bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction of measles virus‐specific cytotoxic T‐cell responses after intranasal immunization with synthetic peptides
Authors: PARTIDOS C. D. , VOHRA P. , STEWARD M. W.
Published: November 2003

We have investigated the structural requirements for the induction of cytotoxic T‐cell responses (CTL) in vivo after intranasal immunization with an immunodominant CTL epitope from the nucleoprotein of measles virus (MV). For the induction of CTL responses, covalent linkage of the CTL epitope to a helper T‐cell epitope was required and the orientation of the epitopes influenced the immunogenicity of the CTL epitope. The presence of two copies as compared with one copy of a T‐helper epitope, rendered the CTL epitope more immunogenic and resulted in the in vivo induction of MV‐specific CTLs without the need for an adjuvant. The role of CTL responses to this epitope in protection after intranasal administration was evaluated in a mouse model against challenge with a neuroadapted strain of MV. Although a decreased mortality in the peptide immunized compared with that in unimmunized mice was observed, the protection achieved was not significant. These findings highlight the importance of the rational design of synthetic immunogens for the induction of CTL responses and the potential of the intranasal route for immunization.
Analysis of immunoglobulin variable region genes of a human IgM anti‐myeloperoxidase antibody derived from a patient with vasculitis
Authors: LONGHURST C. , EHRENSTEIN M. R. , LEAKER B. , STEVENSON F. K. , SPELLERBERG M. , CHAPMAN C. , LATCHMEN D. , ISENBERG D. A. , CAMBRIDGE G.
Published: November 2003

Circulating antibodies to myeloperoxidase (MPO) are associated primarily with pauci‐immune glomerulonephritis and systemic vasculitis. Anti‐MPO antibodies belong to a group of autoantibodies, anti‐neutrophil cytoplasmic antibodies, that may play a pathogenic role in vasculitis. We have generated a human monoclonal anti‐MPO antibody (E3‐MPO) using peripheral blood lymphocytes from a patient with microscopic polyarteritis. Variable region gene analysis of E3‐MPO showed that the VH region had 90% homology with the germ line gene VH4‐21. E3‐MPO was also shown to carry the 9G4 idiotope, which so far has been associated only with human antibodies that utilize the VH4‐21 gene. The 9G4 idiotope was also expressed on anti‐MPO antibodies in sera from the donor patient and from 4/7 additional patients with active, untreated vasculitis. The nucleotide sequences of both the variable heavy and light chains of E3‐MPO showed evidence of an antigen‐driven response.
Impaired immunity and altered pulmonary responses in mice with a disrupted interferon‐γ receptor gene exposed to the irradiated Schistosoma mansoni vaccine
Authors: WILSON R. A. , COULSON P. S. , BETTS C. , DOWLING M‐A. , SMYTHIES L. E.
Published: November 2003

A high level of protection against Schistosoma mansoni is elicited in mice by the irradiated cercaria vaccine and interferon‐γ (IFN‐γ) is a key cytokine in the pulmonary effector response. The role of this cytokine has been investigated in mice with a targeted disruption of the IFN‐γ receptor gene (IFN‐γR−/− mice). The level of protection was impaired relative to that elicited in C57BL/6 and 129 wild‐type (WT) animals. These two groups developed compact effector foci, of largely mononuclear cell composition, around individual challenge parasites migrating through the lungs. In contrast the IFN‐γR−/− mice showed a massive and generalized leucocytic infiltration of the airways and interstitium in which eosinophils were a prominent feature. Cultures of airway leucocytes from C57BL/6 mice produced abundant IFN‐γ whilst those from IFN‐γR−/− mice produced interleukin‐4 (IL‐4), IL‐5 and IL‐10, indicating default to the Th2 pathway; the WT animals showed an intermediate response. The pattern of cytokine gene transcripts in whole lung tissue agreed remarkably well with the level of cytokine protein detected in leucocyte cultures, with the exception of substantial IL‐4 mRNA but negligible protein in C57BL/6 mice. The loose but intense infiltrate of leucocytes in the lungs of IFN‐γR−/− mice was clearly ineffective in eliminating challenge parasites, whereas the level of IFN‐γ protein and mRNA in the lungs of C57BL/6 and WT mice correlated with the size and compactness of effector foci. On the basis of these and earlier observations, we suggest that a primary role for IFN‐γ is to promote intercelluar adhesion between the leucocytes in an effector focus, promoting its ability to block parasite migration.
Modulation of superantigen‐induced T‐cell deletion by antibody anti‐Pgp‐1 (CD44)
Authors: AYROLDI E. , CANNARILE L. , RICCARDI C.
Published: November 2003

We examined the effects of anti‐Pgp‐1 (CD44) antibody on the in vitro deletion of murine CD4 and CD8 single positive T cells induced by Staphylococcal enterotoxin B (SEB). Soluble anti‐Pgp‐1 antibody enhanced the apoptosis and decreased the proliferation of SEB‐responding T cells. In contrast, cross‐linked anti‐Pgp‐1 antibody provided costimulatory signals for the T‐cell activation induced by anti‐CD3 antibody. Hyaluronic acid (HA), a ligand of Pgp‐1, did not affect proliferation and deletion induced by SEB, whereas it mimicked the effects of the cross‐linked antibody in anti‐CD3‐driven proliferation. T‐cell Pgp‐1 surface expression after 48 hr incubation with SEB was unchanged as compared to unstimulated cells. However, when the memory T cells were established, some Vβ8+ (SEB‐specific) T cells Pgp‐1low became Pgp‐1high, displaying a bimodal character. Moreover, the Pgp‐1 increased expression correlated with an increase of Pgp‐1 soluble form in the supernatant. These findings suggested that signals following the triggering of the Pgp‐1 molecule are important in controlling T‐cell survival.
Cleavage of human complement component C5 by cysteine proteinases from Porphyromonas (Bacteroides) gingivalis. Prior oxidation of C5 augments proteinase digestion of C5
Authors: DISCIPIO R. G. , DAFFERN P. J. , KAWAHARA M. , PIKE R. , TRAVIS J. , HUGLI T. E.
Published: November 2003

