bluuebunny/crossref_metadata_2025 huggingface dataset:
γδ T‐cell anergy in human immunodeficiency virus‐infected persons with opportunistic infections and recovery after highly active antiretroviral therapy
Authors: Martini F. , Urso R. , Gioia C. , De Felici A. , Narciso P. , Amendola A. , Paglia M. G. , Colizzi V. , Poccia F.
Published: March 2003

γδ T lymphocytes recognize non‐peptidic microbial antigens without antigen processing and major histocompatibility complex (MHC) restriction, representing an early defence mechanism against invading pathogens. As a defective response to non‐peptidic antigens was observed in human immunodeficiency virus‐positive (HIV+) persons, the aims of this study were twofold: to analyse the incidence of γδ T‐cell anergy in HIV+ patients with opportunistic infections/co‐infections (HIV‐OIC), and to investigate the role of highly active antiretroviral therapy (HAART) on γδ T‐cell functions. Peripheral γδ T‐cell distribution and in vitro reactivity to a non‐peptidic mycobacterial antigen, isopentenyl pyrophosphate (IPP), were analysed. γδ T‐cell subset distribution was altered more in HIV‐OIC patients than in asymptomatic HIV+ subjects (HIV‐ASY). Specifically, the Vδ2/Vδ1 ratio was inverted as a consequence of a decrease in Vδ2 T‐cell number. Moreover, IPP‐stimulated Vδ2 T cells from the HIV‐OIC group displayed a major defect in interferon‐γ (IFN‐γ) production. Interestingly, HAART induced a sustained recovery of naive CD45RA+ and CD62L+ T cells and restored γδ T‐cell function. Accordingly, in vitro CD45RA depletion resulted in γδ T‐cell hyporesponsiveness. Altogether, the incidence of γδ T‐cell anergy was increased in HIV‐OIC patients and dependent on CD45RA helper function. Moreover, HAART was able to restore γδ T‐cell reactivity, extending the immune recovery to non‐peptidic microbial antigens.
Feline leukaemia virus: protective immunity is mediated by virus‐specific cytotoxic T lymphocytes
Authors: Flynn J. N. , Hanlon L. , Jarrett O.
Published: March 2003

Feline leukaemia virus (FeLV) nucleic acid vaccination of domestic cats affords protection against viraemia and the development of latency without inducing antiviral antibodies.1To determine the contribution of cell‐mediated immunity to the control of virus replication and clearance from the host, FeLV‐specific cytotoxic T lymphocyte (CTL) responses were compared in vaccine‐protected, transiently viraemic, and persistently viraemic cats. Vaccinal immunity was associated with the detection of higher levels of virus‐specific effector CTL in the peripheral blood and lymphoid organs to FeLV Gag/Pro and Env antigens than those observed in unvaccinated control, persistently viraemic cats (P < 0·001). Likewise, higher levels of virus‐specific CTLs were also observed in transiently viraemic cats which recovered following exposure to FeLV. In cats that controlled their infection, recognition of Gag/Pro antigens was significantly higher than the recognition of Env antigens. This is the first report highlighting the very significant role that virus‐specific CTL have in determining the outcome of FeLV infection in either vaccinated cats or cats recovering naturally from FeLV exposure.
Immunoglobulin A cell distribution in the human small intestine: phenotypic and functional characteristics
Authors: Farstad I. N. , Carlsen H. , Morton H. C. , Brandtzaeg P.
Published: March 2003

