining whether Nramp1 influences resistance of mice to infection with virulent M. tuberculosis, BALB/c mice homozygous for the susceptibility allele of Nramp1, and DBA/2 mice homozygous for the resistance allele, as well as their F1 and F2 progeny, were typed according to their possession of these alleles using a ‘hot start’ polymerase chain reaction (PCR) procedure. As assessed by the ability of the mice to survive infection, the results show that Nramp1 plays no discernible role in resistance to tuberculosis.
Duck lymphoid organs: their contribution to the ontogeny of IgM and IgY
Authors: BANDO Y. , HIGGINS D. A.
Published: November 2003

The occurrence of mRNAs encoding μ, υ and υ(ΔFc) immunoglobulin heavy chains and λ light chains in organs of duck embryos from 16 days of incubation and ducklings up to 74 days of age was assessed by Northern hybridization. The μ message was first detected in bursa of Fabricius and spleen at 16 days of incubation and in cervical lymph nodes at 23 days of incubation, but in other organs (bone marrow, buffy coat, Harderian gland, liver) not until 7–17 days after hatching; in general, the appearance of the λ message paralleled that of μ. Messenger RNAs encoding one or both of the υ isoforms were first detected in cervical lymph nodes at 25 days of incubation, in spleen and bursa in 1‐day‐old ducklings, in Harderian gland, bone marrow and liver from 10 to 17 days post‐hatching and in buffy coat from 46 days. In most organs, the υ(ΔFc) message was detected prior to the υ message and predominated during the experiment; Harderian gland expressed the υ(ΔFc) message exclusively. These results indicate that bursa of Fabricius, spleen and cervical lymph nodes play early roles in the development of B cells and the ontogeny of duck immunoglobulins while other lymphoid organs support the later differentiation of plasma cells, and that IgY and IgY(ΔFc) are probably not simultaneous products of the same plasma cells.
Phenotypic classification of porcine lymphocyte subpopulations in blood and lymphoid tissues
Authors: YANG H. , PARKHOUSE R. M. E.
Published: November 2003

The pig is a useful model for the heterogeneity of the mammalian immune system and has also recently received attention as a possible source of organs for human transplantation. Here we report a detailed analysis of porcine lymphocyte phenotypes. Peripheral blood αβ T cells consisted of four subsets (CD4+8−, CD4+8lo, CD4−8lo and CD4−8hi) and γδ T cells of three (CD2−4−8−, CD2+4−8lo and CD2+4−8−). There were, in addition, a large proportion of non‐T‐non‐B lymphocytes with CD2+3−4−8lo surface immunoglobulin‐negative phenotype containing natural killer (NK) activity. A striking observation was the relatively low frequency of αβ T cells in the blood of young pigs. Similar phenotypes were also identified in the cells from peripheral lymphoid tissues, though the proportions of the γδ T cells and the non‐T‐non‐B lymphocytes in the lymph nodes and tonsil were much lower and the majority of the γδ T cells in the lymphoid tissues bore CD2 and/or CD8. In thymus, the small thymocytes were predominantly CD3−4+8+while the mature large thymocytes displayed phenotypes similar to those of peripheral T cells. Thus this work has directly defined porcine αβ and γδ T cells, demonstrated the T‐cell nature of the unique CD4+8+ subset of peripheral lymphocytes, revealed the high heterogeneity of the CD8+ cells, and established the phenotype of NK cells. The functional properties of these defined porcine lymphocyte subsets can now be experimentally determined in health and disease.
In vitro immunization of naive human B cells yields high affinity immunoglobulin G antibodies as illustrated by phage display
Authors: DUEÑAS M. , CHIN L.‐T. , MALMBORG A.‐C. , CASALVILLA R. , OHLIN M. , BORREBAECK C. A. K.
Published: November 2003