Since severe periodontitis is characterized by an acute inflammatory response with cellular infiltration and microbial overgrowth, plasma proteins could be exposed to both proteinases and oxidants released from the granulocytes, as well as to proteinases from the microorganisms. When human complement component C5 was digested by cysteine proteinases (i.e. gingipain‐R and gingipain‐K) from Porphyromonas gingivalis, limited cleavage of the C5 molecule was observed. If C5 was first oxidized by hydroxyl radicals, these gingipains converted modified C5 to fragments that exhibited significantly greater pro‐inflammatory activity than did digests of unmodified C5. After cleavage of oxidized C5 by gingipain‐R, the digest exhibited measurably greater neutrophil enzyme release and chemotaxis of human polymorphonuclear leukocytes (PMNs) compared with the activities of unoxidized C5 digests. Gingipain‐K generates virtually no polarization or chemotactic activity of human PMNs from C5, nor is enzyme release stimulated by these C5 digests. However, when oxidized C5 was digested by gingipain‐K, human PMNs were stimulated for polarization, chemotaxis and enzyme release indicating that an active fragment had been generated. Proteolysis of oxidized C5 evokes greater neutrophil activation than does proteolysis of unoxidized protein, a fact which supports the hypothesis that oxidation and proteolysis may be coupled to enhance the destructive effects of the inflammatory process. These results, in which digests of both oxidized and unmodified complement component C5 were evaluated, support the general concept that oxidation and proteolysis may participate cooperatively in amplifying both the severity and duration of the inflammatory reaction.
A subset of γδ T‐cell receptor‐positive cells produce T‐helper type‐2 cytokines and regulate mouse skin graft rejection following portal venous pretransplant preimmunization
Authors: GORCZYNSKI R. M. , CHEN Z. , HOANG Y. , ROSSI‐BERGMAN B.
Published: November 2003

C3H/HeJ mice received B10.BR skin grafts following portal or lateral tail vein infusion of irradiated B10.BR spleen cells. Thereafter mice were injected with anti‐αβ or anti‐γδ T‐cell receptor (TCR) monoclonal antibody (mAb). Anti‐γδ TCR mAb abolished the increased graft survival afforded by portal venous (p.v.) immunization, and reversed the bias towards expression of mRNA for type‐2 cytokines [interleukin‐4 (IL‐4), IL‐10] seen in lymphoid tissue of p.v.‐immunized mice. When γδ TCR+ and αβ TCR+ cells were isolated from the intestinal epithelial compartment (IEL), liver or Peyer's Patch (PP) of p.v.‐immunized mice, the γδ TCR+ cells were found to be enriched in cells producing type‐2 cytokines on rechallenge with irradiated B10.BR cells in vitro. γδ TCR+ cells from p.v.‐immunized mice were further expanded in vitro with anti‐CD3 and cytokines (combined IL‐2 and IL‐4). Following expansion these cells were capable of adoptively transferring increased B10.BR skin graft survival to naive mice, and continued to show a bias in type‐2 cytokine synthesis after allostimulation in vitro. When γδ TCR chain expression was assessed in cells taken from p.v.‐immunized mice, or in cells expanded in culture, our data suggest that p.v. immunization leads to oligoclonal, not polyclonal, expansion of those γδ TCR+ cells involved in inhibition of graft rejection.
Comparative immunophenotypic analysis of human mast cells, blood basophils and monocytes
Authors: AGIS H. , FÜREDER W. , BANKL H. C. , KUNDI M. , SPERR W. R. , WILLHEIM M. , BOLTZ‐NITULESCU G. , BUTTERFIELD J. H. , KISHI K. , LECHNER K. , VALENT P.
Published: November 2003

Mast cells (MC), blood basophils (Ba) and moncoytes (Mo) are of haemopoietic origin. Lineage‐relationships and transdifferentiation between MC and Mo, or MC and Ba, have been considered, based on common expression of antigens. In this study, comparative phenotypic analyses on MC, Ba and Mo and on respective cell lines were performed using monoclonal antibodies (mAb) to previously defined and novel CD antigens (CD1–130). By cluster analysis, the overall (all 130 CD) phenotypic relationships (given as similarity indices, SI), between primary cells (MC, Ba and Mo) and corresponding cell lines (HMC‐1, KU‐812, U937) were 0.716, 0.779 and 0.757, respectively. When primary cells were compared, lower SI values were found (MC versus Ba, 0.509; MC versus Mo, 0.625; Mo versus Ba, 0.698). More distant relationships were found between MC versus Ba and MC versus Mo, compared with Ba versus Mo, for adhesion receptor (R)‐, complement R‐ and cytokine R profiles. Analysis of cytokine R revealed most significant dissimilarities between MC versus Ba and MC versus Mo (SI < 0.2). Moreover, in contrast to other CD subgroups and other lineages, MC and HMC‐1 differed from each other in cytokine R expression (SI = 0.286). Cytokine R detectable on HMC‐1 but not MC were granulocyte–macrophage colony‐stimulating factor (GM‐CSFR)α(CD116), CD40, Apo‐1/FAS(CD95) and gp130(CD130). Cytokine R detectable on Ba but not MC, were interleukin‐3 (IL‐3)Rα(CD123), IL‐1RII(CD121b), IL‐2Rα(CD25) and CD40. In summary, MC, Ba and Mo display a unique CD profile with MC being the most distantly related cell. The most significant mismatch within a given lineage is the loss of cytokine R on mature MC as compared with normal myeloid progenitors and HMC‐1 cells.
Porcine CD3ε: its characterization, expression and involvement in activation of porcine T lymphocytes
Authors: KIRKHAM P. A. , TAKAMATSU H. , YANG H. , PARKHOUSE R. M. E.
Published: November 2003

