bluuebunny/crossref_metadata_2025 huggingface dataset:
Sex hormone levels correlate with the activity of cytokine‐secreting cells in vivo
Authors: Verthelyi D. , Klinman D. M.
Published: March 2003

This work examines the correlation between serum levels of oestrogen, progesterone and dehydroepiandrosterone sulphate (DHEA‐S) and the number of human peripheral blood cells actively secreting interleukin (IL)‐2, IL‐4, IL‐6, IL‐10, tumour necrosis factor‐α (TNF‐α) or interferon‐γ (IFN‐γ) in vivo. Simultaneous assessment of serum hormone levels and cytokine‐secreting cell activity throughout the menstrual cycle showed that the number of peripheral blood mononuclear cells (PBMC) able to secrete IL‐4 in response to stimulation correlated significantly (P < 0·0001) with oestrogen levels and fluctuated with the menstrual cycle in pre‐menopausal women. The activity of IFN‐γ‐secreting cells, on the other hand, varied as a function of serum DHEA‐S levels in pre‐menopausal women (P < 0·0001). Similarly, the number of cells secreting IFN‐γ in men correlated with serum DHEA‐S levels (P < 0·001). In contrast, post‐menopausal women had fewer cells actively secreting cytokines and the activity of these cells did not correlate with sex hormone levels. These results suggest that sex hormones may modulate cytokine production in vivo and contribute to gender‐related differences in normal and pathological immune responses.
T‐cell regulation of peripheral tolerance and immunity: the potential role for  Notch signalling
Authors: Hoyne G. F. , Dallman M. J. , Lamb J. R.
Published: March 2003

Recognition of antigen by T cells in the periphery may lead either to the generation of productive immunity or the induction of tolerance. These two functional outcomes are a consequence of distinct pathways of T‐cell differentiation. T cells are selected to become regulatory cells and their function is to maintain homeostasis with the immune system. In this review we discuss the cell‐fate decisions that T cells might make allowing them to promote immunity or induce tolerance in the context of the role that Notch signalling may play in this process.
The Rubino test for leprosy is a β2‐glycoprotein 1‐dependent antiphospholipid reaction
Authors: Panunto‐Castelo A. , Almeida I. C. , Rosa J. C. , Greene L. J. , Roque‐Barreira M.‐C.
Published: March 2003

We describe the isolation and identification of three components required for the Rubino reaction (RR), which is the rapid sedimentation of formalinized sheep red‐blood cells (SRBC) initiated by serum from leprosy patients with defective Mycobacterium leprae‐specific cell immunity. The Rubino reaction factor (RRF) required for this phenomenon, previously identified as an immunoglobulin M (IgM), was purified from leprosy patient serum by adsorption to formalinized SRBC. Purified RRF IgM, when added to formalinized SRBC, did not produce a positive RR. However, when the contact was carried out in the presence of normal human serum (NHS), cells rapidly sedimented. The purified cofactor from NHS contained two components of 70 000 and 50 000 molecular weight (MW), as determined by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE). The latter was recognized by the RRF IgM on immunoblot and its N‐terminal sequence indicated that it was β2‐glycoprotein 1 (β2‐GP1), an anionic phospholipid‐binding protein. Methanol‐treated formalinized SRBC did not support the RR. Thin‐layer chromatography of an extract of membranes indicated that the SRBC ligand was a cell‐surface phospholipid. Cardiolipin inhibited the RR. These data demonstrate that the RR involves a trimolecular interaction in which IgM, β2‐GP1 and an SRBC phospholipid participate. By analogy with the antiphospholipid antibodies (anti‐PL) that occur in autoimmune processes, serum samples from 29 systemic lupus erythematosus patients with high levels of anticardiolipin antibodies were submitted to the RR. A positive RR was obtained for 45% (13 of 29 patients). These results modify the paradigm of the absolute specificity of the RR for leprosy and demonstrate that RRF IgM is a β2‐GP1‐dependent anti‐PL.
Granulocyte–macrophage colony‐stimulating factor regulates cytokine production in cultured macrophages through CD14‐dependent and ‐independent mechanisms
Authors: Bergamini A. , Bolacchi F. , Bongiovanni B. , Cepparulo M. , Ventura L. , Capozzi M. , Sarrecchia C , Rocchi G.
Published: March 2003

Granulocyte–macrophage colony‐stimulating factor (GM‐CSF) has multiple effects on the antigen phenotype and function of macrophages. In this study we investigated the effect of GM‐CSF on cytokine production by macrophages. We found that GM‐CSF may modify the tumour necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) response to lipopolysaccharide (LPS) through two different mechanisms. Relatively early in culture, GM‐CSF increases the amount of cytokines synthesized by responding cells; this effect appears to be unrelated to modulation of CD14 expression and LPS‐binding capacity. After prolonged incubation, GM‐CSF up‐regulates both CD14 expression and LPS‐binding capacity, and the frequency of cytokine‐producing cells. Release of CD14 in the culture supernatant was decreased in the presence of GM‐CSF, suggesting that a reduced shedding was responsible for the effect of GM‐CSF on CD14 expression. Enhancement of cytokine production was also observed in GM‐CSF‐treated macrophages after stimulation by phorbol 12‐myristate 13‐acetate (PMA), thus indicating that GM‐CSF affects both CD14‐dependent and ‐independent cytokine production. Finally, GM‐CSF did not modulate the LPS‐ and PMA‐induced production of IL‐10 and IL‐12. We conclude that GM‐CSF may play a role in manipulating the activation‐induced expression of pro‐inflammatory cytokines by macrophages. Enhanced production of these cytokines could play an important role in the pathogenesis of Gram‐negative septic shock syndrome and in defence against infectious agents.
Virulence of Sporothrix schenckii conidia and yeast cells, and their susceptibility to nitric oxide
Authors: Fernandes K. S. S. , Coelho A. L. J. , Bezerra L. M. Lopes , Barja‐Fidalgo C.
Published: March 2003