We compared B‐cell phenotypes in Peyer's patches and solitary lymphoid follicles (organized gut‐associated lymphoid tissue, GALT) with those in jejunal or ileal lamina propria. In situ, immunostaining showed that small B cells of naive [surface immunoglobulin D‐positive (sIgD+) CD27–] and memory (sIgD± CD27+) phenotypes occurred almost exclusively in GALT, whereas the lamina propria contained only scattered sIgA+ CD27+ memory cells. In contrast, B‐cell blasts and plasma cells negative for CD20 and often also for CD19 but with strong expression of CD38, CD27 and cytoplasmic IgA (cIgA), dominated in the lamina propria but were scarce in GALT. By flow cytometry, the proportion of dispersed CD19+ B lymphocytes varied from 4 to 42% among jejunal mucosal samples; between 5 and 50% of these were sIgD+, suggesting a variable contamination with GALT cells. B‐cell blasts and plasma cells, identified by their large size and strong expression of CD38, were regularly found (25–35% of the total mononuclear cell population). Distinction between B‐cell blasts and mature plasma cells was made by the presence or absence of human leucocyte antigen (HLA) class II molecules, CD45RA, CD19 and surface immunoglobulin. No CD19+ B cells outside GALT expressed CD5, but a very small portion of the lamina propria B‐cell blasts were positive for CD28. Dispersed sIgA+ lamina propria cells expressed low levels of CD40, proliferated on CD40 ligation and constitutively secreted IgA in vitro. We concluded that the lamina propria B‐cell compartment consists mainly of B‐cell blasts and plasma cells but also has scattered, small sIgA+ cells that can proliferate in response to CD40 ligation and may therefore function as local memory cells for recall antigens.
An αβ T‐cell‐independent immunoprotective response towards gut coccidia is supported by γδ cells
Authors: Smith A. L. , Hayday A. C.
Published: March 2003

Although γδ cells are commonly hypothesized to provide a ‘first line of defence’, γδ‐cell‐deficient mice are generally only marginally more susceptible to pathogens. Because γδ cells are enriched within epithelia, it is important to resolve whether immunoprotective capacity towards epithelial‐tropic pathogens is absent from the γδ‐cell compartment, or whether such activity is present but simply redundant with that of αβ T cells. In this work, following infection of the intestinal epithelium of αβ T‐cell‐deficient mice with the coccidian parasite, Eimeria vermiformis, γδ cells were shown to support the rapid activation of other lymphoid cells and to confer a transferable antipathogen effect that could be eradicated by neutralization of interferon‐γ. However, unlike αβ T cells, these effects of γδ cells showed no evidence of functional immunological memory. These results are directly relevant to coccidiosis, an economically significant disease of livestock, and should have general relevance to infections involving αβ T‐cell deficiencies, e.g. cryptosporidiosis in patients with acquired immune deficiency syndrome (AIDS).
T helper 1‐type cytokine transcription in peripheral blood mononuclear cells of pseudorabies virus (Suid herpesvirus 1)‐primed swine indicates efficient immunization
Authors: Fischer T. , Büttner M. , Rziha H.‐J.
Published: March 2003

The induction of porcine cytokines, which are believed to be important for the regulation of T helper (Th)1‐ and Th2‐specific immune responses of pigs, was analysed after in vitro restimulation with a herpesvirus, Suid herpes 1 (pseudorabies virus [PRV]), in peripheral blood mononuclear cells (PBMC). To this end, quantitative, competitive reverse transcription–polymerase chain reaction (RT–qcPCR) was established using constructed heterologous DNA MIMICS, which contain cytokine‐ or glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH)‐specific primer‐binding sites. This is a simple method that allows reliable determination of the differing regulation of cytokine mRNAs specific for porcine interleukin (IL)‐2, ‐4 and ‐10, interferon gamma (IFN‐γ) and the housekeeping gene, GAPDH, as an endogenous control. PBMC derived from naive (innate response) and PRV‐primed (memory response) outbred swine were analysed comparatively. The results demonstrated that restimulation with PRV significantly enhanced the transcription of Th1‐type cytokines (IL‐2 and IFN‐γ) but not of Th2‐type cytokines (IL‐4 and IL‐10). This virus‐specific cytokine response was only found with PBMC from swine protected against lethal PRV challenge infection, but not with naive PBMC or with PBMC from pigs immunized with plasmid DNA encoding PRV glycoprotein gC. Notably, PBMC derived from immune and naive pigs constitutively produced relatively high amounts of IL‐10‐specific mRNA, exceeding that of GAPDH mRNA, independently of the addition of viral antigen or the mitogen concanavalin A (Con A). The results of this work should help to provide a better understanding of the effector cell/cytokine network response to infection with, or vaccination against, PRV. Additionally, the simple, reliable and sensitive RT–qcPCR, when used to determine the porcine cytokine pattern, might be of prognostic value for the induction of protective immunity.
Natural autoantibodies against heat‐shock proteins hsp70 and gp96: implications for immunotherapy using heat‐shock proteins
Authors: Ménoret A. , Chandawarkar R. Y. , Srivastava P. K.
Published: March 2003

