bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of nitric oxide and prostaglandins in the potentiating effects of calcitonin gene‐related peptide on lipopolysaccharide‐induced interleukin‐6 release from mouse peritoneal macrophages
Authors: TANG  , HAN  , WANG
Published: March 2003

Previous data from our laboratory have shown that calcitonin gene‐related peptide (CGRP) has a potentiating effect on lipopolysaccharide‐(LPS) induced interleukin‐6 (IL‐6) release from mouse macrophages. However, the mechanism of this effect was not clear. Since the nitric oxide (NO) and prostaglandins (PGs) induced by LPS might modulate IL‐6 release, we examined whether NO and PGs were also involved in the potentiating effect of rat CGRP (rCGRP) on LPS‐induced IL‐6 release from mouse macrophages. The IL‐6 level in the medium was measured by enzyme‐linked immunosorbent assay. Accumulation of NO was assessed by measuring the presence of nitrite by the Greiss reaction. PGI2 was assessed by measuring the formation of 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α) by radioimmunoassay. The results showed that the potentiating effect of rCGRP (0·1 nm) on LPS‐induced IL‐6 release was significantly inhibited by either 100 μm NG‐monomethyl‐l‐arginine acetate (l‐NMMA; an inhibitor of NO synthase) or 10 μm indomethacin (an inhibitor of cyclo‐oxygenase). The LPS‐induced NO and PGI2 production from these cells was increased significantly by rCGRP at 0·01–10 nm in a concentration‐dependent manner, which was blocked by l‐NMMA and indomethacin. These results suggest that rCGRP enhances the NO production elicited by LPS and subsequently increases the PGs production which is involved in the potentiating effect of rCGRP on LPS‐induced IL‐6 release from the peritoneal macrophages in the mouse.
The safety and longevity of DNA vaccines for fish
Authors: KANELLOS  , SYLVESTER  , AMBALI  , HOWARD  , RUSSELL
Published: March 2003

A plasmid that contained the cytomegalovirus (CMV)‐promoter‐driven lacZ reporter gene (pCMV‐lacZ) remained in the epaxial muscle of five of eight goldfish as covalently closed circles, the most functional form of plasmid, for at least 70 days at 22°. It was not present in the gills or elsewhere by polymerase chain reaction and was not integrated. Its expressed protein, Escherichia coliβ‐galactosidase (β‐gal), which was in the injected myofibres, was detected in all the fish at 4–21 days and in about half the fish from 28 days until the end of the experiment at 70 days. The numbers of cells that secreted antibody to β‐gal in the kidney peaked at 14 days. Serum antibody and proliferating kidney cells to β‐gal were in all fish from 14 days with a plateau of the responses from 21 days onwards. The plasmid did not induce autoimmune‐like antibodies to itself or to single‐ or double‐stranded salmon testis DNA. Plasmids can therefore induce long‐term foreign protein expression whilst inducing humoral and cell‐mediated immunity without autoimmunity or integration in goldfish.
Calcium‐independent haemolysis via the lectin pathway of complement activation in the guinea‐pig and other species*
Authors: Zhang  , Suankratay  , Zhang  , Jones  , Lint  , Gewurz
Published: March 2003

We previously reported that complement‐dependent haemolysis of sheep erythrocytes (E) coated with mannan (M) and sensitized with human mannan‐binding lectin (MBL) via the lectin pathway in man occurs in Mg‐EGTA and requires alternative pathway amplification. Calcium was required for MBL binding to E‐M, but once the E‐M‐MBL intermediate was formed, MBL was retained and haemolysis occurred in the absence of calcium. Comparable or greater lectin pathway haemolysis in the absence of calcium was observed upon incubation of E‐M‐MBL in guinea‐pig, rat, dog and pig sera, and was further investigated in the guinea‐pig, in which titres were much higher (~14‐fold) than in man, and in contrast to humans, greater than classical pathway haemolytic activity. As in human serum, no lysis was observed in C4‐ or C2‐deficient guinea‐pig serum until purified C4 or C2, respectively, were restored. However, lectin pathway haemolytic activity in the guinea‐pig did not require the alternative pathway. Removal (>98%) of factor D activity by three sequential passages through Sephadex G‐75, resulting in serum which retained a normal classical pathway but no alternative pathway haemolytic activity, did not reduce the ability of guinea‐pig serum to mediate haemolysis via the lectin pathway. Further, the C3‐convertase formed via the lectin pathway (E‐M‐MBL‐C4,2) lysed in C2‐deficient guinea‐pig but not human serum chelated with EDTA, a condition which precludes alternative pathway amplification. Thus, lectin pathway haemolysis occurs efficiently in guinea‐pig serum, in the absence of calcium and without requirement for alternative pathway amplification. The guinea‐pig provides a model for studying the assembly and haemolytic function of a lectin pathway which contrasts with the lectin pathway of man, and allows for comparisons that may help clarify the role of this pathway in complement biology.
Glomerular overexpression and increased tyrosine phosphorylation of focal adhesion kinase p125FAK in lupus‐prone MRL/MP‐lpr/lpr mice
Authors: Morino  , Matsumoto  , Ueki  , Mimura  , Hamasaki  , Kanda  , Naruse  , Yazaki  , Nojima
Published: March 2003