The involvement of nitric oxide (NO) in macrophage (Mφ) fungicidal activity against Sporothrix schenckii, and the relationship between NO susceptibility and the differential virulence of conidia and yeast cells, were investigated. Confirming a previously reported correlation between the length of time in culture and virulence of S. schenckii, conidia isolated from 12‐day mycelial cultures (Ss‐12) were less virulent to mice than conidia from 7‐day cultures (Ss‐7) or yeast cells. Indicative of NO production, infected animals showed a significant increase in serum levels of nitrite that was lower in mice infected with Ss‐12 than in mice infected with Ss‐7 or yeast. Stimulation of murine Mφ with interferon‐γ (IFN‐γ) induced NO production and inhibition of fungal growth. The cytotoxic activity of Mφ against Ss‐12 was significantly greater than against Ss‐7 or yeast cells, the highly virulent fungal forms. The addition of NO synthase inhibitors abrogated Mφ cytotoxic activity against all fungal forms. The phagocytic activity of Mφ against Ss‐7 was significantly lower than against Ss‐12 or yeast cells. Although the ingestion of fungal cells triggered the oxidative burst in Mφ, the fungicidal activity was not altered in the presence of superoxide dismutase (SOD) and catalase. In addition, Ss‐12 and yeast cells were more susceptible than Ss‐7 to the direct fungicidal activity of the NO donors S‐nitroso‐N‐acetyl‐dl‐penicillamine (SNAP), S‐nitrosoglutathione (GSNO) and 3‐morpholinosydnonimine (SIN‐1). The results of this study indicate that NO is a key cytotoxic mediator involved in the murine Mφ defence against S. schenckii, and that the virulence of Ss‐7, Ss‐12 and yeast cells may be related to a differential susceptibility to NO.
Differential expression of ovine CD1
Authors: Rhind S. M. , Hopkins J. , Grant E. S.
Published: March 2003

Differential expression of ovine CD1 was examined using a combination of reverse transcription–polymerase chain reaction (RT–PCR) with sequence‐specific primers and Northern and in situ hybridization techniques. The aim of the study was to establish the patterns of CD1 expression at the molecular level and address questions posed by previous studies in other species regarding expression patterns of CD1. A ‘pan‐CD1’ probe based on the exon 4 (alpha 3) region was used in addition to isotype‐specific probes for SCD1B (the exon 3 region of clone SCD1B42) and SCD1D (the exon 3 region of clone SCD1D). Widespread expression of CD1 (including thymus, peripheral blood lymphocytes, lung and intestine) was identified using both the exon 4 and SCD1D probe. SCD1B expression was more restricted, being identified in equivalent levels only in the thymus and in scattered populations of dendritic cells. These results highlight the difference in expression patterns between group 1 and group 2 CD1 family members and establish SCD1D as the CD1 family member with the widest pattern of expression, consistent with a differential role for the different CD1 family members.
Modulatory effect of prostaglandins on human monocyte activation for killing of high‐ and low‐virulence strains of Paracoccidioides brasiliensis
Authors: Soares A. M. V. C. , Calvi S. A. , Peraçoli M. T. S. , Fernandez A. C. , Dias L. A. , Dos Anjos A. R.
Published: March 2003

The effect of indomethacin (Indo), a cyclo‐oxygenase inhibitor, on the monocyte‐mediated killing of a low‐ (Pb265) and a high‐ (Pb18) virulence strain of Paracoccidioides brasiliensis was examined. The Pb18 strain was not killed by either non‐activated or interferon‐γ (IFN‐γ) ‐activated human monocytes but these cells did show fungicidal activity if pretreated with Indo. In contrast with IFN‐γ, tumour necrosis factor‐α (TNF‐α) was very effective at stimulating the fungicidal activity of monocytes. While the low‐virulence strain, Pb265, could not be killed by monocytes, cells preincubated with IFN‐γ demonstrated fungicidal activity. The killing of this strain was also induced by pretreatment of monocytes with Indo. The results suggest a negative role for prostaglandins, which are synthesized via the cyclo‐oxygenase pathway, in the regulation of monocyte‐mediated killing of virulent and avirulent strains of P. brasiliensis and that TNF‐α generation during the fungus–monocyte interaction is more important in the killing of Pb265 than Pb18.
Metalloprotease inhibitor‐mediated inhibition of mouse immunoglobulin production
Authors: Kilmon M. A. , Mayer R. J. , Marshall L. A. , Conrad D. H.
Published: March 2003

High levels of membrane CD23 have been shown to decrease immunoglobulin E (IgE). CD23 is a very labile molecule and is cleaved from the cell surface by an unknown metalloprotease. Two metalloprotease inhibitors, compound A (N‐[4‐hydoxyamino‐2‐(R)‐isobutyl‐3‐(S)propargylthiomethylsuccinyl]‐(S)‐phenylalnine‐N′‐methyl‐amide) and compound B (N‐[3‐(S)‐hydroxy‐4‐hydroxyamino‐2‐(R)‐(2‐naphthylmethyl) succinyl]‐(S)‐tert‐leucinamide), were chosen for their ability to inhibit human CD23 cleavage and selectively inhibit IgE production. The ability of these inhibitors to block cleavage of murine CD23 and immunoglobulin production in an in vitro system was examined. The inhibitors blocked sCD23 release from B cells. The inhibitors also decreased IgE production by B cells; however, 20–30 times more inhibitor was needed to give a similar amount of inhibition as compared with sCD23 release. The effects on immunoglobulin production did not require the presence of CD23 in that these inhibitors also blocked in vitro immunoglobulin production when B cells from CD23–/– mice were used. The inhibitors decreased production of all other immunoglobulin isotypes examined and reduced the number of IgE antibody‐forming cells (AFC) while having no effect on cell proliferation or viability. The level of Iε transcripts in cells treated with compounds A and B were not different as compared with control cells. These results suggest that while these inhibitors effectively inhibit IgE production in a CD23‐specific manner in the human, these compounds, in the mouse, inhibit immunoglobulin production by an unknown mechanism that is unrelated to CD23.
Complement activation by apoptotic endothelial cells following hypoxia/reoxygenation
Authors: Mold C. , Morris C. A.
Published: March 2003