The cloning, characterization and expression of porcine CD3ε and establishment of its role in T‐cell activation using an anti‐porcine CD3ε monoclonal antibody, as described here, provides a first step towards a greater understanding of the porcine immune response. Porcine CD3ε was cloned from a porcine T‐cell cDNA library by polymerase chain reaction and found to have up to 72% identity with other CD3ε chains, retaining all the necessary primary structural motifs for correct functioning of porcine CD3ε. When expressed in COS7 cells porcine CD3ε was an intracellularly localized, monomeric 23 000 MW protein exhibiting no evidence of N‐glycosylation. A monoclonal antibody, PPT3, recognized expressed porcine CD3ε and activated porcine T cells as demonstrated by stimulation of calcium mobilization, an increase in protein tyrosine phosphorylation and proliferation. These results further reaffirm and identify CD3ε as an important cell surface protein involved in signal transduction of activation signals in porcine T cells.
Constitutive and inducible expression of interleukin‐6 by Langerhans cells and lymph node dendritic cells
Authors: CUMBERBATCH M. , DEARMAN R. J. , KIMBER I.
Published: November 2003

During the induction phase of contact sensitization and other cutaneous immune responses a proportion of epidermal Langerhans cells (LC) is induced to leave the skin and migrate via afferent lymphatics to lymph nodes draining the site of exposure. The cells that accumulate in draining nodes have acquired the characteristics of immunostimulatory dendritic cells and effectively present antigen to responsive T lymphocytes. In the present study we have questioned whether LC in the epidermis and the lymph node dendritic cells into which they develop express interleukin‐6 (IL‐6), a cytokine that has been shown to serve as an important costimulator of T lymphocyte activation. In situ immunocytochemical analyses using a biotin–streptavidin staining technique revealed that dendritic cells resident in the epidermis of untreated mice constitutively express this cytokine. Keratinocytes expressed detectable IL‐6 only following local exposure to the contact allergen oxazolone. Such treatment also appeared to enhance the expression by epidermal dendritic cells of this cytokine. Analyses of unfractionated and LC‐enriched and ‐depleted populations of epidermal cells revealed a close correlation between major histocompatibility complex (MHC) class II (Ia) antigen expression and staining for IL‐6, implicating LC as the sole or major source of this cytokine in unstimulated epidermis. Finally, compared with tissue isolated from mice treated with vehicle alone, draining lymph nodes prepared from animals 18 hr following sensitization with oxazolone displayed a substantial increase in both the frequency of dendritic cells and the number of IL‐6+ cells within the paracortex. These data demonstrate that resident epidermal LC and the dendritic cells into which they develop are important sources of IL‐6. Their constitutive and inducible expression of this cytokine will facilitate the induction of cutaneous immune responses.
Effects of antigen presentation on superantigen‐induced apoptosis mediated by Fas/Fas ligand interactions in human T cells
Authors: BOSHELL M. , McLEOD J. , WALKER L. , HALL N. , PATEL Y. , SANSOM D.
Published: November 2003

Stimulation of T cells with bacterial superantigens has several distinct functional outcomes including proliferation, anergy and apoptosis. At present however, the mechanisms that dictate whether activation, anergy, or apoptosis predominate are unclear. In this study we have investigated the effects of superantigen presentation to mature superantigen‐reactive human T‐cell lines. Despite expressing major histocompatibility complex (MHC) class II molecules, these lines failed to proliferate in response to superantigen in the absence of antigen‐presenting cells (APC) but proliferated when minimal APC were added. In the absence of APC a significant proportion of the T cells underwent apoptosis. This response was rapid, antigen dependent and largely abolished by the addition of cyclosporin A. Interestingly the response was not blocked by the addition of a number of antibodies to cell surface molecules including MHC class II and intracellular adhesion molecule‐1. Using both a bioassay and messenger RNA analysis we were able to demonstrate that stimulation of these T cells with superantigen resulted in the induction of Fas‐ligand expression on the T cells and furthermore, the ability of these cells to induce apoptosis was inhibited by the addition of blocking Fas antibodies as well as a Fas–Fc fusion protein. These data demonstrate that stimulation of T cells with staphylococcal enterotoxin B induces expression of Fas‐ligand resulting in T‐cell apoptosis; however, the final outcome of proliferation or apoptosis is determined by the presence of APC.
Stimulation of human peripheral blood mononuclear cells with live Mycobacterium bovis BCG activates cytolytic CD8+ T cells in vitro
Authors: TURNER J. , DOCKRELL H. M.
Published: November 2003

Experimental data have shown that Mycobacterium tuberculosis can survive within the host cell and in doing so may release secreted antigen into the endogenous antigen‐processing pathway. If mycobacterial antigen can gain access to MHC class I molecules then CD8+ T cells may play a role in host defence against M. tuberculosis infection. To identify whether there is a role for the CD8+ T cell in mycobacterial infection we have stimulated peripheral blood mononuclear cells (PBMC) from bacillus Calmette–Guérin (BCG) vaccinated individuals with live M. bovis BCG. The activation state of the T cells was established by staining for the interleukin‐2 (IL‐2) receptor (CD25), HLA‐DR or the transferrin receptor (CD71). Using FACScan analysis we have shown that, in vitro, live M. bovis BCG activates significantly more CD8+ T cells in comparison to the soluble antigen purified protein derivative (PPD). In addition, live M. bovis BCG activates more CD8+ T cells than a non‐viable preparation of the same M. bovis BCG following irradiation. The function of the activated CD8+ T cells was addressed using positively selected cells in a cytotoxic T‐cell assay. CD8+ T cells isolated from a 7‐day M. bovis BCG‐stimulated PBMC culture were shown to be cytolytic against target cells infected with live M. bovis BCG, dead M. bovis BCG, and to a lesser extent, PPD. These results suggest that CD8+ T cells may be activated by stimulation with live mycobacteria, and that this subset can play a cytolytic role in the immune response to mycobacterial infections.
Dynamic production of tumour necrosis factor‐α (TNF‐α) messenger RNA, intracellular and extracellular TNF‐α by murine macrophages and possible association with protein tyrosine phosphorylation of STAT1α and ERK2 as an early signal
Authors: ZHENG Z. M. , SPECTER S.
Published: November 2003