Immunization of mice with cognate cancer‐derived heat‐shock protein (hsp) preparations leads to protection from cancer growth. As hsp used for vaccination or therapy are derived from autologous cancers, questions of pathological autoimmunity are of immense significance for the ongoing translation of this approach to therapy of human cancer. Employing the sera of normal adult mice as the first antibody, highly sensitive immunoblotting revealed the presence of anti‐hsp natural autoantibodies in healthy animals. Natural autoantibodies of the immunoglobulin D (IgD) isotype bind to gp96, whereas hsp70 was recognized by IgD and IgM autoantibodies. Neither hsp was recognized by the IgA, IgE or IgG immunoglobulins contained in the serum. The antigen–antibody recognition was titratable and dependent on the integrity of the IgD molecule. Sera from only a subset of the animals tested were found to be positive for autoantibodies against gp96 and hsp70, and individual and strain‐specific variations were detected. Injection of gp96 into healthy mice did not show sustained or consistent anti‐gp96 IgD antibody response, class switching, toxicity or pathological autoimmunity. IgD autoantibodies against gp96 and hsp70 were also not detected in the autoimmune lpr mice. These observations show the existence of a measured and tightly regulated natural immune response to hsp.
Modulation of delayed‐type hypersensitivity during the time course of immune response to a protein antigen
Authors: Jacysyn J. F. , Abrahamsohn I. A. , Macedo M. S.
Published: March 2003

Delayed‐type hypersensitivity reactions elicited in the footpad of ovalbumin‐sensitized mice after challenge with aggregated ovalbumin on day 4 or 8 of immunization are distinct. The former was characterized by a dense mononuclear infiltrate and, macroscopically, the reaction peaked at 48 hr after antigen challenge; the latter was preceded by immediate‐type reactions, reached the maximum at 24 hr and faded drastically later. Histologically, oedema and a mixed granulocytic–lymphocytic infiltrate was found at this time‐point. Immunoglobulin G1 (IgG1), IgG2a and IgE antibodies were detected only in plasma obtained after 8 days of immunization. Regarding the cytokines produced by draining lymph node cells after in vitro restimulation, interleukin‐4 (IL‐4) and IL‐10 were predominant after 4 days and interferon‐γ and IL‐2 after 8 days of immunization. These two types of delayed‐type hypersensitivity (DTH) were used to study the influence of antibody‐mediated responses on the inductive and effector phases of cell‐mediated immunity. The effector phase of DTH was not affected by immediate‐type reactions, as abrogation of these reactions by mediators' antagonists on day 8 or induction of passive reactions by transfer of immune serum on day 4 did not change the extent or kinetics of either type of DTH. Only transfer, before immunization, of whole or T‐cell‐enriched spleen cells, but not sera, from hyperimmunized donors (high antibody producers) abolished the induction of pure DTH in 4‐day immunized recipient mice and changed their cytokine profile to a T helper 2 type. These results indicate that in a non‐polarized immune response to a protein antigen there is initially a bias towards cell‐mediated immunity, which is gradually dampened by the development of antibody‐mediated immunity.
Local versus systemic control of numbers of endometrial T cells during pregnancy in sheep
Authors: Majewski A. C. , Tekin S. , Hansen P. J.
Published: March 2003

Pregnancy in sheep is associated with changes in numbers of specific T‐lymphocyte populations in the uterine endometrium. These changes probably contribute to evasion by the conceptus of maternal immunological rejection and indicate a possible role for T cells in placental growth, parturition and post‐parturient uterine defence against infection. The purpose of the present experiment was to evaluate the relative importance of systemic signals (i.e. those present throughout the uterus or from the circulation, including conceptus hormones secreted into the maternal blood) versus locally acting conceptus signals for regulating changes in numbers of endometrial lymphocytes during pregnancy. The approach taken was to surgically confine pregnancy to one uterine horn and compare differences in lymphocyte numbers between the two uterine horns as well as between both horns of pregnant ewes with those of ovariectomized ewes. As compared with ovariectomized ewes, there was a decline in numbers of CD45R+ lymphocytes within glandular epithelium and an increase in γδ T‐cell number within the luminal epithelium. These changes occurred in both the pregnant and non‐pregnant uterine horns of unilaterally pregnant ewes. Moreover, there were no significant differences in lymphocyte numbers between the two uterine horns of unilaterally pregnant ewes. Expression of CD25 was absent in tissues from both uterine horns. In conclusion, changes in numbers of endometrial lymphocytes during pregnancy, rather than due to locally acting signals of conceptus origin, are the result of hormonal signals of maternal or conceptus origin that either act directly on endometrial lymphocytes or stimulate the uterine endometrium to induce synthesis of regulatory molecules that affect lymphocyte dynamics.
Functional and phenotypic characterization of distinct porcine dendritic cells derived from peripheral blood monocytes
Authors: Paillot R. , Laval F. , Audonnet J.‐C. , Andreoni C. , Juillard V.
Published: March 2003