Reperfusion of ischaemic tissue initiates an inflammatory reaction that increases tissue injury. Complement activation at the endothelium contributes to this inflammation. This study investigated the mechanism of complement activation following reoxygenation of hypoxic human umbilical vein endothelial cells (HUVEC) as a model for complement activation observed on endothelium in reperfused ischaemic tissue. HUVEC cultured in 1% oxygen followed by reoxygenation activated the classical complement pathway resulting in C3 deposition. There was an increase in apoptotic cells in these cultures that was demonstrated by binding of fluorescein isothiocyanate‐Annexin V and staining for hypodiploid nuclei. To determine if apoptotic HUVEC activate complement, uniformly apoptotic cells were produced by serum and growth factor deprivation. These cells, but not the control HUVEC, activated the classical complement pathway in the absence of antibody or other serum factors. To determine if apoptotic cells in the reoxygenated cultures were activating complement, fluorescent analysis was done. Annexin V binding and C3d deposition on cells from reoxygenated cultures showed complete concordance on the subpopulation of apoptotic cells. In addition, complement activation following reoxygenation of HUVEC was eliminated by treatment of the cultures with a caspase inhibitor during reoxygenation. These results suggest that oxidative damage to endothelial cells during reoxygenation initiates apoptosis with exposure of phosphatidylserine. Apoptotic cells directly activate the classical pathway of complement by binding C1. Activation of complement at the endothelium may contribute to the inflammatory response as well as clearance and repair.
Integrating signals from T‐cell receptor and serum by T cells enhance translation of tumour necrosis factor‐α
Authors: Buxadé M. , Ramírez‐Alvarado M. , Fernández‐Troy N. , MacKenzie S. , Casaroli‐Marano R. P. , Vilella R. , Espel E.
Published: March 2003

Tumour necrosis factor‐α (TNF‐α) is a proinflammatory cytokine produced by several cell types, including T cells upon antigen stimulation. Its production is crucial for the development of an early defence against many pathogens, but its beneficial effects are dependent on the strength and duration of its expression. In this paper we present evidence indicating that serum increases translational efficiency of TNF‐α in human peripheral blood mononuclear cells stimulated with superantigen. The increase in translation of TNF‐α due to serum could be inhibited by the phosphatidylinositol (PI) 3‐K inhibitors, wortmannin and LY294002, suggesting that PI 3‐K is involved in the translational control of TNF‐α by serum. Similarly to primary T cells, stimulation of Jurkat T cells with superantigen led to TNF‐α secretion and this was up‐regulated by serum. Transfection of Jurkat cells with a constitutively active form of PI 3‐Kα increased the production of TNF‐α in cells stimulated with superantigen. Additionally, we used the specific inhibitors targeting ERK kinase and p38 mitogen‐activated protein kinase (MAPK), potentially downstream of PI 3‐kinase, PD98059 and SB203580. Differently from with PI 3‐K inhibitors, the accumulation of TNF‐α mRNA was inhibited by PD98059 or SB203580. These results suggest that, in T cells, activation of PI 3‐K is an important step in controlling TNF‐α protein synthesis in response to growth factors.
The role of the macrophage scavenger receptor in immune stimulation by bacterial DNA and synthetic oligonucleotides
Authors: Zhu Fu‐Gang , Reich Charles F. , Pisetsky David S.
Published: March 2003

To assess the role of the macrophage scavenger receptor type A (SRA) in immune activation by CpG DNA, cytokine induction and DNA uptake were tested in vitro and in vivo using SRA knockout (SRA−/−) and wild type (WT) mice. As a source of CpG DNA, Escherichia coli DNA (EC DNA) and a 20‐mer phosphorothioate oligodeoxynucleotide with two CpG motifs (CpG ODN) were used. In vitro, both EC DNA and the CpG ODN induced dose‐dependent increases of interleukin (IL)‐12 production by spleen cells and bone‐marrow‐derived macrophages (BMMΦ) from both SRA−/− and WT mice. The levels of cytokines produced by SRA−/− spleen cells and BMMΦ were similar to those of WT spleen cells and BMMΦ. When injected intravenously with CpG ODN and EC DNA, both SRA−/− and WT mice showed elevated serum levels of IL‐12. To investigate further the role of the SRA, flow cytometry and confocal microscopy were performed to examine the uptake of fluorescently labelled oligonucleotides. SRA−/− and WT BMMΦ showed similarity in the extent of uptake and distribution of oligonucleotides as assessed by these two techniques. Together, these findings indicate that, while the SRA may bind DNA, this receptor is not essential for the uptake of CpG DNA or its immunostimulatory activity.
Partial tolerance of subcutaneously transplanted xenogeneic tumour cell graft by Fas‐mediated immunosuppression
Authors: Sawada Takahiro , Koji Takehiko , Hishikawa Yoshitaka , Kishimoto Koji , Nagayasu Takeshi , Takahashi Takao , Oka Tadayuki , Ayabe Hiroyoshi
Published: March 2003