Tumour necrosis factor‐α (TNF‐α), an important mediator in both immune and inflammation responses, is one of the major cytokines released by activated macrophages. The present study shows that, during macrophage activation, protein tyrosine phosphorylation of STAT1α and ERK2 occurred as an immediate early signal, whereas maximum TNF‐α mRNA transcription appeared at 3 hr, precursor TNF‐α formation at 3 to 4 hr, and TNF‐α release at 5 to 6 hr after stimulation of an RPMI‐1640‐based induction medium containing lipopolysaccharide (100 ng/ml), interferon‐γ (100 U/ml), and 0.5% bovine serum albumin. Herbimycin A, a tyrosine kinase inhibitor, suppresses protein tyrosine phosphorylation of STAT1α and ERK2 and also blocks TNF‐α production by resident peritoneal macrophages from BALB/c mice, suggesting a possible association between protein tyrosine phosphorylation of STAT1α and ERK2 and macrophage activation resulting in TNF‐α production.
Chimpanzee dendritic cells with potent immunostimulatory function can be propagated from peripheral blood
Authors: BARRATT‐BOYES S. M. , HENDERSON R. A. , FINN O. J.
Published: November 2003

We have established dendritic cell (DC) cultures from chimpanzee peripheral blood mononuclear cells (PBMC) by using recombinant human (rh) granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and rh interleukin‐4 (IL‐4) and demonstrate that these cells have all the characteristics of DC as described for other species. We consistently can obtain 1×107 DC per 100 ml of blood, a yield of 5% DC as compared to 0.1 to 0.5% DC reported in fresh human PBMC. The cultured DC have a varied morphology with typical cytoplasmic extensions. Phenotypically, the blood‐derived DC lack expression of most lineage antigens, but express CD83, an antigen specifically expressed on human blood DC. Chimpanzee DC express very high levels of major histocompatability complex class II antigens, adhesion and costimulatory molecules. Consistent with this phenotype of a powerful antigen‐presenting cell, chimpanzee DC generate allogeneic mixed leukocyte responses 15 to 20 times more potent than that elicited by macrophages, Epstein–Barr virus‐transformed lymphoblasts and fresh PBMC. In addition, chimpanzee DC very efficiently present tetanus toxoid to PBMC‐derived CD4+ T cells as compared to macrophages and PBMC. The DC generated by culturing chimpanzee PBMC with rhGM‐CSF and rhIL‐4 thus closely resemble human blood‐derived DC propagated in the same manner. This technology provides a powerful animal model with which to apply DC to clinical studies with relevance to human disease. In particular, chimpanzee DC can be tested as immunotherapeutic agents for cancer, and be studied in relation to the pathogenesis of human immunodeficiency virus (HIV) infection.
Upregulation of MHC class II antigen on dendritic cells from hepatitis B virus transgenic mice by interferon‐γ: abrogation of immune response defect to a T‐cell‐dependent antigen
Authors: FAZLE AKBAR S. M. , INABA K. , ONJI M.
Published: November 2003

The experiments presented here were performed to see whether the level of expression of major histocompatibility complex (MHC) class II antigen (Ia antigen) on dendritic cells, one of the most critical antigen presenting cells (APC), influences the humoral immune response in hepatitis B virus (HBV) transgenic mice. We have reported that transgenic mice had a low responsiveness in specific antibody production to keyhole limpet haemocyanin (KLH), a T‐cell dependent, HBV‐unrelated antigen compared with the age, sex, and major histocompatibility‐matched normal mice,1 due to a significantly lower T‐cell stimulatory capacity of transgenic mice‐derived dendritic cells, possibly as a result of significantly lower level of Ia antigen.1 Immunohistochemical staining has shown that treatment of transgenic mice with mouse recombinant interferon‐γ (IFN‐γ), daily for six consecutive days resulted in an increased expression of Ia antigen on splenic dendritic cells. Again, flow cytometric analyses have further confirmed the significant increase in the expression of Ia antigen on dendritic cells, isolated from transgenic mice treated with IFN‐γ compared with the same from the untreated or phosphate‐buffered saline (PBS)‐treated transgenic mice. Transgenic mice immunized with two optimum doses of KLH (5 μg/mouse) could not produce anti‐KLH antibodies in sera, but injecting transgenic mice with the same doses of KLH together with IFN‐γ resulted in the production of anti‐KLH antibodies in sera. Again, KLH‐primed normal mice‐derived T/B lymphocytes produced anti‐KLH antibody, when cultured with dendritic cells from IFN‐γ‐treated transgenic mice expressing a higher level of Ia antigen, but not with the same from PBS‐treated or untreated transgenic mice. Treatment of transgenic mice with IFN‐γ resulted in a reduced level of hepatitis B virus (HBV) DNA in liver and in sera. These experiments have shown that the level of expression of Ia antigen on dendritic cells is a critical factor for its APC capability and its modulation of IFN‐γ may be used for immune therapy in HBV carriers.
Cyclosporin A abrogates the acquired immunity to cutaneous reinfection with the parapoxvirus orf virus
Authors: McK. HAIG D. , McINNES C. J. , HUTCHISON G. , SEOW H.‐F. , REID H. W.
Published: November 2003