Much progress has been made in understanding how mammalian cells receive a diverse array of external stimuli and convert them into intracellular biochemical signals. Such efforts have identified a large number of signalling molecules. However, our knowledge is limited as to their pathophysiological role in particular diseases. We demonstrate herein that an integrin‐linked signalling molecule, focal adhesion kinase p125FAK (FAK), is overexpressed in glomeruli of lupus‐prone MRL/MP‐lpr/lpr (MRL‐lpr) mouse as compared to its congeneic MRL‐+/+ strain. Increased expression was specifically demonstrated in glomeruli but not in other tissues examined. The overexpression was observed in 16‐week‐old MRL‐lpr mice with active nephritis, as well as in younger animals at 4 weeks of age. Thus, the upregulation of FAK clearly preceded the clinical onset of nephritis. FAK in MRL‐lpr glomeruli is highly tyrosine phosphorylated and is associated with adapter protein Grb2. Previous in vitro studies have shown that the association of FAK/Grb2 links cell adhesion to the Ras pathway, which ultimately stimulates mitogen‐activated protein (MAP) kinase, an important regulator of cell proliferation. In accordance, we observed constitutive MAP kinase activation in MRL‐lpr glomeruli. Our findings suggest that signalling pathways involving FAK are activated in MRL‐lpr glomeruli, and are likely to play a role in the development and progression of autoimmune‐mediated murine nephritis.
Impaired contact hypersensitivity to trinitrochlorobenzene in interleukin‐4‐deficient mice
Authors: Dieli  , Sireci  , Scirè  , Salerno  , Bellavia
Published: March 2003

We have examined the role of endogenously produced interleukin‐4 (IL‐4) in the contact hypersensitivity (CH) reaction to the haptene trinitrochlorobenzene (TNCB). The CH reaction was abolished in IL‐4 genetically deficient mice (IL‐4 KO), when compared to wild‐type (wt) mice. The CH reaction was restored by treatment with IL‐4 and further analysis revealed that IL‐4 exerted its action both at the induction and effector stages of the CH reaction. Despite failure to develop a CH reaction, IL‐4 KO mice developed a T helper type 1 (Th1) response to TNCB, in terms of lymphokine production in vitro. Furthermore, the number of Vγ3+ cells accumulating in the lymph nodes of TNCB‐immune IL‐4 KO mice was normal. The recruitment of mononuclear cells and vascular leakage at the challenge site were consistently reduced in IL‐4 KO mice and were restored by injection of IL‐4. This suggests that IL‐4 acts as a proinflammatory mediator in CH, perhaps favouring the accumulation of mononuclear cells at the site of inflammation. Among Th2‐type cytokines, IL‐13, but not IL‐10, was shown to restore the CH reaction to TNCB in IL‐4 KO mice. However, IL‐4 KO mice developed a normal CH response to oxazolone, indicating that IL‐4 was required for the CH reaction to TNCB, but not for that to oxazolone.
Nitric oxide and macrophage antiviral extrinsic activity
Authors: Benencia  , Courreges
Published: March 2003

In this study we evaluated the relationship between nitric oxide (NO) and macrophage antiviral extrinsic activity. Macrophages activated by intraperitoneal injection of herpes simplex virus‐2 (HSV‐2), showed both extrinsic antiviral activity and high nitrite production in contrast to non‐activated, resident macrophages. The extrinsic antiviral activity was observed in cultures of Vero cells infected with HSV‐1 and HSV‐2. The NO inhibitor N‐monomethyl‐l‐arginine acetate (l‐NMA) impaired the antiviral activity of HSV‐elicited macrophages. The effect was dose dependent and correlated with a reduction of nitrite in the culture media. The effect of l‐NMA was reversed by the addition of l‐arginine. These data indicate that NO could be responsible for the described activity. Furthermore, l‐NMA treatment resulted in the aggravation of HSV‐1‐induced keratitis in the mouse model, supporting a defensive role of NO in the pathogenesis of HSV‐1 corneal infection.
Adhesion of human mast cells to extracellular matrix provides a co‐stimulatory signal for cytokine production
Authors: Krüger‐Krasagakes  , Grützkau  , Krasagakis  , Hoffmann  , Henz
Published: March 2003

Engagement of integrin receptors during cell adhesion leads to changes in the morphology and the state of activation of cells. We therefore examined whether mast cell adhesion to extracellular matrix proteins affects the synthesis and release of various proinflammatory cytokines. Cells of the human mast cell line HMC‐1 were added to fibronectin (FN)‐, vitronectin (VN)‐ or, as a control, bovine serum albumin (BSA)‐coated wells and were stimulated with phorbol 12‐myristate 13‐acetate (PMA) and/or calcium ionophore A23187 (ionophore). Cytokine production was evaluated using semiquantitative reverse transcription–polymerase chain reaction (RT–PCR) analysis of cell extracts and enzyme‐linked immunosorbent assay (ELISA) analysis of cell supernatants. After a 4‐hr incubation, mRNA expression of interleukin (IL)‐8 (and weakly of IL‐6) was up‐regulated in matrix‐adherent cells, with further increase in the presence of PMA and/or ionophore, compared with unstimulated cells. High‐level de novo expression of IL‐3 and of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) was observed mainly in matrix‐adherent cells. These changes were paralleled by the secretory pattern of HMC‐1 cells after a 24‐hr stimulation. Unstimulated cells adherent to FN or VN had already released small amounts of IL‐8, and both VN‐ and FN‐adherent cells produced, almost invariably, a higher level of cytokines than BSA‐exposed cells after additional stimulation. These results show that mast cell adhesion to matrix proteins by itself has only selected and minor effects, but additional activation of mast cells by secretory stimuli causes significantly enhanced cytokine gene expression and secretion, suggesting that mast cells are far more active in their natural tissue environment than hitherto suggested from data in suspension cultures.
Replenishment of RANTES mRNA expression in activated eosinophils fromatopic asthmatics
Authors: Velazquez J. R. , Lacy P. , Moqbel R.
Published: March 2003