Certain anti‐Fas antibodies, such as RMF2, induce apoptosis of Fas‐expressing cells. We applied the Fas/anti‐Fas system to induce killing of Fas‐expressing immunocytes with resultant immunosuppression. W7TM‐1 tumour cells, a rat T‐cell line, were inoculated subcutaneously in BALB/c mice and tumour growth was monitored in untreated mice and in mice treated with RMF2. Prior to treatment with RMF2, we examined the expression of Fas in isolated splenocytes and in tumour‐infiltrating lymphocytes by flow cytometry and immunohistochemistry, respectively. There was a remarkable increase in Fas‐positive lymphocytes, including natural killer (NK) cells, among splenocytes at day 5 after tumour cell inoculation. The number of Fas‐positive infiltrating lymphocytes also increased markedly, from day 5 to day 10. We then examined whether RMF2 could induce apoptosis of Fas‐positive activated lymphocytes isolated from the spleen at day 5 in vitro. Terminal deoxy (d) ‐UTP nick end labelling (TUNEL) and Annexin V staining methods showed apoptosis of isolated cells when incubated with RMF2, and typical apoptotic features were confirmed by 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI) staining. Furthermore, suppression of cellular and humoral immunity was noted in RMF2‐treated mice by mixed lymphocyte reaction and assay of serum levels of immunoglobulin G, respectively. Finally, treatment of animals with RMF2 daily from day 5 to day 9 could maintain the tumour size, while the tumour mass began to diminish in untreated mice immediately after reaching a maximum size. We confirmed the enhancing effects of long‐term treatment with RMF2, through the induction of immunosuppression, on the growth of unvascularized xenogeneic tumour cell grafts.
Refocusing of B‐cell responses following a single amino acid substitution in an antigen
Authors: Chiesa Marta Dalla , Martensen Pia M. , Simmons Cameron , Porakishvili Nino , Justesen Just , Dougan Gordon , Roitt Ivan M. , Delves Peter J. , Lund Torben
Published: March 2003

Intranasal immunization of BALB/c strain mice was carried out using baculovirus‐derived human chorionic gonadotrophin (hCG) β‐chain, together with Escherichia coli heat‐labile enterotoxin. Gonadotrophin‐reactive immunoglobulin A (IgA) was induced in a remote mucosal site, the lung, in addition to a systemic IgG response. The extensive sequence homology with luteinizing hormone (LH) results in the production of LH cross‐reactive antibodies when holo‐hCG is used as an immunogen. In contrast to wild‐type hCGβ, a mutated hCGβ‐chain containing an arginine to glutamic acid substitution at position 68 did not induce the production of antibodies which cross‐react with LH. Furthermore, the epitopes utilized in the B‐cell response to the mutated hCGβ shifted away from the immunodominant region of the parent wild‐type molecule towards epitopes within the normally weakly immunogenic C terminus. This shift in epitope usage was also seen following intramuscular immunization of rabbits. Thus, a single amino acid change, which does not disrupt the overall structure of the molecule, refocuses the immune response away from a disadvantageous cross‐reactive epitope region and towards a normally weakly immunogenic but antigen‐unique area. Similar mutational strategies for epitope‐refocusing may be applicable to other vaccine candidate molecules.
Intraspecies heterogeneity of immunoglobulin α‐chain constant region genes in rhesus macaques
Authors: Scinicariello Franco , Attanasio Roberta
Published: March 2003

Immunoglobulin A (IgA) is the major antibody class present in external secretions and is also an important component of serum immunoglobulins. On mucosal surfaces, IgA represents a first line of defence by neutralizing invading pathogens. The number of IgA constant‐region genes (Cα) present in different mammalian species is variable. Immunoglobulin Cα genes differ mainly in the sequences located in the hinge region. IgA molecules, whose hinge regions are remarkably similar to those of the respective human molecules, are present in hominoid primates. In this report, we show that two alleles of a single immunoglobulin Cα are present in rhesus macaques (Macaca mulatta). In addition, we show that intraspecies immunoglobulin Cα allelic polymorphism is very high in this non‐human primate species. Specifically, five different hinge regions, some of which are proline‐rich, were identified from a total of eight rhesus macaque immunoglobulin Cα‐chains. The five hinge regions were different from those present in hominoid primates, both in length and in sequence. These results represent the first example of high levels of intraspecies immunoglobulin constant‐region variability and suggest that IgAs of variable structure and function may be present in rhesus macaques. As rhesus macaques are widely used as animal models for the development of vaccines for acquired immune deficiency syndrome (AIDS), the possible presence of structurally and functionally variable IgA molecules in different animals should be taken into account when designing experimental strategies to induce mucosal antibody responses to human immunodeficiency virus (HIV).
Sulphasalazine inhibits macrophage activation: inhibitory effects on inducible nitric oxide synthase expression, interleukin‐12 production and major histocompatibility complex II expression
Authors: Haskó György , Szabó Csaba , Németh Zoltán H. , Deitch Edwin A.
Published: March 2003

The anti‐inflammatory agent sulphasalazine is an important component of several treatment regimens in the therapy of ulcerative colitis, Crohn's disease and rheumatoid arthritis. Sulphasalazine has many immunomodulatory actions, including modulation of the function of a variety of cell types, such as lymphocytes, natural killer cells, epithelial cells and mast cells. However, the effect of this agent on macrophage (Mφ) function has not been characterized in detail. In the present study, we investigated the effect of sulphasalazine and two related compounds – sulphapyridine and 5‐aminosalicylic acid – on Mφ activation induced by bacterial lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ). In J774 Mφ stimulated with LPS (10 µg/ml) and IFN‐γ (100 U/ml), sulphasalazine (50–500 µm) suppressed nitric oxide (NO) production in a concentration‐dependent manner. The expression of the inducible NO synthase (iNOS) was suppressed by sulphasalazine at 500 µm. Sulphasalazine inhibited the LPS/IFN‐γ‐induced production of both interleukin‐12 (IL‐12) p40 and p70. The suppression of both NO and IL‐12 production by sulphasalazine was superior to that by either sulphapyridine or 5‐aminosalicylic acid. Although the combination of LPS and IFN‐γ induced a rapid expression of the active forms of p38 and p42/44 mitogen‐activated protein kinases and c‐Jun terminal kinase, sulphasalazine failed to interfere with the activation of any of these kinases. Finally, sulphasalazine suppressed the IFN‐γ‐induced expression of major histocompatibility complex class II. These results demonstrate that the Mφ is an important target of the immunosuppressive effect of sulphasalazine.
Regulation of T‐cell interaction with fibronectin by transforming growth factor‐β is associated with altered Pyk2 phosphorylation
Authors: Brill Alexander , Franitza Susanne , Lider Ofer , Hershkoviz Rami
Published: March 2003