The effect of cyclosporin A (CsA) on host immunity to cutaneous reinfection with the parapoxvirus orf virus was studied in 6‐month‐old lambs. In control reinfected animals, clinical lesions and viral replication (measured by the presence of vesicular/pustular lesions and viral antigen) in regenerating epidermal cells were at a maximum on day 4 with resolution by day 9. Lesion histology revealed recruitment of T cells, B cells and dermal dendritic cells (DDC) which increased and decreased in parallel with the clinical course of the reinfection. In animals treated with CsA (25 mg/kg/day) 1 day before and for 8 days after reinfection, more severe clinical lesions and viral replication typical of primary infections were recorded and had not resolved by 28 days following reinfection. During CsA treatment, the recruitment of T cells, B cells and DDC was inhibited. With cessation of CsA treatment there was dramatic recruitment of CD4 T cells followed by DDC then B cells to the lesion site but rapid onset of acquired immunity was not recorded. Reverse transcription–polymerase chain reaction (RT–PCR) analysis of cytokine mRNAs from lesion biopsies showed individual sheep variations. However, interleukin‐2 (IL‐2) and interferon‐γ (IFN‐γ) mRNAs were detected in the control reinfected animals on days 3 and/or 9 after reinfection but not on these days in animals undergoing treatment with CsA. In the untreated lambs there was an inexplicable lack of IL‐2 and IFN‐γ mRNAs on day 6 after reinfection. Tumour necrosis factor‐α (TNF‐α) and vascular endothelial growth factor (VEGF) mRNAs were unaffected by CsA treatment. The data suggest that CsA abrogates acquired immunity to orf virus reinfection by targetting T‐cell lymphokine production.
Naive human αβ T cells respond to membrane‐associated components of malaria‐infected erythrocytes by proliferation and production of interferon‐γ
Authors: DICK S. , WATERFALL M. , CURRIE J. , MADDY A. , RILEY E.
Published: November 2003

Crude extracts of Plasmodium falciparum schizont‐infected erythrocytes (PfSE) induce polyclonal activation of peripheral blood T lymphocytes from naive (malaria unexposed) humans. We demonstrate that the active component of PfSE is membrane bound, soluble in sodium dodecyl sulphate (SDS) and partially heat stable, but distinct from the tumour necrosis factor (TNF)‐inducing, exoantigen‐like activity of schizont extracts. Malaria pigment induces little or no T‐cell activation. The responding cells are predominately CD4+, CD45RO+, T‐cell receptor (TCR)αβ+. Contrary to previous reports, expansion of the TCRγδ+ subset was observed in cells from only one of eight donors. Proliferating cells secrete interferon‐γ (IFN‐γ) and release large amounts of soluble interleukin‐2R (sIL‐2R) into the culture supernatant but produce no detectable interleukin‐4 (IL‐4), a phenotype typical of the T‐helper (Th)1 subset of CD4+ T cells. We propose that these activated T cells may initiate the inflammatory response to malaria infection in non‐immunes and may contribute to the pathology of the disease.
Mac‐1 (CD11b/CD18) is the predominant β2 (CD18) integrin mediating human neutrophil migration through synovial and dermal fibroblast barriers
Authors: GAO J.‐X. , ISSEKUTZ A. C.
Published: November 2003

Recently we reported that polymorphonuclear leucocyte (PMNL) migration in vitro through a barrier of human synovial fibroblasts (HSF) involves both β2 (CD18) and β1 (CD29) integrins on the PMNL. Here we investigated the role of the β2 integrin family members, lymphocyte function‐associated (LFA)‐1 (αLβ2 or CD11a/CD18) and Mac‐1 (αMβ2 or CD11b/CD18), in PMNL migration through HSF and human dermal fibroblast (HDF) monolayers. Treatment of PMNL with monoclonal antibody (mAb) to LFA‐1 (anti‐αL) did not inhibit PMNL migration through either monolayer in response to C5a. In contrast, mAb to Mac‐1 (CD11b) inhibited (by 30–40%) PMNL migration, and by virtually the same extent as mAb to the β2 integrin chain (CD18) (40% inhibition). Addition of mAb to LFA‐1 to mAb to Mac‐1 did not result in greater inhibition. This was in contrast to PMNL migration through human umbilical vein endothelium (HUVE) monolayers, where mAb to LFA‐1 or to Mac‐1 each partially inhibited PMNL transendothelial migration, and when these mAbs were combined, synergistic inhibition of migration was observed, reaching 90–95% inhibition. Intercellular adhesion molecule 1 (ICAM‐1) was not required for Mac‐1 mediated migration through HSF or HDF, because treatment of the fibroblasts with mAb R6.5 (F(ab)2) to ICAM‐1, which blocks the Mac‐1 binding site on ICAM‐1, did not inhibit PMNL migration. An LFA‐1–ICAM‐1 mechanism of PMNL migration through HSF and HDF monolayers could be detected after treatment (4 hr) of the monolayers with TNF‐α plus IFN‐γ, which upregulated ICAM‐1 on the fibroblasts. The results demonstrate the β2 integrin dependent PMNL migration in connective tissue may involve primarily Mac‐1, with little involvement of LFA‐1 or ICAM‐1, a situation in marked contrast to PMNL migration across endothelium. However, in inflammatory conditions in which TNF‐α and/or IFN‐γ may be generated, a role for LFA‐1–ICAM‐1 may be induced.
Role of LFA‐1, ICAM‐1, VLA‐4 and VCAM‐1 in lymphocyte migration across retinal pigment epithelial monolayers in vitro
Authors: DEVINE L. , LIGHTMAN S. L. , GREENWOOD J.
Published: November 2003