Dendritic cells (DCs) are bone marrow‐derived antigen‐presenting cells that have an exquisite capacity to interact with T cells and modulate their responses. Little is known about porcine DCs despite the fact that they represent an important target in strategies that are aimed at modulating resistance to infection in pigs and may be of major importance in transplantation biology. We generated immature monocyte‐derived porcine dendritic cells (MoDCs) directly from adherent peripheral blood cells treated with porcine granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐4 (IL‐4). The cells were observed via electron microscopy and their phenotype was characterized using monoclonal antibodies. The functionality of the porcine MoDCs was demonstrated showing that the cells were capable of different specialized functions relevant to antigen capture and were potent stimulators in a primary allo‐mixed leucocyte reaction. Treatment of the MoDCs with porcine cell line‐derived necrotic factors resulted in the phenotypic and functional maturation of MoDCs. We confirmed also that monocyte‐derived DCs were differentially regulated by cytokines, showing that transforming growth factor‐β1 (TGF‐β1) is able to redirect monocytic precursors into the differentiation pathway of Langerhans' cells presenting typical Birbeck granules. Interestingly, and in contrast to the human and murine model, we showed that the monocyte‐derived porcine Langerhans'‐type cells (MoLCs) were much more potent activators of allogeneic T cells than MoDCs obtained without TGF‐β1.
Differential endocytotic characteristics of a novel human B/DC cell line HBM‐Noda: effective macropinocytic and phagocytic function rather than scavenging function
Authors: Torii Ikuko , Morikawa Shigeru , Nagasaki Makoto , Nokano Akinobu , Morikawa Keiko
Published: March 2003

In order to characterize a novel human B cell‐lineage dendritic cell line (B/DC line) as an antigen‐presenting cell (APC), we compared three types of endocytosis (micropinocytosis via a clathrin‐coated pit, macropinocytosis via membrane ruffling, and phagocytosis) among myeloid‐related, macrophage (Mφ) cell lines and a B/DC line. In the present examination, we used a unique human dendritic cell (DC) line, HBM‐Noda (Noda). Flow cytometric and immunocytochemical analyses revealed that Noda not only expresses some DC markers, but also it expresses some B‐cell associated markers. Noda shows strong capacities to stimulate allogenic T cells, to produce immunoglobulin G (IgG), and to perform immunoglobulin gene rearrangment. These data strongly suggest that Noda is a B‐cell lineage DC line. The endocytic differences among these cell lines were as follows. (1) The level of micropinocytosis of Noda was significantly less than that of conventional human Mφ cell lines, and the formation of a clathrin‐coated pit was not observed in Noda. (2) The level of macropinocytosis of Noda was also smaller than that of conventional Mφ cells indicating that the active membrane ruffling of Noda induces rapid recycling. (3) Phagocytosis of opsonized sheep red blood cells (SRBC) was performed more efficiently in Noda than in other Mφ cell lines. Collectively, these data suggest that in human bone marrow cells, we can identify a unique DC subtype, B/DC line, which develops through a lymphoid DC‐differentiation pathway, and DC in this lineage plays an important role in the host immune response because of its effective uptake of a variety of size of antigens by using the skilful membrane ruffling and surface receptors
Third complementarity‐determining region of mutated VH immunoglobulin genes contains shorter V, D, J, P, and N components than non‐mutated genes
Authors: Rosner Karli , Winter David B. , Tarone Robert E. , Skovgaard Gunhild Lange , Bohr Vilhelm A. , Gearhart Patricia J.
Published: March 2003