Although the involvement of transforming growth factor‐β (TGF‐β) in inflammatory reactions has been extensively studied, its mode of action in the context of the extracellular matrix (ECM) is still not fully understood. We undertook this study in an attempt to reveal the putative roles of TGF‐β in T‐cell adhesion and migration. We found that a 60‐min treatment of T cells with TGF‐β regulates T‐cell adhesion to fibronectin (FN), a prototype cell adhesion protein of the ECM, depending on the presence of other activators. At 5 pg/ml to 1 ng/ml, TGF‐β alone induced T‐cell adhesion to FN in an integrin α4/β1‐ and integrin α5/β1‐dependent manner. TGF‐β also attenuated T‐cell migration on the stromal cell‐derived factor (SDF)‐1α gradients. These effects of TGF‐β were not accompanied by alteration in the expression of very‐late activation antigen type 4 (VLA‐4) and VLA‐5, nor were they mediated by the cyclo‐oxygenase pathway. The cellular mechanism underlying the adhesion‐regulating activities of TGF‐β involves adhesion‐associated cytoskeletal elements. TGF‐β induced the phosphorylation of focal adhesion kinase Pyk2, but not extracellular signal‐regulated kinase (ERK), and this effect was markedly increased in the presence of immobilized FN, suggesting a collaborative role for FN‐specific integrins. Indeed, TGF‐β‐induced Pyk2 phosphorylation was inhibited by monoclonal antibodies against VLA‐4, VLA‐5 and CD29. Thus, TGF‐β, which may appear at extravascular sites during inflammation, affects the adhesion of T cells to ECM glycoproteins and their migration by its ability to differentially induce or inhibit the phosphorylation of Pyk2.
Size of the population of CD4+ natural killer T cells in the liver is maintained without supply by the thymus during adult life
Authors: Kameyama Hitoshi , Kawamura Toshihiko , Naito Tetsuya , Bannai Makoto , Shimamura Kazuhiko , Hatakeyama Katsuyoshi , Abo Toru
Published: March 2003

Given that there are few natural killer T (NKT) cells in the liver of athymic nude mice and in neonatally thymectomized mice, it is still controversial whether all NKT cells existing in the liver are supplied by the thymus or if some such cells develop in the liver. To determine whether or not NKT cells are consistently supplied from the thymus during adult life, thymectomy was conducted in mice at the age of 8 weeks. Interestingly, the proportion and number of CD4+ NKT cells increased or remained unchanged in the liver after adult thymectomy and this phenomenon continued for up to 6 months after thymectomy. The administration of α‐galactosylceramide induced severe cytopenia (due to apoptosis) of CD4+ NKT cells in the liver on day 1, but subsequent expansion of these NKT cells occurred in thymectomized mice similar to the case in normal mice. However, in thymectomized mice given lethal irradiation (9·5 Gy) and subsequent bone marrow transfer, the population of CD4+ NKT cells no longer expanded in the liver, although that of CD8+ NKT cells did. These results suggest that thymic CD4+ NKT cells, or their progenitors, may migrate to the liver at a neonatal stage but are not supplied from the thymus in the adult stage under usual conditions. CD8+ NKT cells can be generated in the liver.
Porcine dendritic cells generated in vitro: morphological, phenotypic and functional properties
Authors: Carrasco Carlos P. , Rigden Rachael C. , Schaffner Rene , Gerber Heidi , Neuhaus Viviane , Inumaru Shigeki , Takamatsu Haru , Bertoni Guiseppe , McCullough Kenneth C. , Summerfield Artur
Published: March 2003

Despite the central role that dendritic cells (DC) play in immune regulation and antigen presentation, little is known about porcine DC. In this study, two sources of DC were employed. Bone marrow haematopoietic cell‐derived DC (BM‐DC) were generated using granulocyte–macrophage colony‐stimulating factor (GM‐CSF) in the presence or absence of tumour necrosis factor‐α (TNF‐α). Monocyte‐derived DC (Mο‐DC) were generated with GM‐CSF and interleukin‐4 (IL‐4). In both systems, non‐adherent cells developed with dendritic morphology, expressing high levels of major histocompatibility complex (MHC) class II. The presence of TNF‐α increased the BM‐DC yield, and enhanced T‐cell stimulatory capacity. Both BM‐DC and Mο‐DC expressed the pan‐myeloid marker SWC3, as well as CD1 and CD80/86, but were also CD14+ and CD16+. The CD16 molecule was functional, acting as a low‐affinity Fc receptor. In contrast, the CD14 on DC appeared to differ functionally from monocyte CD14: attempts to block CD14, in terms of lipopolysaccharide (LPS)‐induced procoagulant activity (PCA), failed. The use of TNF‐α or LPS for DC maturation induced up‐regulation of MHC class II and/or CD80/86, but also CD14. Allogeneic mixed leucocyte reactions and staphylococcal enterotoxin B antigen presentation assays demonstrated that these DC possessed potent T‐cell stimulatory capacity. No T helper cell polarization was noted. Both the BM‐DC and the Mο‐DC induced a strong interferon‐γ and IL‐4 response. Taken together, porcine DC generated in vitro possess certain characteristics relating them to DC from other species including humans, but the continued presence of CD14 and CD16 on mature and immature porcine DC was a notable difference.
Human leucocyte antigen‐A2 restricted and Mycobacterium tuberculosis 19‐kDa antigen‐specific CD8+ T‐cell responses are oligoclonal and exhibit a T‐cell cytotoxic type 2 response cytokine‐secretion pattern
Authors: Höhn Hanni , Kortsik Cornelius , Nilges Katja , Necker Antje , Freitag Kirsten , Tully Glenn , Neukirch Claudia , Maeurer Markus J.
Published: March 2003