The blood–retinal barrier (BRB), which is composed of the retinal pigment epithelium (RPE) and retinal vascular endothelium, normally restricts the traffic of lymphocytes into the retina. During ocular inflammatory conditions such as posterior uveitis there is a large increase in lymphocyte migration across the BRB. The differential role played by the two barrier sites, however, remains unclear. To evaluate the role of the posterior BRB, the migration of a CD4+ antigen‐specific T‐cell line through rat RPE cell monolayers was investigated in vitro using time‐lapse videomicroscopy. The adhesion molecules involved in controlling transepithelial migration across normal and interferon‐γ (IFN‐γ)‐activated RPE was assessed with monoclonal antibodies directed against cell adhesion molecules. Lymphocytes were treated with antibodies specific for CD11a (αL subunit of LFA‐1), CD18 (β2 subunit of the leucam family) and CD49 d (α4 subunit of very late activation antigen‐4, VLA‐4), and the RPE with antibodies specific for CD54 (intracellular adhesion molecule‐1, ICAM‐1) and CD106 (vascular cell adhesion molecule‐1, VCAM‐1). Migration across unstimulated RPE was inhibited by antibodies to ICAM‐1 (48.6 ± 3.5% reduction), leucocyte functional antigen‐1 (LFA‐1)α (61 ± 5.2%) and LFA‐1β (63.2 ± 4.7%), but not by antibodies to VLA‐4. VCAM‐1 was not expressed on untreated RPE. Following activation of the RPE monolayers for 72 hr with IFN‐γ, antibodies to LFA‐1α, LFA‐1β and ICAM‐1 inhibited migration by 49.9 ± 9.4%, 63.6 ± 5.5% and 47.7 ± 4.2% respectively. Antibodies to VLA‐4 and VCAM‐1 blocked migration by 21.5 ± 8.4% and 32.3 ± 6.2%, respectively, which correlated with the induction of VCAM‐1 expression on RPE and increased migration. Under these conditions blocking both VCAM‐1 and ICAM‐1 reduced migration by 70.9 ± 2.3%, which was greater than the effect of blocking either of these molecules alone. These results demonstrate that the posterior barrier of the BRB utilizes the same principle receptor–ligand pairings in controlling lymphocyte traffic into the retina as the vascular endothelium of the anterior BRB.
Cross‐antigenicity of horse serum albumin with dog and cat albumins: study of three short peptides with significant inhibitory activity towards specific human IgE and IgG antibodies
Authors: GOUBRAN BOTROS H. , GREGOIRE C. , RABILLON J. , DAVID B. , DANDEU J.‐P.
Published: November 2003

Horse serum albumin is present in the near vicinity of the animal, while dog and cat serum albumins are very common allergens present in house dust. Human patients clinically defined as allergic to horse could react with horse serum albumin by means of IgE or IgG antibodies. Studies regarding the specificities of these antibodies by inhibition enzyme‐linked immunosorbent assay (ELISA) and depletion experiments have demonstrated that they are directed against dog serum albumin and cross‐react not only with horse serum albumin but with other serum albumins from different origins. To investigate these observations further, we isolated and characterized three tryptic peptides (P1, P2 and P3) from horse serum albumin. The peptide P1 contains loops 1 and 2 of the first domain, P2 is derived from loop 4 of the second domain, and P3 contains the disulphide loop 9 of the third domain. These were able to inhibit the binding of the patients' IgE and IgG antibodies to horse albumin as well as to dog and cat serum albumins. This indicates that these peptides are involved in the observed cross‐reactions. They also shared common epitopes, as revealed by human IgE antibodies. After reduction and alkylation, they totally lost their inhibitory capacity, suggesting that the intra‐chain disulphide bridges, essential for the preservation of the loop structure, probably maintain their allergenic/antigenic reactivity.
Immunoglobulin D enhances the release of tumour necrosis factor‐α, and interleukin‐1β as well as interleukin‐1 receptor antagonist from human mononuclear cells
Authors: DRENTH J. P. H. , GÖERTZ J. , DAHA M. R. , VAN DER MEER J. W. M.
Published: November 2003

Immunoglobulin D (IgD) is normally present in only low concentrations in serum. In the hyper‐IgD and periodic fever syndrome (HIDS), however, serum levels exceed 140 mg/l. This syndrome is further characterized by recurrent inflammatory febrile attacks together with an acute phase response and appearance of cytokines in the circulation. The role of IgD in the pathogenesis of HIDS and its relation to the increased cytokine concentrations is unclear. Therefore, we tested whether IgD, IgG and α1‐acid glycoprotein (AGP) isolated from human serum influence the synthesis of interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α), and IL‐1ra, as measured by specific radioimmunoassays, in human peripheral blood mononuclear cells (PBMC). Incubation of PBMC with IgD and AGP for 24 hr led to increased release of IL‐1β, TNF‐α, and IL‐1ra. The magnitude of stimulation of IgD exceeded that of AGP; the effect by IgD was dose‐dependent and showed a 30‐fold (TNF‐α) to almost 150‐fold (IL‐1β) increase at the highest concentration (50 mg/l), while AGP (750 μg/ml) only increased the cytokine secretion fourfold (TNF‐α) to almost 30‐fold (IL‐1β). The effect of IgD on IL‐1ra was less dramatic but a fivefold increase was observed at 50 mg/l compared with a 2.5‐fold increase with AGP. IgD potentiated the effect of lipopolysaccharide (LPS) on secretion of both IL‐1β and TNF‐α, although the effect was most apparent for TNF‐α. Apart from inducing IL‐1ra synthesis, IgG did not influence cytokine release in human PBMC. These data indicate that IgD is a potent inducer of TNF‐α, IL‐1β and IL‐1ra and thus may contribute to the pathogenesis of HIDS.
Immunogenicity of peptides for B cells is not impaired by overlapping T‐cell epitope topology
Authors: HARRIS D. P. , VORDERMEIER H.‐M. , ARYA A. , BOGDAN K. , MORENO C. , IVANYI J.
Published: November 2003