Eosinophils have been shown to express the gene encoding regulated upon activation, normal T‐cell expressed and secreted (RANTES), a potent eosinophilotactic chemokine. RANTES protein expression in eosinophils has previously been shown to be up‐regulated by a number of agonists, including complement‐dependent factors (C3b/iC3b) and interferon‐γ (IFN‐γ). We hypothesized that gene expression of RANTES is regulated in these cells by eosinophil‐specific agonists. We analysed RANTES mRNA expression by reverse‐transcription polymerase chain reaction (RT‐PCR) in human peripheral blood eosinophils obtained from mild atopic asthmatics following stimulation over time. In resting eosinophils, a low level of RANTES mRNA was found to be constitutively expressed in all the atopic donors tested in this study (n = 6). Following stimulation with C3b/iC3b (serum‐coated surfaces), eosinophils released measurable levels of RANTES, while sustained transcript expression was detected for up to 24 hr of stimulation. In contrast, IFN‐γ (5 ng/ml) transiently and significantly (P < 0·05, n = 3) depleted relative amounts of RANTES PCR product (compared with β2‐microglobulin) after 1–4 hr of stimulation. RANTES transcript was again detectable after 24 hr of IFN‐γ incubation, suggesting that the pool of RANTES mRNA had been replenished. Other eosinophil‐active cytokines, interleukin‐3 (IL‐3), IL‐4, IL‐5 and granulocyte–macrophage colony‐stimulating factor, did not appear to modulate RANTES mRNA expression after 1 hr of incubation. The effect of IFN‐γ on RANTES mRNA was reversed by cycloheximide, suggesting that IFN‐γ may act by increasing the rate of translation of RANTES mRNA. These findings indicate that IFN‐γ may induce a rapid and transient effect on the translation and replenishment of RANTES mRNA in eosinophils. This novel observation supports the notion that eosinophils have the potential to replenish their stored and released bioactive proteins.
Interactions between hormone‐mediated and vaccine‐mediated immunotherapy for pulmonary tuberculosis in BALB/c mice
Authors: Hernandez‐Pando R. , Pavon L. , Orozco E. H. , Rangel J. , Rook G. A. W.
Published: March 2003

Problems of logistics, compliance and drug resistance point to an urgent need for immunotherapeutic strategies capable of shortening the current 6‐month chemotherapy regimens used to treat tuberculosis, or of supplementing ineffective therapy. In this study we sought to define the mechanism of action of two immunotherapies, both of which have previously been shown to prolong survival. Secondly, we wished to identify any clinically useful synergy between these therapies. In BALB/c mice infected via the trachea with Mycobacterium tuberculosis H37Rv there is an initial phase of partial resistance dominated by type 1 cytokines plus tumour necrosis factor‐α (TNF‐α) and interleukin‐1 (IL‐1), followed by a phase of progressive disease. This progressive phase is accompanied by increasing expression of IL‐4, and diminished expression of IL‐1 and TNF‐α. Animals in this late progressive phase of the disease (day 60) were treated with two injections (day 60 and day 90) of 0·1 or 1·0 mg of heat‐killed Mycobacterium vaccae, or with 3β,17β‐androstenediol (AED; 25 µg subcutaneously three times/week), or with both therapies. We show here using four techniques in parallel (morphometry, immunohistochemistry with automated cell counting, semiquantitative reverse transcription–polymerase chain reaction and enzyme‐linked immunosorbent assays of cytokines in lung extracts) that treatment with M. vaccae causes a switch back towards a type 1 cytokine profile, restoration of expression of IL‐1α and TNF‐α, and a switch from pneumonia to granuloma. This is very similar to the changes previously seen after treatment with AED. However, there was no evidence for synergy between M. vaccae and AED.
Poly‐guanosine motifs costimulate antigen‐reactive CD8 T cells while bacterial CpG‐DNA affect T‐cell activation via antigen‐presenting cell‐derived cytokines
Authors: Lipford G. B. , Bendigs S. , Heeg K. , Wagner H.
Published: March 2003

Pathogen‐derived pattern recognition ligands like lipopolysaccharide (LPS) and bacterial cytidine–guanosine (CpG)‐DNA not only activate dendritic cells and macrophages but are also mitogenic for B cells. Less clear are the claimed effects of CpG‐DNA on T cells, which range from direct activation, costimulation, or indirect transient activation via antigen‐presenting cell (APC)‐derived interferon type I (IFN type I). Here we demonstrate that CpG‐DNA sequence specifically triggers macrophages to produce IFN type I, interleukin (IL)‐12, IL‐6 and tumour necrosis factor (TNF), but lacks the ability to directly costimulate T cells. Strikingly, poly‐guanosine (poly‐G) extensions to CpG‐containing oligonucleotides (ODN) abolished the macrophage stimulatory potential yet generated T‐cell costimulatory activities. In fact, independently of CpG‐motifs, poly‐G‐ODN displayed the ability to costimulate T cells. Costimulation was operative on CD8 T cells but not CD4 T cells. Poly‐G‐mediated costimulation resulted in IL‐2‐driven T‐cell proliferation and induced cytolytic T cells. Overall the data imply that poly‐G motifs costimulate antigen reactive CD8 T cells, while CpG‐DNA motifs fail to do so but may affect T‐cell activation via APC derived cytokines such as IFN type I.
Structure/function studies of human decay‐accelerating factor
Authors: Brodbeck W. G. , Kuttner‐Kondo L. , Mold C. , Medof M. E.
Published: March 2003