CD8+ T cells can be grouped into two different types of secretory T lymphocytes, based on the cytokine‐secretion pattern upon antigen exposure: those with a T‐cell cytotoxic type 1 response (Tc1), which secrete interferon‐γ (IFN‐γ), or those with a T‐cell cytotoxic type 2 response, which secrete interleukin (IL)‐4 and IL‐10. We examined the CD8+ T‐cell response directed against an immunodominant human leucocyte antigen (HLA)‐A2‐presented peptide derived from a 19‐kDa Mycobacterium tuberculosis‐associated antigen. T cells were examined by functional analysis and by T‐cell receptor (TCR) complementarity‐determining region 3 (CDR3)‐spectratyping, which defines the complexity of a T‐cell response. T‐cell stimulation with the immunodominant VLTDGNPPEV epitope yielded a Tc2 (IL‐4) cytokine‐secretion pattern and resulted in oligoclonal expansion of TCR‐variable beta chain (VB) families, which differed from patient to patient. Generation of T‐cell clones corroborated the notion that the CD8+ T‐cell response directed against the HLA‐A2‐presented VLTDGNPPEV epitope leads to a Tc2 cytokine‐secretion pattern in CD8+ T cells, as defined by IL‐4 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) release. Characterization of the cytokine‐secretion profile in HLA‐A2/VLTDGNPPEV‐tetramer sorted T cells from patients with active tuberculosis supported this observation: peptide‐specific T cells from three of three patients secreted IL‐4 and only one of three patients produced IFN‐γ in response to the nominal target epitope. Permutation of this T‐cell epitope may aid to elicit a qualitatively different CD8+ T‐cell response in patients with M. tuberculosis infection.
B‐cell precursors differentiated from cord blood CD34+ cells are more immature than those derived from granulocyte colony‐stimulating factor‐mobilized peripheral blood CD34+ cells
Authors: Hirose Yuka , Kiyoi Hitoshi , Itoh Katsuhiko , Kato Koji , Saito Hidehiko , Naoe Tomoki
Published: March 2003

Umbilical cord blood (CB) has been widely used instead of bone marrow (BM) and peripheral blood (PB) for stem cell transplantation (SCT). However, problems of sustained immunodeficiency after CB transplantation remain to be resolved. To elucidate the mechanism of immunodeficiency, we compared the characteristics of B cells differentiated in vitro from CD34+ cells of CB with those of PB. Purified CD34+ cells from CB and PB were cultured on murine stroma cell‐line MS‐5 with stem cell factor and granulocyte colony‐stimulating factor for 6 weeks. The B‐cell precursors (pre‐B cells) that differentiated in this culture system, were analysed as to their immunoglobulin heavy chain (IgH) variable region gene repertoire and the expression of B‐cell differentiation‐related genes. CD10+ CD19+ pre‐B cells were differentiated from both PB and CB. Although the usages of IgH gene segments in pre‐B cells differentiated from CB and PB were similar, the N region was significantly shorter in CB‐derived than PB‐derived cells. Productive rearrangements were significantly fewer in cells of CB than PB in the third week. Among a number of B‐cell differentiation‐related genes, the terminal deoxynucleotidyl transferase (TdT) gene was not expressed in CB‐derived cells during the culture. These results indicated that immature features of pre‐B cells from CB, such as lack of TdT expression, and a short N region and few productive rearrangements in the IgH gene, might cause the delay in mature B‐cell production.
Neonatal mycobacterial specific cytotoxic T‐lymphocyte and cytokine profiles in response to distinct BCG vaccination strategies
Authors: Hussey Gregory D. , Watkins Marcia L. V. , Goddard Elizabeth A. , Gottschalk Sean , Hughes Elizabeth J. , Iloni Karen , Kibel Maurice A. , Ress Stanley R.
Published: March 2003

This study evaluated whether different bacillus Calmette–Gue´rin (BCG) strains, routes of administration, vaccination age and percutaneous tools influenced immune responses to BCG vaccination in infants. Proliferative responses, cytokine production and cell‐mediated cytotoxicity obtained in post‐vaccinated children were compared to baseline cord bloods and unvaccinated 10‐week‐old infants. BCG vaccination generally induced strong lymphoproliferative and T helper type 1 (Th1)‐type cytokine responses. There was a trend for greater responsiveness following the intradermal route of vaccination, with Japanese‐172 strain and with delaying vaccination until 10 weeks. Cord mononuclear cells differentially stimulated the Th2‐type cytokines interleukin‐5 (IL‐5) and IL‐10 selectively in response to BCG, as compared to H37Rv or purified protein derivative stimulation. We document for the first time the generation of mycobacterium‐specific cytotoxic T lymphocytes in neonates, following BCG vaccination. Cytotoxic activity correlated with the ratio of interferon‐γ to IL‐5, aside from a single instance where use of the Biovac® tool resulted in a striking dissociation selectively against H37Rv targets. These data have implications for correlates of protective immunity in design of vaccine studies.
Tissue‐specific expression of mast cell granule serine proteinases and their role in inflammation in the lung and gut
Authors: Miller Hugh R. P. , Pemberton Alan D.
Published: March 2003