In vitro antibody responses to a synthetic immunogen, consisting of both a B cell [V3 loop of gp120 from human immunodeficiency virus type 1 (HIV‐1)] and a T‐helper epitope (15 amino acids of tetanus toxoid) was studied. The in vitro activation was performed by primary and secondary in vitro immunizations, using lymphocytes obtained from uninfected, seronegative donors. Analysis of the in vitro immune response demonstrated an antigen‐specific isotype switch, which was dependent on the presence of antigen‐specific T‐helper cells, CD40 ligation and antigen. Antibody libraries were constructed from cells derived directly from the naive donors, or from primary or secondary in vitro immunized B cells. Five libraries were displayed on filamentous phage and selected for anti‐V3‐specific Fab fragments, using a selection approach that linked recognition and phage replication. A panel of 19 recombinant antigen‐specific Fab, representing different phases of the humoral in vitro immune response, were sequenced, expressed and analysed using a biosensor. Recombinant Fab fragments derived from cultures on day 12 exhibited an increase in affinity of close to two orders of magnitude compared to those obtained from cells primary immunized for 7 days. This study provides the first evidence that an antigen‐specific in vitro immune response can yield high‐affinity immunoglobulinG antibodies.
Molecular cloning of the feline CD8β‐chain
Authors: PECORARO M. R. , SHIMOJIMA M. , MAEDA K. , INOSHIMA Y. , KAWAGUCHI Y. , KAI C. , MIKAMI T.
Published: November 2003

Human, mouse and rat CD8 have been described as being disulphide‐linked heterodimers of α and β chains. More recently the chicken α and β chains were described. In the bovine and feline immune system only the α‐chain was reported. In this study we have cloned and determined the nucleotide sequence of a cDNA encoding the β‐chain of the feline T‐cell surface antigen CD8. Using a nested polymerase chain reaction (PCR) and two primer pairs designed from the human CD8β cDNA nucleotide sequence, we amplified a 430 base pair fragment from a feline thymus cDNA library which was used as a probe for screening the feline library at high stringency. After three rounds of screening, five clones were isolated. A clone, named FTb‐6, containing a 3.8 kilobase pair insert was mapped, sequenced and compared with the published sequences of the genes encoding the human, mouse, rat and avian CD8β. We have determined the primary structure of the feline CD8β. The feline CD8β has an open reading frame, 630 nucleotides in length encoding a protein with 210 amino acid residues and its composition showed that the feline molecule is a member of the immunoglobulin gene super family.
Integrin α4β7 mediates human eosinophil interaction with MAdCAM‐1, VCAM‐1 and fibronectin
Authors: WALSH G. M. , SYMON F. A. , LAZAROVITS A. I. , WARDLAW A. J.
Published: November 2003

We have investigated the contribution of integrin α4β7 to human peripheral blood eosinophil adhesive interactions. Immunofluorescence and flow cytometry demonstrated constitutive expression of α4β7 by eosinophils. Expression of α4β1 or α4β7 was not enhanced by eosinophil activation with platelet‐activating factor (PAF). Expression of α4β7 was confirmed by immunoprecipitation of 125I‐labelled lysates analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). Approximately 20% of unstimulated eosinophils were adherent to L1‐2 cells transfected with vascular cell adhesion molecule‐1 (VCAM‐1) cDNA, while very few resting eosinophils adhered to mouse mucosal adressin cell adhesion molecule‐1 (MAdCAM‐1) transfectants. Binding of unstimulated eosinophils to VCAM‐1 transfectants was inhibited by HP1/2 (an antibody that blocks both α4β1 and α4β7 functions), but not Act‐1, an α4β7 monoclonal antibody (mAb). PAF stimulation resulted in increased binding of eosinophils to MAdCAM‐1 transfectants, which was inhibited by both HP1/2 and Act‐1. In contrast, PAF did not enhance binding to VCAM‐1 transfectants, although binding of PAF‐stimulated eosinophils to VCAM‐1 could be partially inhibited by Act‐1. Stimulation of eosinophils with the β1‐activating mAb TS2/16 resulted in enhanced binding of eosinophils to both VCAM‐1 and MAdCAM‐1 transfectants. The increased binding was largely α4β7‐dependent. Unstimulated eosinophils bound to soluble recombinant human (rh)VCAM‐1 and fibronectin (Fn), coated on 96‐well plates in a dose‐dependent manner. Binding was inhibited by HP1/2 and 4b4, an anti‐β1 mAb, but not by Act‐1. TS2/16 treatment increased adherent cell numbers and this enhanced binding was inhibited by Act‐1. We have therefore confirmed that α4β1 is functionally active on unstimulated eosinophils. In contrast, PAF‐induced enhancement of eosinophils binding to VCAM‐1 or MAdCAM‐1 was α4β7‐dependent. In addition, treatment with TS2/16 resulted in a α4β7‐dependent enhancement of eosinophil binding to VCAM‐1, MAdCAM‐1 and Fn. We therefore hypothesize that α4β7 may have an important role in eosinophil localization in diseases such as asthma and inflammatory bowel disease.
IDENTIFICATION OF A NOVEL DENDRITIC CELL SURFACE ANTIGEN DEFINED BY CARBOHYDRATE SPECIFIC CD24 ANTIBODY CROSS‐REACTIVITY
Authors: Williams L. A. , McLellan A. D. , Summers K. L. , Sorg R. V. , Fearnley D. B. , Hart D. N. J.
Published: November 2003

Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T‐lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co‐stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B‐lymphocyte signalling but co‐stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC; however, cross‐reactive, sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down‐regulated following activation/differentiation in vitro.
Autologous anti‐idiotypic antibody response is regulated by the level of circulating complementary idiotype
Authors: BORGHESI C. , NICOLETTI C.
Published: November 2003

BALB/c mice injected with lyophilized vaccine from Streptococcus pneumoniae R36a (Pn) predominantly responded with antibody molecules the vast majority of which expressed the public idiotype T15 and were directed to the immunodominant epitope phosphorylcholine (PC). However, after a single immunization with Pn vaccine young (3‐month‐old) BALB/c mice did not produce any specific anti‐T15 antibody response. In contrast, young D1.LP mice were able to mount a specific anti‐T15 response upon primary immunization with pneumococcal vaccine. The anti‐PC response in the two mouse strains differed in that the proportion of antibody molecules that expressed the T15 idiotype for Pn‐primed D1.LP mice showed a smaller proportion of PC‐specific antibody expressing the T15 idiotype. Neonatal injection of anti‐T15 monoclonal antibodies led to a long‐term suppression of the PC‐specific T15+ B‐cell clones but at young/adult age these mice maintained the ability to produce a normal amount of PC‐specific antibody. Interestingly, the idiotypically‐suppressed BALB/c mice mounted a significant anti‐T15 response during the primary response to Pn. We interpreted these data as showing that the level of circulating idiotype may regulate the production of the complementary anti‐idiotypic antibody. In addition, in vitro experiments demonstrated that the lack of the anti‐T15 response during primary antibody response in BALB/c mice is probably because of a state of tolerance that is regulated by T cells.
Production of the third and fourth component of complement (C3, C4) by smooth muscle cells
Authors: UEDA Y. , NAGASAWA K. , TSUKAMOTO H. , HORIUCHI T. , NISHIZAKA H. , IKEDA K. , NIHO Y.
Published: November 2003

Production of the third and fourth components of complement (C3, C4) by smooth muscle cells was investigated by using normal human aortic smooth muscle cells (AoSMC), human smooth muscle cell line (G402) and vascular smooth muscle cells obtained from human umbilical cord vein (UVSMC). AoSMC spontaneously produced both C3 and C4 at 15 ng/106 cells/72 hr and 22 ng/106 cells/72 hr, respectively, and both were enhanced by interferon‐γ (IFN‐γ). Although phorbol 12‐myristate 13‐acetate (PMA) and tumour necrosis factor‐α (TNF‐α) enhanced C3 production, C4 production was reduced by these agents. On the other hand, G402 produced C4 but not C3 in a dose‐dependent manner when cultured with IFN‐γ. UVSMC produced only a small amount of C3 and C4 compared with AoSMC or G402. C3 and C4 produced by AoSMC were confirmed to be identical with their human serum counterparts as determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and measurement of haemolytic activity. Northern blotting analysis showed that the expression of mRNA of C3 and C4 was enhanced by TNF‐α and IFN‐γ, respectively, in AoSMC. Our findings suggest the importance of smooth muscle cells as a source of components of complement in vascular diseases including vasculitis.
Antigen‐driven shedding of L‐selectin from human γδ T cells
Authors: SANCHEZ‐GARCIA J. , ATKINS C. , PASVOL G. , WILKINSON R. J. , COLSTON M. J.
Published: November 2003