The epitope specificity of T‐cell help to B cells and of surface immunoglobulin‐mediated B‐cell‐binding of antigens usually involves topographically distinct antigenic determinants. The possibility of cross‐recognition of the same peptide sequence by both T cells and antibodies has been a matter of conflicting opinions. We investigated this subject by detailed mapping of T‐ and B‐cell epitopes within four immunogenic mycobacterial peptides. The identified core sequences of T‐ and B‐cell epitopes showed different topology within each peptide: they were partially overlapping or adjacent in two P38‐derived peptides, but entirely overlapping in two P19‐derived peptides. The critically important result using the two truncated peptides (P19/67–78 and P19/146–155) containing only the fully overlapping epitope cores was, that they retained full potency for inducing antibody responses. However, despite this desirable overlap of determinants, anti‐peptide sera failed to block the proliferation of corresponding T‐cell hybridomas. We conclude, that our study, in contrast to previous findings, suggests that overlapping topology of T‐ and B‐cell epitopes within synthetic peptides does not necessarily impair B‐cell immunogenicity.
Molecular characterization of VDJ transcripts from a newborn piglet
Authors: SUN J. , BUTLER J. E.
Published: November 2003

The sequences of 42 transcripts, expressed with IgM, IgG and IgA and cloned from the mesenteric lymph node of a newborn piglet, are described. Forty transcripts used either DHA and DHB and their FR4 were identical to the single swine germline JH. The low frequency of somatic mutation made it possible to identify 35/41 as originating from five putative germline VH genes, of which VHA, VHB and VHC accounted for > 85%. The remaining six transcripts were hybrids of these five germline genes. The most 3′ functional VH gene (VH2 = VHB) was the only one exclusively expressed with IgM although VHA, of unknown location in the genome, accounted for half of all transcripts. Junctional diversity in CDR3 was extensive and asymmetrical, in that D–J joining contributed more diversity than V–DJ joining. Reading frame II was used twice as frequently as frame III and the CDR3 generated using the former would have a higher expected R/S ratio. This study indicated that the expressed VH repertoire of the newborn piglet is restricted and nearly germline although junctional diversity is mature and better developed than in fetal mice. The hybrid clones suggest that swine compensate for their < 20 VH genes and single JH by using somatic gene conversion. There was no evidence for exclusive or preferential expression of the most 3′ VH gene as occurs in chickens and rabbits respectively, and switching to downstream constant regions probably occurs in utero, even in the theoretical absence of environmental antigens and maternal regulatory molecules. Preferential VHA expression is probably a selection phenomenon.
Local immune response in Helicobacter pylori‐infected cats and identification of H. pylori in saliva, gastric fluid and faeces
Authors: FOX J. G. , PERKINS S. , YAN L. , SHEN Z. , ATTARDO L. , PAPPO J.
Published: November 2003

Helicobacter pylori‐infected cats were screened by culture and polymerase chain reaction (PCR) for the presence of H. pylori in salivary secretions, gastric juice, gastric tissue and faeces. H. pylori was cultured from salivary secretions in six of 12 (50%) cats and from gastric fluid samples in 11 of 12 (91%) cats. A 298 base pair polymerase chain reactions (PCR) product specific for an H. pylori 26 000 MW surface protein was amplified from dental plaque samples from five of 12 (42%) cats and from the faeces of four of five (80%) cats studied. Analyses of serum and mucosal secretions by enzyme‐linked immunosorbent assay (ELISA) revealed an H. pylori‐specific immunoglobulin G (IgG) response, and elevated IgA anti‐H. pylori antibody levels in salivary and local gastric secretions. Immunohistochemical analyses of gastric tissue revealed the presence of IgM+ B cells assembled into multiple lymphoid follicles surrounded by clusters of CD4+ and CD8+ T cells. The lamina propria also contained single cells or aggregates of IgA+ and IgM+ B cells. These observations show that H. pylori can be identified in feline mucosal secretions, and that a localized IgA immune response develops in gastric tissue of H. pylori‐infected cats. The findings suggest a zoonotic risk from exposure to personnel handling H. pylori‐infected cats in vivaria.
Preparation of monoclonal anti‐porcine CD3 antibodies and preliminary characterization of porcine T lymphocytes
Authors: YANG H. , OURA C. A. L. , KIRKHAM P. A. , PARKHOUSE R. M. E.
Published: November 2003

The CD3–T‐cell receptor complex is the clonotypic surface structure by which T lymphocytes recognize foreign antigens and are subsequently activated. Because of the low immunogenicity of the CD3 molecules, anti‐CD3 monoclonal antibodies (mAb) are difficult to prepare and have not been available in several species. Following isolation of porcine CD3, 14 anti‐porcine CD3 mAb were prepared, which define six groups of CD3‐ε epitopes, coprecipitate two types of TCR and reveal considerable heterogeneity of CD3 expression amongst lymphocyte subpopulations. Thus, both CD3 positive and negative subpopulations of CD2 or CD8 positive cells were found in the blood. The density of CD3 on CD2+ or CD8+ cells was relatively low and heterogeneous, whereas the CD2−, CD8− or MAC320+ T cells expressed CD3 at a higher and more homogeneous level. Finally, in the thymus, staining with anti‐CD3 resolved large thymocytes into two subsets: one expressing a high level of CD3 and the other being negative. In contrast, small thymocytes expressed CD3 at a low and more homogeneous level. Immunohistological studies confirmed the presence of clearly detectable CD3 in thymus medulla and the T‐cell regions of peripheral lymphoid tissues. Most of the mAb were mitogenic, when presented to peripheral blood mononuclear cells in immobilized form. The anti‐CD3 mAb also induced redirected cytotoxicity which was shown to be Fc receptor dependent.
Expansion of cytotoxic CD8+ CD28− T cells in healthy ageing people, including centenarians
Authors: FAGNONI F. F. , VESCOVINI R. , MAZZOLA M. , BOLOGNA G. , NIGRO E. , LAVAGETTO G. , FRANCESCHI C. , PASSERI M. , SANSONI P.
Published: November 2003