The third complementarity‐determining region (CDR3) of immunoglobulin variable genes for the heavy chain (VH) has been shown to be shorter in length in hypermutated antibodies than in non‐hypermutated antibodies. To determine which components of CDR3 contribute to the shorter length, and if there is an effect of age on the length, we analysed 235 cDNA clones from human peripheral blood of VH6 genes rearranged to immunoglobulin M (IgM) constant genes. There was similar use of diversity (D) and joining (JH) gene segments between clones from young and old donors, and there was similar use of D segments among the mutated and non‐mutated heavy chains. However, in the mutated heavy chains, there was increased use of shorter JH4 segments and decreased use of longer JH6 segments compared to the non‐mutated proteins. The overall length of CDR3 did not change with age within the mutated and non‐mutated categories, but was significantly shorter by three amino acids in the mutated clones compared to the non‐mutated clones. Analyses of the individual components that comprise CDR3 indicated that they were all shorter in the mutated clones. Thus, there were more nucleotides deleted from the ends of VH, D, and JH gene segments, and fewer P and N nucleotides added. The results suggest that B cells bearing immunoglobulin receptors with shorter CDR3s have been selected for binding to antigen. A smaller CDR3 may allow room in the antibody binding pocket for antigen to interact with CDRs 1 and 2 as well, so that as the VDJ gene undergoes hypermutation, substitutions in all three CDRs can further contribute to the binding energy.
The pan‐chemokine inhibitor NR58‐3.14.3 abolishes tumour necrosis factor‐α accumulation and leucocyte recruitment induced by lipopolysaccharide in vivo
Authors: Reckless Jill , Tatalick Lauren M. , Grainger David J.
Published: March 2003

Chemokines participate in the regulation of leucocyte recruitment in a wide variety of inflammatory processes, including host defence and diseases such as asthma, atherosclerosis and autoimmune disorders. We have previously described the properties of Peptide 3, the first broad‐specificity chemokine inhibitor in vitro. Here, we report the properties of NR58‐3.14.3, a retroinverso analogue of Peptide 3. NR58‐3.14.3 inhibited leucocyte migration induced by a range of chemokines, including monocyte chemoattractant protein‐1 (MCP‐1) (2·5 nm), macrophage inflammatory protein‐1α (MIP‐1α) (5 nm), regulated on activation, normal T‐cell expressed and presumably secreted (RANTES) (20 nm), stromal cell‐derived factor‐1α (SDF‐1α) (25 nm) and interleukin‐8 (IL‐8) (30 nm), but did not affect migration induced by N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) or complement C5a (> 100 µm). NR58‐3.14.3 is therefore ≈ 1000‐fold more potent than Peptide 3 but retains the broad‐spectrum chemokine inhibitory activity of the parent peptide. In vivo, pretreatment with a systemic dose of 10 mg of NR58‐3.14.3, but not the inactive derivative NR58‐3.14.4, abolished leucocyte recruitment in response to intradermal injection of 500 ng of MCP‐1 into rat skin. This suggests that NR58‐3.14.3 is a functional chemokine inhibitor in vivo as well as in vitro. We utilized NR58‐3.14.3 as a tool to investigate the role of chemokine activity during leucocyte recruitment in response to lipopolysaccharide (LPS) in vivo. NR58‐3.14.3, but not NR58‐3.14.4, abolished leucocyte recruitment in response to intradermal injection of 50 ng of LPS into rat skin. Furthermore, NR58‐3.14.3 completely inhibited LPS‐induced accumulation of tumour necrosis factor‐α (TNF‐α). This data is consistent with a model in which multiple chemokines act in parallel upstream of TNF‐α. NR58‐3.14.3 is therefore a powerful anti‐inflammatory agent in vivo, suppressing proinflammatory cytokine production and leucocyte recruitment in response to endotoxin stimulus in rat skin.
Allergen‐induced murine upper airway inflammation: local and systemic changes in murine experimental allergic rhinitis
Authors: Saito Hiroko , Howie Karen , Wattie Jennifer , Denburg Avram , Ellis Russ , Inman Mark D. , Denburg Judah A.
Published: March 2003