The decay‐accelerating factor (DAF) contains four complement control protein repeats (CCPs) with a single N‐linked glycan positioned between CCPs 1 and 2. In previous studies we found that the classical pathway regulatory activity of DAF resides in CCPs 2 and 3 while its alternative pathway regulatory activity resides in CCPs 2, 3 and 4. Molecular modelling of the protein predicted that a positively charged surface area on CCPs 2 and 3 (including KKK125–127) and nearby exposed hydrophobic residues (L147F148) on CCP3 may function as ligand‐binding sites. To assess the roles of the N‐linked glycan and the above two sets of amino acids in the function of DAF, we mutated N61 to Q, KKK125–127 to TTT and L147F148 to SS. Following expression of the mutated cDNAs in Chinese hamster ovary cells, the glycosylphosphatidylinositol (GPI)‐anchored mutant proteins were affinity purified and their functions were assessed. In initial assays, the proteins were incorporated into sheep and rabbit erythrocytes and the effects of the mutations on regulation of classical and alternative C3 convertase activity were quantified by measuring C3b deposition. Since DAF also functions on C5 convertases, comparative haemolytic assays of cells bearing each mutant protein were performed. Finally, to establish if spatial orientation between DAF and the convertases on the cell surface played any role in the observed effects, fluid‐phase C3a generation assays were performed. All three assays gave equivalent results and showed that the N‐linked glycan of DAF is not involved in its regulatory function; that L147F148 in a hydrophobic area of CCP3 is essential in both classical and alternative pathway C3 convertase regulation; and that KKK125–127 in the positively charged pocket between CCPs 2 and 3 is necessary for the regulatory activity of DAF on the alternative pathway C3 convertase but plays a lesser role in its activity on the classical pathway enzyme.
Interleukin‐8 fails to induce human immunodeficiency virus‐1 expression in chronically infected promonocytic U1 cells but differentially modulates induction by proinflammatory cytokines
Authors: Tiemessen C. T. , Kilroe B. , Martin D. J.
Published: March 2003

This study addresses the role of interleukin (IL)‐8, a CXC‐chemokine, the level of which is reported to be raised in the peripheral circulation of human immunodeficiency virus‐1 (HIV‐1)‐infected individuals, during the induction of HIV‐1 expression from latency and during cytokine‐mediated HIV‐1 up‐regulation. IL‐8 at the higher concentrations tested (≥ 100 ng/ml) was unable to induce HIV‐1 expression in the chronically infected promonocytic U1 cell line, as measured by p24 antigen enzyme‐linked immunosorbent assay (ELISA), whereas at lower concentrations of 1 and 10 ng/ml, constitutive HIV‐1 expression was only marginally reduced. HIV‐1 replication in acutely infected U937 cells was also significantly reduced by IL‐8. The potent up‐regulation of HIV‐1 expression in U1 cells by tumour necrosis factor‐α (TNF‐α) remained unaffected by the addition of IL‐8. HIV‐1 induction by IL‐1β, IL‐6 and TNF‐β, cytokines grouped here as intermediate HIV‐1 inducers, was suppressed by IL‐8 at concentrations of 1 and 10 ng/ml. However, IL‐8 at 100 ng/ml did not significantly alter the effect of IL‐1β, synergized with IL‐6 in enhancing, and marginally suppressed TNF‐β‐induced HIV‐1 expression. IL‐8 suppressed granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and enhanced interferon‐γ (IFN‐γ)‐induced HIV‐1 expression in a dose‐dependent manner. Pretreatment of U1 cells with IL‐8 did not alter the IL‐8‐mediated effects on cytokine‐induced HIV‐1 expression, suggesting that this chemokine exerts its effect at the time of HIV‐1 induction or at a postinduction stage. Furthermore, IL‐8 was itself induced by cytokines that up‐regulate HIV‐1 expression in U1 cells and the levels produced correlated directly with the levels of p24 antigen produced, suggesting common pathways for cytokine induction of both HIV‐1 and IL‐8. These results show that IL‐8, typically a non‐inducer, can differentially modulate HIV‐1 expression in U1 cells and that this is dependent on the inducing cytokine and on the concentration of IL‐8.
Soluble isoforms of CEACAM1 containing the A2 domain: increased serum levels in patients with obstructive jaundice and differences in 3‐fucosyl‐N‐acetyl‐lactosamine moiety
Authors: Dráberová L. , Černá H. , Brodská H. , Boubelík M. , Watt S. M. , Stanners C. P. , Dráber P.
Published: March 2003

CEACAM1 (biliary glycoprotein or CD66a) is a member of the carcinoembryonic antigen (CEA) subgroup of the CEA family. Eleven RNA isoforms derived from the splicing of a single CEACAM1 gene have been described. Some of the CEACAM1 isoforms have been recognized by the CD66 antibodies in T and B lymphocytes, natural killer cells, granulocytes and epithelial cells in several human tissues. Although it is also present in soluble form in bile and serum, and elevated levels have been found in the serum of patients with liver diseases, it is not known which isoforms are primarily involved. In order to learn more about the distribution and properties of particular CEACAM1 isoforms, we have prepared a monoclonal antibody specific for the A2 domain of CEACAM1, designated TEC‐11. This antibody does not cross‐react with other members of the CEA family. Immunoblotting analysis revealed that the TEC‐11 epitope was present in all cell types expressing CEACAM1 containing the A2 domain [CEACAM1(A2)], including granulocytes (160 000 MW isoform) and sperm cells (140 000 MW isoform). A 115 000 MW isoform of CEACAM1(A2) was present in human serum, bile, saliva and seminal fluid. Human bile, saliva and seminal fluid also contained the 160 000 MW CEACAM1(A2) isoform. Significantly higher serum levels of the 115 000 MW CEACAM1(A2) isoform were detected in patients with obstructive jaundice. The 160 000 MW isoform of CEACAM1(A2) in bile, but not a 115 000 MW isoform in serum and bile, carried the 3‐fucosyl‐N‐acetyl‐lactosamine moiety. The combined data indicate that various isoforms of CEACAM1(A2) are present in different body fluids where they could take part in different CEACAM1‐mediated functions.
The C5a receptor is expressed in normal renal proximal tubular but not in normal pulmonary or hepatic epithelial cells
Authors: Fayyazi A. , Scheel O. , Werfel T. , Schweyer S. , Oppermann M. , Götze O. , Radzun H. J. , Zwirner J.
Published: March 2003