Ageing is associated with complex remodelling in the phenotypic and functional profiles of T lymphocytes. We investigated whether expression of CD28 antigen on T cells is conserved throughout adulthood and ageing in humans. For this purpose we analysed T cells obtained from peripheral blood of 102 healthy people of ages ranging from 20 to 105 years. We found an age‐related increase of CD28− T cells in percentage and absolute number, predominantly among CD8+ T cells. CD28− T cells from aged donors analysed by flow cytometry appeared as resting cells (not expressing CD25, CD38, CD69, CD71, DR), bearing markers of cytotoxic activity (CD11b and CD57) and with a phenotype compatible with ‘memory’ cells (up‐regulated CD2 and CD11a; CD62L absent). At the functional level, freshly isolated purified CD28−CD8+ T cells showed high anti‐CD3 redirected cytotoxic activity against Fc‐bearing P815 cells. The same activity tested on freshly isolated bulk T lymphocytes was significantly augmented with age. We found a positive correlation between age, number of CD8+ CD28− T cells and anti‐CD3 redirected cytotoxicity by freshly isolated T cells. These data suggest that an activation of unknown nature within the cytotoxic arm of the immune system occurs with age. We speculate that these cytotoxic T lymphocytes (CTL) in vivo may constitute armed effector cells for immediate killing of targets bearing peptides from pathogens of intracellular origin.
Synthetic peptides non‐covalently bound to bacterial hsp 70 elicit peptide‐specific T‐cell responses in vivo
Authors: ROMÁN E. , MORENO C.
Published: November 2003

We have examined the immunogenicity of complexes formed by non‐covalent association of a synthetic peptide corresponding to influenza A virus nucleoprotein, residues 206–229 (pNP) and Mycobacterium tuberculosis heat‐shock protein 70 (hsp 70). One or two injections of these complexes given to BALB/c mice without any additional adjuvant, were capable of eliciting very strong peptide‐specific proliferative T‐cell responses in the spleen. These responses were dependent on the stability of the complex since immunogenicity was lost when dissociated with ATP prior to immunization. T‐cell responses to hsp 70 were easily generated by immunization with the purified chaperone alone, either after primary or secondary immunization. Injection of pNP–hsp 70 complexes, however, although generating good primary responses, resulted in very much decreased proliferative responses to the hsp 70.
Expression and modulation of C5a receptor (CD88) on skin dendritic cells. Chemotactic effect of C5a on skin migratory dendritic cells
Authors: MORELLI A. , LARREGINA A. , CHULUYÁN E. , KOLKOWSKI E. , FAINBOIM L.
Published: November 2003

Although it is known that dendritic cells (DC) migrate in response to inflammatory stimuli, there is little information about the expression of receptors for chemotactic factors on DC. The present study has demonstrated by double immunostaining and flow cytometry of Langerhan's cell (LC)‐enriched epidermal cell suspensions that a small subpopulation (5–6%) of epidermal resident LC (rLC) expresses receptors for C5a (C5aR). Epidermal rLC positive for C5aR show a round‐shape morphology, were located next to the basement membrane, and express HLA‐DR molecules higher than C5aR negative rLC. These observations suggest that rLC would express C5aR as part of their process of maturation during tissue trafficking. To investigate whether epidermal LC up‐regulate C5aR along their differentiation pathway, LC were differentiated in vitro after culture in epidermal cell suspensions supplemented with granulocyte–macrophage colony‐stimulating factor (GM‐CSF). As a result, in vitro differentiated LC increased the expression of C5aR up to 69% of the DC population. In accordance with this observation, interdigitating DC of secondary lymphoid organs (lymph node and tonsil) also expressed C5aR. Migratory CD1a positive DC that spontaneously migrated out of dermal or split‐skin organ explants were also positive for C5aR and were used for chemotaxis and chemokinesis assays in response to human recombinant C5a (rC5a). Optimum migration to rC5a was observed at 10−8 M with a sigmoidal dose–response curve. Checkboard analysis demonstrated that locomotion in response to rC5a was chemotaxis and not chemokinesis.
Expression and function of α4/β7 integrin on human natural killer cells
Authors: PÉREZ‐VILLAR J. J. , ZAPATA J. M. , MELERO I. , POSTIGO A. , SÁNCHEZ‐MADRID F. , LÓPEZ‐BOTET M.
Published: November 2003

In this report, we have analysed the expression and function of the α4/β7 heterodimer in human natural killer (NK) cells. The expression of α4β7 is induced in NK cells upon activation, as the anti‐α4β7 ACT‐1 monoclonal antibody (mAb) faintly stained a minority of peripheral blood NK cells, whereas it strongly reacted with in vitro long‐term interleukin‐2 (IL‐2)‐activated NK cells. Incubation with ACT‐1 or its F(ab′)2 fragments induced a strong homotypic adhesion of NK cells, comparable to that stimulated by the anti‐α4 HP1/7 mAb. Cell–cell interaction induced by the ACT‐1 mAb was only prevented by another anti‐α4 mAb (HP2/1) that recognizes a different epitope. In α4β7‐mediated cell aggregation, the α4β7 heterodimer was redistributed to intercellular contact sites, thus, suggesting a direct involvement of this integrin in the formation of cell clusters. In NK cells attached to Fibronectin (FN38) or vascular cell adhesion molecule‐1 (VCAM‐1), both α4β7 and α4β1 integrins were redistributed at the ventral cellular membrane forming discrete contact sites. The ACT‐1 mAb only partially blocked NK cell binding to FN38, but in combination with the anti‐β1 mAb LIA1/2, NK cell binding to FN38 was completely inhibited. In contrast, ACT‐1 did not modify NK cell adhesion to VCAM‐1, thus supporting the theory that the α4β7 binding sites for both ligands appear to be different. Our results indicate that upon IL‐2‐activation, expression of functional α4/β7 integrin is induced on NK cells, potentially participating in their interaction with both extracellular matrix and endothelial cells.