Activation of lymphocytes leads to the modulation of a number of surface molecules. We have investigated the expression of one such molecule, L‐selectin, following activation of γδ T cells with Mycobacterium tuberculosis. L‐selectin is modulated during lymphocyte entry into lymph nodes; this modulation reflects the recirculation and homing potential of lymphocytes. We find that stimulation of γδ T cells by M. tuberculosis antigens results in shedding of L‐selectin from γδ T cells. Re‐expression of L‐selectin occurs on removal of antigen suggesting that the regulation of expression is controlled by the presence or absence of antigen. The γδ T‐cell receptor (TCR)‐positive, L‐selectin negative population of peripheral blood lymphocytes appears to be resting cells, as assessed by forward‐ and light‐scatter analysis. We further find that γδ T cells isolated from a site of infection, the pleural fluid of a tuberculosis patient, are L‐selectin negative, and that L‐selectin is re‐expressed following culture of the pleural fluid γδ T cells in the absence of antigen. These results demonstrate that, in addition to stimulation with polyclonal mitogens, antigen stimulation can also promote the surface shedding of L‐selectin and that γδ T cells have the potential to home to sites of infection supporting their role in the immunological defence against infectious micro‐organisms.
Alloproliferation of purified CD4+ T cells to adult human heart endothelial cells, and study of second‐signal requirements
Authors: MCDOUALL R. M. , PAGE C. S. , HAFIZI S. , YACOUB M. H. , ROSE M. L.
Published: November 2003

Human endothelial cells have been shown to be capable of causing direct allostimulation of T cells. However, the majority of immunological studies of human endothelial cells have been performed on cells of fetal origin. Here we use endothelial cells isolated from the adult human heart, both large vessel (coronary artery, pulmonary artery and aorta) and also microvascular. We have examined the ability of all these endothelial cells to cause direct allostimulation of T cells, and show that purified CD4+ T cells can proliferate in response to adult human heart endothelial cells, the response being dependent on pretreatment of the endothelial cells with interferon‐γ (IFN‐γ) and inhibited by anti‐HLA‐DR monoclonal antibody. The proliferative responses of CD8+ T cells to adult but not fetal endothelial cells was inconsistent and weak. Proliferative responses were not blocked by CTLA4‐Ig, which inhibits T‐cell responses to ‘classical’ antigen‐presenting cells (APC), but >50% inhibition was achieved with monoclonal antibody to lymphocyte function‐associated antigen‐3 (LFA‐3). These results show that adult human cardiovascular endothelial cells are capable of causing allostimulation of resting CD4+ T cells, using a different second signal to classical APC. In view of these findings endothelial cells should be considered as APC following solid organ transplantation.
Enhancement of CD3‐mediated thymocyte apoptosis by the cross‐linkage of heat‐stable antigen
Authors: HITSUMOTO Y. , SONG D.‐S. , OKADA M. , HAMADA F. , SAHEKI S. , TAKEUCHI N.
Published: November 2003

Heat‐stable antigen (HSA) is a murine differentiating antigen that is expressed on both CD4−CD8− double‐negative and CD4+CD8+ double‐positive thymocytes but not CD4+ or CD8+ single‐positive thymocytes. Effects of anti‐HSA monoclonal antibody, R13, on thymocyte apoptosis induced by various stimulations were investigated by a single‐cell suspension culture system. Immobilized R13 enhanced the CD3‐mediated DNA fragmentation and killing of thymocytes but not the dexamethasone‐induced or phorbol myristate acetate‐induced killing of thymocytes. Immobilized R13 by itself could not induce thymocyte apoptosis. Soluble R13 enhanced CD3‐mediated apoptosis when HSA and T‐cell receptor (TCR)/CD3 were co‐cross‐linked by a cross‐reactive secondary antibody. Even without the cross‐reactive secondary antibody, soluble R13 enhanced CD3‐mediated apoptosis, although a greater than 100‐fold increase in the amount of R13 was needed to give a similar enhancement compared with immobilized R13. Neither R13 by itself nor R13 plus secondary antibody induced cytosolic calcium influx, whereas R13 enhanced CD3‐mediated cytosolic calcium increase. These results suggest a functional role of HSA in promoting the activation‐induced apoptosis of thymocytes and the involvement of HSA in negative selection.