C5a, a 74 amino acid peptide cleaved from the complement protein C5, is an extremely potent anaphylatoxin. Expression of the receptor for the anaphylatoxin C5a (C5aR) has been thought to be restricted to cells of myeloid origin. However, recent evidence suggests that the C5aR is also expressed in hepatocytes as well as in pulmonary epithelial, endothelial and smooth muscle cells. In the present study, we investigated the tissue distribution of C5aR by immunohistochemistry in normal human lung, liver, intestine and kidney using well‐defined monoclonal antibodies (mAbs) directed against the extracellular N‐terminus of the receptor. In all tissues examined, macrophages displayed an abundant expression of C5aR protein. However, in the normal human lung, C5aR expression was not detectable in bronchial and alveolar epithelial cells or in vascular smooth muscle or endothelial cells. In the normal human liver, no C5aR protein was detected in hepatocytes, whereas Kupffer cells strongly expressed the C5aR. In normal human kidney, the C5aR was detectable only in proximal tubular cells. C5aR gene transcription in Kupffer cells and proximal tubular cells was confirmed by in situ hybridization. Thus, our results point to an as yet unknown role of the C5aR in normal renal physiology. In the normal lung and liver, however, previous evidence for the ubiquitous expression of C5aR in epithelial, endothelial and smooth muscle cells in situ should be re‐evaluated.
Vγ1+γδ T cells play protective roles at an early phase of murine cytomegalovirus infection through production of interferon‐γ
Authors: Ninomiya T. , Takimoto H. , Matsuzaki G. , Hamano S. , Yoshida H. , Yoshikai Y. , Kimura G. , Nomoto K.
Published: March 2003

Cytomegalovirus (CMV) causes severe opportunistic infection in immunocompromised hosts. The importance of conventional αβ T cells in protection against CMV infection has been well documented. However, the role of the second T‐cell population (which express the γδ T‐cell receptor) in CMV infection is not known. In the present study, we analysed the function and protective role of γδ T cells in a murine cytomegalovirus (MCMV) infection model. After intraperitoneal infection with MCMV, the number of γδ T cells increased in the liver and peritoneal cavity from day 3, and reached a peak on day 5. The γδ T cells showed an activated T‐cell phenotype and predominantly expressed Vγ1, which is known to be expressed by heat‐shock protein 65 (hsp 65)‐specific γδ T cells. Analysis of cytokine expression demonstrated that the MCMV‐induced γδ T cells expressed interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) but not interleukin‐4 (IL‐4), implying their participation in the cell‐mediated immune response against MCMV. Depletion of γδ T cells by anti‐T‐cell receptor (TCR) γδ monoclonal antibody (mAb) treatment resulted in significant increase of virus titre and decrease of IFN‐γ in the liver on day 3 after MCMV infection, which further supports the importance of γδ T cells in early protection against infection. Finally, the MCMV‐induced γδ T cells produced IFN‐γin vitro in response to hsp 65. Our results suggest that γδ T cells participate in early protection against MCMV infection through recognition of hsp 65 and production of IFN‐γ.
T‐cell apoptosis and differential human leucocyte antigen class II expression in human thymus
Authors: Douek D. C. , Altmann D. M.
Published: March 2003

Relatively little is known of the details of human leucocyte antigen (HLA) expression and thymocyte selection in human thymus. In both humans and mice major histocompatibility complex (MHC) molecules have been described which show a highly restricted thymic expression. Such patterns may offer clues about cellular interactions in thymic selection because transgenic mice with MHC expression targeted to specific thymic sites show altered T‐cell receptor (TCR) repertoire selection. We have analysed human thymic HLA class II expression, relating the expression pattern to sites of thymocyte apoptosis. While HLA‐DQ is poorly expressed by most peripheral antigen‐presenting cells (APC), thymus stains strongly for HLA‐DQ as well as for HLA‐DR. HLA‐DM is abundant in medulla but weakly expressed by cortical cells. Class II expression in Hassall’s corpuscles (HC) is unusual in several respects: we have previously shown them to be encircled by HLA‐DO+ epithelial cells and here further demonstrate that HC are negative for HLA‐DR and HLA‐DP, but often positive for HLA‐DQ and HLA‐DM. Transcriptional control of HLA class II products at this site is thus unlike cells that have previously been studied. Apoptotic thymocytes are restricted to the cortex and the corticomedullary junction. However, a minority of apoptotic cells are visible in the medulla, these being found in the HLA‐DQ positive HC. The apoptotic thymocytes in HC can be CD4+ single positive (SP), CD8+ SP or CD4+CD8+ double‐positive (DP). This study thus shows that the HC within human thymic medulla are noteworthy both for their unusual hierarchy of HLA class II expression and because they are the only medullary site of thymocyte apoptosis. We propose that HC are a site at which mature thymocytes receive activation/tolerization signals from peptides reprocessed from apoptotic cells. The differential HLA transcriptional control at this site may indicate that specific T‐cell subpopulations are affected.
Immunostimulatory bacterial DNA sequences activate dendritic cells and promote priming and differentiation of CD8+ T cells
Authors: Tascon R. E. , Ragno S. , Lowrie D. B. , Colston M. J.
Published: March 2003