The role of inflammatory effector cells in the pathogenesis of airway allergy has been the subject of much investigation. However, whether systemic factors are involved in the development of local responses in both upper and lower airways has not been fully clarified. The present study was performed to investigate aspects of the pathogenesis of isolated allergic rhinitis in a murine model sensitized to ovalbumin (OVA). Both upper‐ and lower‐airway physiological responsiveness and inflammatory changes were assessed, as well as bone marrow progenitor responses, by culture and immunohistological methods. Significant nasal symptoms and hyper‐responsiveness appeared after intranasal OVA challenge (P < 0·0001 and P < 0·01, respectively), accompanied with significant nasal mucosal changes in CD4+ cells (P < 0·001), interleukin (IL)‐4+ cells (P < 0·01), IL‐5+ cells (P < 0·01), basophilic cells (P < 0·02) and eosinophils (P < 0·001), in the complete absence of hyper‐responsiveness or inflammatory changes in the lower airway. In the bone marrow, there were significant increases in CD34+ cells, as well as in eosinophils and basophilic cells. In the presence in vitro of mouse recombinant IL‐5, IL‐3 or granulocyte–macrophage colony‐stimulating factor (GM‐CSF), the level of bone marrow eosinophil/basophil (Eo/Baso) colony‐forming cells increased significantly in the OVA‐sensitized group. We conclude that, in this murine model of allergic rhinitis, haemopoietic progenitors are upregulated, which is consistent with the involvement of bone marrow in the pathogenesis of nasal mucosal inflammation. Both local and systemic events, initiated in response to allergen provocation, may be required for the pathogenesis of allergic rhinitis. Understanding these events and their regulation could provide new therapeutic targets for rhinitis and asthma.
Interleukin‐10‐secreting Peyer's patch cells are responsible for active suppression in low‐dose oral tolerance
Authors: Tsuji Noriko M. , Mizumachi Koko , Kurisaki Jun‐ichi
Published: March 2003

We demonstrate the induction of antigen‐specific interleukin‐10 (IL‐10)‐secreting cells in murine Peyer's patches (PPs) after low‐dose β‐lactoglobulin (BLG) feeding. In addition, we show that PP cells can inhibit the T‐cell proliferative response in vitro as well as T‐cell‐mediated inflammation in vivo. The active suppression mediated by these regulatory cells was seen only within a narrow range of antigen dosage (feeding), with the most prominent effect at 5 × 1 mg BLG. On either side of this range, T‐helper 1‐like cytokine responses were observed when PP cells were stimulated with antigen in vitro. This result correlated with reduced production of regulatory cytokines as well as reduced activity of bystander suppression. We found that changes in IL‐10 production correlated inversely with changes in interferon‐γ production. Inhibitory effects mediated by CD4+ PP cells were partially neutralized by antibodies to IL‐10 and transforming growth factor‐β. Interestingly, the generation of such regulatory cells after low‐dose BLG feeding exhibited organ dependence. Among spleen, lymph node and PP cells derived from orally tolerized mice, PP cells were the most effective in promoting bystander suppression in the presence of BLG, indicating the significance of PPs as an inductive site for antigen‐specific regulatory cells upon induction of low‐dose oral tolerance. Moreover, PP cells from mice fed 5 × 1 mg BLG were shown to suppress a BLG‐specific delayed‐type hypersensitivity response induced in footpads, suggesting that IL‐10‐secreting PP cells regulate systemic inflammation.
Multiple cytokines sharing the common receptor γ chain can induce CD154/CD40 ligand expression by human CD4+ T lymphocytes via a cyclosporin A‐resistant pathway
Authors: Fayen John D.
Published: March 2003