Serine proteinases with trypsin‐like (tryptase) and chymotrypsin‐like (chymase) properties are major constituents of mast cell granules. Several tetrameric tryptases with differing specificities have been characterized in humans, but only a single chymase. In other species there are larger families of chymases with distinct and narrow proteolytic specificities. Expression of chymases and tryptases varies between tissues. Human pulmonary and gastrointestinal mast cells express chymase at lower levels than tryptase, whereas rodent and ruminant gastrointestinal mast cells express uniquely mucosa‐specific chymases. Local and systemic release of chymases and tryptases can be quantified by immunoassay, providing highly specific markers of mast cell activation. The expression and constitutive extracellular secretion of the mucosa‐specific chymase, mouse mast cell proteinase‐1 (mMCP‐1), is regulated by transforming growth factor‐β1 (TGF‐β1) in vitro, but it is not clear how the differential expression of chymases and tryptases is regulated in other species. Few native inhibitors have been identified for tryptases but the tetramers dissociate into inactive subunits in the absence of heparin. Chymases are variably inhibited by plasma proteinase inhibitors and by secretory leucocyte protease inhibitor (SLPI) that is expressed in the airways. Tryptases and chymases promote vascular permeability via indirect and possibly direct mechanisms. They contribute to tissue remodelling through selective proteolysis of matrix proteins and through activation of proteinase‐activated receptors and of matrix metalloproteinases. Chymase may modulate vascular tissues through its ability to process angiotensin‐I to angiotensin‐II. Mucosa‐specific chymases promote epithelial permeability and are involved in the immune expulsion of intestinal nematodes. Importantly, granule proteinases released extracellularly contribute to the recruitment of inflammatory cells and may thus be involved in innate responses to infection.
Peroxisome‐proliferator‐activated‐receptor gamma (PPARγ) independent induction of CD36 in THP‐1 monocytes by retinoic acid
Authors: Han Shouwei , Sidell Neil
Published: March 2003

Retinoic acid (RA) has been shown to regulate cellular growth and differentiation of a variety of cell types, including cells of the myelomonocytic lineage. We used the monocytic leukaemia cell line THP‐1, which differentiates to macrophages in response to phorbol 12‐myristate 13‐acetate (PMA), to investigate the regulation by RA of genes in the scavenger receptor type B family (CD36) in human monocyte/macrophages. Reverse transcription–polymerase chain reaction and flow cytometry demonstrated that, like PMA and the natural peroxisome‐proliferator‐activated receptor‐γ (PPARγ) ligand 15d‐PGJ2, RA induced CD36 gene expression in these cells. Moreover, RA plus 15d‐PGJ2 further enhanced CD36 protein and mRNA levels over that seen with the RA or PPARγ compounds alone. The PPARγ antagonist GW9662 was shown to block completely PPARγ‐ligand induction of CD36 gene expression, but had little effect on the action of RA. Our data indicated that RXR‐ and RAR‐specific ligands (LG153 and TTNPB, respectively) were each alone able to increase CD36 mRNA and surface protein levels. By using calphostin C, a specific protein kinase C (PKC) inhibitor, we demonstrated that induction of CD36 by PMA, as well as by PPARγ and RXR ligands were dependent upon PKC activation. In contrast, activation of CD36 through the RAR pathway was not affected by inhibition of PKC activity. Taken together, these data demonstrate that RA can up‐regulate CD36 expression in human monocytes/macrophages. This regulation appears to be predominantly mediated through the RAR/RXR pathway of action and, unlike previously described methods of CD36 modulation, is independent of PPARγ and PKC signalling. This study suggests a possible role for RA in physiological processes involving the scavenger receptor function in cells of the monocyte/macrophage lineage.
Regulation of major histocompatibility complex class II synthesis by interleukin‐10
Authors: Morel Anne‐Sophie , Coulton Gary , Londei Marco
Published: March 2003

We have shown previously that interleukin‐10 (IL‐10) blocks the development and T‐cell stimulatory capacity of human monocyte‐derived dendritic cells, without apparently down‐regulating the surface expression of co‐stimulatory molecules or human leucocyte antigen (HLA) molecules. In the majority of donors (60%), the cell surface levels of HLA‐DR actually increased upon IL‐10 treatment. Here we have shown that IL‐10 does not regulate HLA‐DR transcription as assessed by polymerase chain reation. Epifluorescence microscopy analysis showed that IL‐10 primarily increased the intracellular pool of HLA‐DR. In fact, IL‐10 directly increased HLA‐DR protein synthesis. However, IL‐10 did not significantly alter the synthesis of invariant chain (Ii), which plays a crucial role in the assembly, transport and loading of newly formed HLA class II molecules, nor the amount of Ii reaching the cell‐surface. In contrast, IL‐10 increased the amount of HLA‐DR‐bound Iip33 shortly after the HLA‐DR complex assembly. We postulate that, upon IL‐10 treatment, immature Ii‐associated HLA II molecules can still transit to the cell surface as they do in immature dendritic cells and recycle to the intracellular space, where they accumulate. A higher proportion of Ii‐associated HLA‐DR, coupled to increased membrane recycling, may contribute to the lower T‐cell stimulatory capacity of IL‐10‐treated dendritic cells.
Modulation of the CD40–CD40 ligand interaction using human anti‐CD40 single‐chain antibody fragments obtained from the n‐CoDeR phage display library
Authors: Ellmark Peter , Ottosson Camilla , Borrebaeck Carl A. K. , Malmborg Hager Ann‐Christin , Furebring Christina
Published: March 2003