CD8+ T lymphocytes producing high levels of interferon‐γ (IFN‐γ) and expressing antigen specific cytotoxic activity are effectively induced after plasmid DNA vaccination and mediate protection against several intracellular micro‐organisms. Recent evidence suggests that the priming of CD8+ T‐cell responses following DNA injection involves antigen presentation mediated by dendritic cells. Here, we show that bacterial DNA and synthetic oligonucleotides containing dinucleotide (CpG) motifs activate cytokine expression in dendritic cells and modulate in vivo CD8+ T‐cell priming and differentiation.
Interaction of merosin (laminin 2) with very late activation antigen‐6 is necessary for the survival of CD4+ CD8+ immature thymocytes
Authors: Iwao M. , Fukada S. , Harada T. , Tsujikawa K. , Yagita H. , Hiramine C. , Miyagoe Y. , Takeda S. , Yamamoto H.
Published: March 2003

The laminin α2‐chain is a component of merosin, a member of the laminin family molecules, which is mainly expressed in the basement membranes of striated muscle. It is known that laminin α2 gene (lama2) null mutant mice (dy3k/dy3k) exhibit congenital muscular dystrophy (CMD). Because the laminin α2‐chain is also expressed in the thymus, the role of merosin in the thymus was examined. In association with the onset of muscular dystrophy, CD4+ CD8+ double‐positive (DP) thymocytes disappear by apoptotic cell death, while CD4+ CD8– or CD4– CD8+ thymocytes remain. In order to study the mechanisms leading to the selective death of DP cells in the absence of merosin, the role of the interaction between very late activation antigen‐6 (VLA‐6), a candidate merosin ligand in the thymus, and merosin was examined. The in vitro survival of thymocytes from normal mice was maintained by the addition of either anti‐VLA‐6 monoclonal antibodies (mAbs) or merosin. Furthermore, when the normal thymocytes were cultured on thymic epithelial cell lines, viable DP cell recoveries on wild‐type epithelial cells were better than on cells from null mutant mice. The results suggest that DP cells are more sensitive to an uncharacterized apoptotic death signal, and that survival is supported by the interaction between VLA‐6 and merosin.
Up‐regulation of β‐chemokines and down‐modulation of CCR5 co‐receptors inhibit simian immunodeficiency virus transmission in non‐human primates
Authors: Lehner T. , Wang Y. , Cranage M. , Tao L. , Mitchell E. , Bravery C. , Doyle C. , Pratt K. , Hall G. , Dennis M. , Villinger L. , Bergmeier L.
Published: March 2003

A non‐cognate mechanism of protection against human immunodeficiency virus‐1 (HIV‐1) infection involves up‐regulation of β‐chemokines, which bind and may down‐modulate the CCR5 co‐receptors, thereby preventing transmission of M‐tropic HIV‐1. The objective of this investigation was to evaluate this mechanism in vivo in non‐human primates. Rhesus macaques were immunized by a modified targeted lymph nodes (TLN) route with recombinant simian immunodeficiency virus (SIV) glycoprotein 120 (gp120) and p27 in alum, and adsorbed recombinant granulocyte–macrophage colony‐stimulating factor (GM‐CSF) with either interleukin (IL)‐2 or IL‐4. Immunization induced significant increases in the concentrations of CD8 cell‐derived suppressor factor (CD8‐SF), regulated on activation normal T cells expressed and secreted (RANTES), macrophage inflammatory protein (MIP)‐1α and MIP‐1β, and down‐modulation of the proportion of cells expressing CCR5 (r = 0·737, P < 0·05). The macaques were then challenged with SIVmac 220 by the rectal mucosal route. The plasma SIVmac RNA showed a significant inverse correlation with the CD8‐SF or the concentration of the three β‐chemokines (r = 0·831 and 0·824, P < 0·01), but a positive correlation between the proportion of CCR5+ cells and SIVmac RNA (r = 0·613, P = 0·05). These results demonstrate for the first time in vivo that immunization up‐regulates β‐chemokines, which may down‐modulate CCR5 co‐receptors, and both functions are significantly correlated with the viral load. Hence, the non‐cognate β‐chemokine–CCR5 mechanism should be considered as complementary to specific immunity in vaccination against HIV.
Fas–FasL interaction modulates nitric oxide production in Trypanosoma cruzi‐infected mice
Authors: Martins Gislâine A. , Petkova Stefka B. , Machado Fabiana S. , Kitsis Richard N. , Weiss Louis M. , Wittner Murray , Tanowitz Herbert B. , Silva João S.
Published: March 2003

During acute Trypanosoma cruzi infection in mice, many leucocytes undergo apoptosis. Although apoptosis has been ascribed to increased levels of nitric oxide (NO) and Fas–FasL interaction, the importance of this phenomenon in modulating the host response against T. cruzi is unknown. Herein, the role of NO‐ and Fas–FasL‐induced apoptosis in modulating the immune response to T. cruzi was evaluated using mice deficient in Fas expression (MRL/MpJ‐Fas lpr) and inducible nitric oxide synthase (iNOS) knockout mice (iNOS–/–). The results showed that besides decreasing apoptosis induction after infection, impairment of the Fas–FasL interaction resulted in decreased NO production, as a consequence of enhanced T helper 2 (Th2) cytokine production. Differently, blockage of NO‐induced apoptosis resulted in uncontrolled cytokine production, rather than a biased Th2 cytokine pattern. Together, these results suggested that Fas and FasL‐induced apoptosis could be implied in modulation of the immune response against T. cruzi by interfering with cytokine and NO production during the acute phase of the infection.
Studies on transcriptional regulation of the mucosal T‐cell integrin αEβ7 (CD103)
Authors: Robinson Paul W. , Green Sally J. , Carter Christine , Coadwell John , Kilshaw Peter J.
Published: March 2003

Integrin αEβ7 is expressed almost exclusively by mucosal T cells and mucosal dendritic antigen‐presenting cells (APCs) and is thought to be induced locally by transforming growth factor‐β (TGF‐β). In mice, mRNA for the αE subunit was found to be abundant in mucosal T cells but absent from other tissues. Exposure of a T‐cell line to TGF‐β strongly up‐regulated αE mRNA levels within 30 min, and nuclear run‐on experiments established that regulation occurred at the level of transcription. The organization of the human αE gene and a very closely linked novel gene, ELG, was determined. The αE promoter was tested in T cells and fibroblasts and functioned equally well in both cell types and did not confer TGF‐β responsiveness. Regions of the promoter providing enhancer activity and phorbol 12‐myristate 13‐acetate (PMA) responsiveness were identified by deletion studies. DNAse 1 hypersensitivity analysis of 36 kb of the αE gene revealed one hypersensitive site, found only in αE+ cells, located near the transcription start points. These results show that, unlike the situation with other integrins, lineage specificity and cytokine responsiveness of αE transcription are not conferred by the proximal promoter. Specificity may depend on distant control elements that have not yet been identified.
Evidence of the extrathymic development of tyrosinase‐related protein‐2‐recognizing CD8+ T cells with low avidity
Authors: Harada, Mamoru , Yamada Hisakata , Tatsugami Katsunori , Nomoto Kikuo
Published: March 2003

The majority of the human tumour‐associated antigens characterized to date are derived from non–mutated self‐proteins. However, nothing is known about the development of autoreactive and tumour‐associated antigen‐recognizing T cells. Tyrosinase‐related protein (TRP)‐2 is a non‐mutated melanocyte differentiation antigen and TRP‐2‐recognizing CD8+ T cells are known to show responses to melanoma both in humans and mice. In addition, TRP‐2‐reactive T cells with low avidity have been suggested to be readily induced from the spleen cells of naïve mice. On the other hand, recent reports suggest that self antigen‐reactive CD8+ T cells can be positively selected in the periphery. In this study, we tested the possibility that TRP‐2‐reactive CD8+ T cells in naïve mice could develop via the extrathymic pathway. As a consequence, TRP‐2‐reactive CD8+ T cell precursors in naïve C57BL/6 mice were suggested to express both interleukin‐2 (IL‐2) receptor β chain (IL‐2Rβ) and CD44 molecules, in a manner similar to that of extrathymically developed T cells. Furthermore, IL‐2Rβ+ CD44+ CD8+ T cells were detected in the adult thymectomized and bone marrow‐reconstituted mice, and functional TRP‐2‐reactive T cells were generated from their spleen cells. Overall, these results suggest that low avidity CD8+ T cells recognizing TRP‐2 can be developed extrathymically.
Murine gammaherpesvirus‐68‐induced interleukin‐10 increases viral burden, but limits virus‐induced splenomegaly and leukocytosis
Authors: Peacock James W. , Bost Kenneth L.
Published: March 2003

Based on its genomic sequence and its pathogenesis, murine gammaherpesvirus‐68 (γHV‐68) has been established as a tractable model for the study of viral infections caused by the human gammaherpesviruses, Epstein–Barr virus or human herpesvirus‐8. Despite significant advances, the mechanisms responsible for γHV‐68‐induced alterations in the protective host response, and the accompanying virus‐induced leukocytosis, are not clear. In the present study, we questioned whether viral infection resulted in endogenous interleukin‐10 (IL‐10) production that might alter the host response. Infection of C57BL/6 mice resulted in increased IL‐10 expression, demonstrating that γHV‐68 could induce endogenous production of this cytokine. Infected C57BL/6 mice demonstrated the characteristic splenomegaly associated with this viral infection, however, we were surprised to discover that the splenomegaly was greater in syngeneic mice genetically deficient in IL‐10 (IL‐10−/−). These results strongly suggested that endogenously produced IL‐10 might serve to limit leukocytosis in wild‐type mice. Quantification of viral burden demonstrated a significant elevation in C57BL/6 versus IL‐10−/− mice, with increases in virus being observed in both the macrophage and B‐lymphocyte populations. The decreased viral load in syngeneic IL‐10−/− mice correlated with an increased expression of endogenous IL‐12, suggesting a mechanism of protection that was IL‐12 dependent. Taken together, these studies demonstrate a surprising dichotomy for endogenous IL‐10 production during γHV‐68 infection. While the lack of IL‐10 results in increased IL‐12 expression and a lower viral burden, IL‐10−/− mice also experience an increased leukocytosis.
Diacylglycerol kinase α activity promotes survival of CD4+ 8+ double positive cells during thymocyte development
Authors: Outram Susan V. , Crompton Tessa , Merida Isabel , Varas Alberto , Martinez‐A Carlos
Published: March 2003

The diacylglycerol kinases (DGK) form a family of isoenzymes that catalyse the conversion of diacylglycerol (DAG) to phosphatidic acid (PA), both powerful second messengers in the cell. DGKα is expressed in brain, peripheral T cells and thymocytes and has been shown to translocate to the nuclear matrix upon T‐cell receptor (TCR) engagement. Here, we show that high level expression of DGKα is induced following a signal transmitted through the pre‐TCR and the protein tyrosine kinase, lck. Activity of DGKα contributes to survival in CD4+ 8+ (DP) thymocytes as pharmacological inhibition of DGK activity results in death of this cell population both in cell suspension and thymic explants. DGKα promotes survival in these thymocytes through a Bcl‐regulated pathway. A consequence of inhibition of DGKα is the specific down‐regulation of Bcl‐xl, whereas in transgenic mice that over‐express Bcl‐2, death induced by the inhibitor is partially blocked. Thus we report a novel activity of DGKα in survival of thymocytes immediately after entry into the DP stage in development.
Age‐related telomere length dynamics in peripheral blood mononuclear cells of healthy cynomolgus monkeys measured by Flow FISH
Authors: Lee Won‐Woo , Nam Ki‐Hoan , Terao Keiji , Yoshikawa Yasuhiro
Published: March 2003