CD40 plays a central regulatory role in the immune system and antibodies able to modulate CD40 signalling may consequently have a potential in immunotherapy, in particular for treatment of lymphomas and autoimmune disease like multiple sclerosis. As a first step to achieve this goal, we describe the selection and characterization of a novel set of fully human anti‐CD40 antibody fragments (scFv) from a phage display library (n‐CoDeR). In order to determine their biological potential, these antibody fragments have been analysed for their ability to promote B‐cell activation, rescue from apoptosis and to block the CD40–CD40 ligand (CD40L) interaction. The selected cohort of human scFv could be subcategorized, each expressing a distinct functional signature. Thus scFv were generated that induced B‐cell proliferation, rescued B cells from apoptosis and blocked the CD40–CD40L interaction to different extents. In particular, one of the scFv clones (F33) had the ability to abrogate completely this interaction. The epitope recognition patterns as well as individual rate constants were also determined and the affinity was shown to vary from low to high nanomolar range. In conclusion, this panel of human anti‐CD40 scFv fragments displays a number of distinct properties, which may constitute a valuable source when evaluating candidates for in vivo trials.
Enhancement of neutrophil infiltration in histidine decarboxylase‐deficient mice
Authors: Hirasawa Noriyasu , Ohtsu Hiroshi , Watanabe Takehiko , Ohuchi Kazuo
Published: March 2003

The roles of histamine in the anaphylactic increase in vascular permeability and leucocyte infiltration were analysed in an air pouch‐type allergic inflammation model in histidine decarboxylase‐deficient (HDC−/−) mice and wild‐type mice. In the immunized wild‐type mice, histamine content in the pouch fluid and vascular permeability in the anaphylaxis phase were increased by injection of the antigen solution into the air pouch. However, in the immunized HDC−/− mice, the antigen challenge did not increase histamine content in the pouch fluid and vascular permeability in the anaphylaxis phase. Number of leucocytes (more than 83% are neutrophils) in the pouch fluid 4–24 hr after the antigen challenge in the HDC−/− mice was significantly higher than that in the wild‐type mice. Simultaneous injection of histamine with the antigen solution into the air pouch of the immunized HDC−/− mice reduced the antigen‐induced leucocyte infiltration at 4 hr. Simultaneous injection of the H2 antagonist cimetidine but not the H1 antagonist pyrilamine with the antigen solution into the air pouch of the immunized wild‐type mice further increased leucocyte infiltration at 4 hr. The levels of macrophage inflammatory protein‐2 at 2 hr and of tumour necrosis factor‐α at 4 hr in the pouch fluid of the HDC−/− mice were significantly higher than those of the wild‐type mice. These findings indicate that histamine plays significant roles not only in the anaphylactic increase in vascular permeability via H1 receptors but also in the negative regulation of neutrophil infiltration via H2 receptors in allergic inflammation.
Functional and phenotypic studies of two variants of a human mast cell line with a distinct set of mutations in the c‐kit proto‐oncogene
Authors: Sundström Magnus , Vliagoftis Harissios , Karlberg Peter , Butterfield Joseph H. , Nilsson Kenneth , Metcalfe Dean D. , Nilsson Gunnar
Published: March 2003

The human mast cell line (HMC)‐1 cell line is growth‐factor independent because of a constitutive activity of the receptor tyrosine kinase Kit. Such deregulated Kit activity has also been suggested causative in gastrointestinal stromal tumours (GISTs) and mastocytosis. HMC‐1 is the only established continuously growing human mast cell line and has therefore been widely employed for in vitro studies of human mast cell biology. In this paper we describe two sublines of HMC‐1, named HMC‐1560 and HMC‐1560,816, with different phenotypes and designated by the locations of specific mutations in the c‐kit proto‐oncogene. Activating mutations in the Kit receptor were characterized using the pyrosequencing™ method. Both sublines have a heterozygous T to G mutation at codon 560 in the juxtamembrane region of the c‐kit gene causing an amino acid substitution of Gly‐560 for Val. In contrast, only HMC‐1560,816 cells have the c‐kitV816 mutation found in mast cell neoplasms causing an Asp→Val substitution in the intracellular kinase domain. Kit was constitutively phosphorylated on tyrosine residues and associated with phosphatidylinositol 3′‐kinase (PI 3‐kinase) in both variants of HMC‐1, but this did not lead to a constitutive phosphorylation of Akt or extracellular regulated protein kinase (ERK), which are signalling molecules normally activated by the interaction of stem cell factor (SCF) with Kit. The documentation and characterization of two sublines of HMC‐1 cells provides both information on the biological consequences of mutations in Kit and recognition of the availability of what in reality are two distinct cultured human mast cell lines.
CD1d‐restricted natural killer T cells are potent targets for human immunodeficiency virus infection
Authors: Fleuridor Richardson , Wilson Brian , Hou Runhua , Landay Alan , Kessler Harold , Al‐Harthi Lena
Published: March 2003

Invariant human natural killer T cells (NKT) express a restricted T‐cell receptor (TCR) Vα24Vβ11 repertoire. These cells share both phenotypic and functional similarities between NK and T cells. Given the emerging role of NKT cells as critical cells in bridging the gap between innate and adaptive immunity, we examined their susceptibility to productive human immunodeficiency virus (HIV) infection by T‐tropic, M‐tropic, and primary isolates of HIV. We generated three human NKT cell clones (CA5, CA29, and CA31). Phenotypic characterization of these Vα24+ Vβ11+ clones indicated that they were predominately positive for CD4, CD161, HLA‐DR, CD38, CD45RO, and CD95 expression. The NKT cell clones expressed significantly more surface CCR5 molecules/cell and lower CXCR4 molecules/cell than phytohaemagglutinin‐stimulated peripheral blood mononuclear cells (PBMC). Consistent with the surface expression of CCR5 and CXCR4, the NKT clones were also selectively susceptible to HIV M‐tropic, T‐tropic, and primary isolate infection, as evaluated by both HIV p24 enzyme‐linked immunosorbent assay and intracellular staining of HIV proteins. The amount of p24 production was dependent on the NKT clone studied and the HIV strain used. Clones CA29 and CA31 were also susceptible to HIV IIIB infection. The virions produced by these clones were able to productively infect PHA‐stimulated PBMCs with the same kinetics as for primary infection of CD4+ blast. Collectively, this data demonstrates that NKT cells can be a target for productive HIV infection but with a lag in the time to peak p24 production.