Telomere length is a good biomarker to study the cellular senescence as well as aging of an organism, because it regulates the replicative capacity of vertebrate somatic cells. To demonstrate age‐related telomere length dynamics in the peripheral blood mononuclear cells (PBMC) of the cynomolgus monkey, we introduced a novel method of measuring telomere length by fluorescence in situ hybridization with a Peptide Nucleic Acid (PNA) labelled probe and flow cytometry (Flow FISH). A highly significant correlation was observed between the intensity of telomere‐specific fluorescence by Flow FISH and telomere length by Southern blot analysis (R = 0·923, n = 22). The intensity of telomere fluorescence in PBMC significantly decreased with age in 55 monkeys aged from 0 to 34 years and this decrease corresponded to the loss of 62·7 base pairs per year (R = − 0·52, P < 0·00004). We also analysed the expression of naive cell‐associated markers, CD28, CD62L and CD45RA/CD62L in T lymphocytes of 47 cynomolgus monkeys. An age‐related increase in the CD28− subset was observed in CD8+ T lymphocytes in monkeys less than 11 years old and in CD4+ T lymphocytes in monkeys over 23 years old, respectively. The percentage of CD62L+ subsets was significantly decreased with age in both CD4+ (R = − 0·55) and CD8+ T lymphocytes (R = − 0·73). From the comparison of telomere length among PBMC, CD62L+ and CD62L− T lymphocytes, it was clearly evident that loss of naive subsets results in the shortening of telomere length in vivo. These results show that this method can be applicable to studying the turnover and precursor‐progeny of PBMC in cynomolgus monkeys as an animal model of aging.
Characterization of human CD25+ CD4+ T cells in thymus, cord and adult blood
Authors: Wing Kajsa , Ekmark Ann , Karlsson Helen , Rudin Anna , Suri‐Payer Elisabeth
Published: March 2003

CD4+ CD25+ regulatory T cells prevent organ‐specific autoimmune diseases in various animal models. We analysed human lymphoid tissues to identify similar CD25+ regulatory T cells. Adult peripheral blood contained two populations of CD4+ T cells that expressed CD25 at different densities. The larger population (≈ 40%) expressed intermediate levels of CD25 (CD25+) and displayed a memory T‐cell phenotype (CD45RA−/RO+, CD45RBlow, CD95+, CD62Llow, CD38low). The smaller population of cells (≈ 2%) expressed very high levels of CD25 (CD25++). In addition to the activation/memory T‐cell antigens mentioned above they also expressed intracellular CD152 (CTLA‐4) as well as enhanced levels of cell‐surface CD122, similar to the murine CD4+ CD25+ regulatory counterpart. To exclude that the CD25++ cells had not been recently primed by external antigen we analysed cord blood and thymus. CD25++, CD152+ and CD122++ cells were present in paediatric thymus (10% of CD4+ CD8− thymocytes) expressing signs of recent selection (CD69+) and in cord blood (5% of CD4+ cells) where they showed a naive phenotype. In addition, cord blood contained a small population of CD25+ cells (≈ 2% of CD4 T cells) that were CD152− and CD122low and displayed signs of activation. Together with published data that CD25+ CD25++ cells from the thymus and peripheral blood are regulatory, our results suggest that regulatory CD25+ T cells leave the thymus in a naïve state and become activated in the periphery.
Complex expression of natural killer receptor genes in single natural killer cells
Authors: Husain Zaheed , Alper Chester A. , Yunis Edmond J. , Dubey Devendra P.
Published: March 2003

Human natural killer (NK) cells express several inhibitory and non‐inhibitory NK receptors per cell. Understanding the expression patterns of these receptor genes in individual cells is important to understanding their function. Using a single‐cell reverse transcription–polymerase chain reaction (RT‐PCR) method, we analysed the expression of nine NK receptor genes in 38 resting CD56+ NK cells from peripheral blood of normal donors. We observed highly diverse patterns of receptor expression in these cells. No NK receptor is expressed universally in every CD56+ NK cell. The expressed receptor types per cell varied from two to eight. We specifically analysed the distribution of inhibitory (DL) and non‐inhibitory (DS) killer immunoglobulin‐like receptors (KIR). The frequency of individual receptor expression varied from 26% for 2DS2 to 68% for both 2DL1 and 2DL4. A comparison of the coexpression of DL and DS receptors showed a significant association in the expression of 2DL2 and 2DS2 (χ2=16·6; P<0·001) genes but no association between 2DL1 and 2DS1 or between 3DL1 and 3DS1 genes. Coexpression analysis of the 2DL1 and 2DL2 genes in 2DL4+ and 2DL4− cells showed a strong association in 2DL4+ but not in 2DL4− cells, suggesting a differential effect of the 2DL4 gene on the expression of 2DL1 and 2DL2 genes. Single‐cell RT‐PCR is a powerful tool to study multiple receptor gene expression ex vivo in individual NK cells and provides information about the expression pattern of KIR receptors that may suggest mechanisms of gene expression responsible for generation of the KIR repertoire.
Immunological weapons against acute myeloid leukaemia
Authors: Galea‐Lauri Joanna
Published: March 2003

A better understanding of the biology of malignant cells and of the host immune system together with dramatic advances in technology have led to the design of innovative immune‐mediated approaches to control neoplastic clones, including various haematological malignancies. One of the major problems with conventional cancer therapies is their inability to eradicate residual cancer cells that are resistant to therapy, hence immune intervention might improve the clinical outcome of patients. This mini‐review will focus mainly on immunological approaches to the therapy of acute myeloid leukaemia (AML), a subset of a much larger family of leukaemias. Immune‐mediated approaches ranging from allogeneic lymphocyte transplants to cytokine therapy, immune‐gene therapy and vaccination by dendritic‐cell‐based vaccines will be discussed.