Expression of CD154/CD40 ligand (CD154/CD40L), an important molecular component of CD4+ T‐cell help, can be triggered by T‐cell receptor (TCR) stimulation. Dephosphorylation of the transcriptional element Nuclear Factor of Activated T cells‐1 (NFAT1) is a critical activation step in the TCR‐initiated signal transduction cascade which promotes CD154/CD40L expression. Cyclosporin A (CsA), which interferes with NFAT1 activation, has been shown to be an effective inhibitor of TCR‐triggered CD154/CD40L expression by resting T cells. We now report that recombinant interleukin‐2 (rIL‐2) is also capable of inducing CD154/CD40L on CD4+ T lymphoblasts via a pathway triggered independently of the CD3/TCR receptor complex. Recombinant IL‐2‐mediated CD154/CD40L expression, in contrast to that triggered by CD3/TCR stimulation, is only partially inhibited by CsA. The capacity of rIL‐2 to induce CD154/CD40L expression by T lymphoblasts also extends to a restricted number of cytokines sharing the cytokine receptor common γ chain, including IL‐15, and, to a lesser extent, IL‐7, but not IL‐4. A similar CsA‐resistant CD154/CD40L induction pathway can be triggered in primary T cells by the combination of anti‐CD3 stimulation and recombinant lymphokines. In contrast to T lymphoblasts, the CsA‐resistant CD154/CD40L induction in primary lymphocytes can be efficiently triggered by multiple cytokines which bind the common γ chain receptor family. The data outline a novel pathway of CD154/CD40L induction which is, at least in part, independent of NFAT1 and resistant to CsA. A more complete understanding of the mechanisms governing CD154/CD40L expression may facilitate the rational design of specifically targeted immunotherapeutic agents.
Allograft inflammatory factor‐1 augments production of interleukin‐6, ‐10and ‐12 by a mouse macrophage line
Authors: Watano Keiko , Iwabuchi Kazuya , Fujii Satoshi , Ishimori Naoki , Mitsuhashi Shinya , Ato Manabu , Kitabatake Akira , Onoé Kazunori
Published: March 2003

Mouse allograft inflammatory factor‐1 (AIF‐1) cDNA was cloned and the AIF‐1‐specific monoclonal antibodies were established to examine its tissue distribution. The mouse AIF‐1 was highly conserved among all reported AIF‐1 from a variety of species, from invertebrates to mammals, and the cloned cDNA was in good accordance with putative expressed regions of genomic sequences in the mouse major histocompatibility complex (MHC) class III region. The messages of mouse AIF‐1 were abundantly expressed in the testis, moderately in the spleen and lymph nodes and slightly in the liver and thymus of normal BALB/c mice. Immunohistological examination revealed that differentiating germ cells in the testis and presumably macrophages in the red pulp of the spleen were positive for AIF‐1. To analyse the function of the AIF‐1, a macrophage cell line, RAW 264.7, was transfected with mouse AIF‐1 cDNA. Upon stimulation with bacterial lipopolysaccharide, the transfectants that overexpressed AIF‐1 showed marked morphological changes and produced significantly large amounts of interleukin (IL)‐6, IL‐10 and IL‐12p40 but not IL‐12p70 compared with control cells. No difference was noted in production of tumour necrosis factor‐α, transforming growth factor‐β1 and IL‐1α. These results suggest that AIF‐1 plays an important role in cells of a monocyte/macrophage lineage upon stimulation with inflammatory stimuli by augmenting particular cytokine production.
Failure to induce enhanced protection against tuberculosis by increasing T‐cell‐dependent interferon‐γ generation
Authors: Leal Irene S. , Smedegård Birgitte , Andersen Peter , Appelberg Rui
Published: March 2003

We evaluated the use of recombinant human interleukin‐6 (rhIL‐6) and a monoclonal antibody specific for interferon‐γ (IFN‐γ) as co‐adjuvants in a subunit vaccine against tuberculosis consisting of the culture filtrate proteins of Mycobacterium tuberculosis (ST‐CF) emulsified in the adjuvant dimethyl‐dioctadecylammonium bromide (DDA). Both the addition of rhIL‐6 and the neutralization of IFN‐γ resulted in an increased T helper type 1 (Th1) response characterized by enhanced IFN‐γ production and cell proliferation. Nevertheless, this did not result in the enhancement of protection against either an intravenous or an aerosol M. tuberculosis challenge. Our data stress the need to identify further correlates of protection in addition to IFN‐γ production to screen vaccines against tuberculosis infection.
Human immunodeficiency virus type 1 Tat binds to Candida albicans, inducing hyphae but augmenting phagocytosis in vitro
Authors: Gruber Andreas , Lell Claudia P. , Speth Cornelia , Stoiber Heribert , Lass‐Flörl Cornelia , Sonneborn Anja , Ernst Joachim F. , Dierich Manfred P. , Würzner Reinhard
Published: March 2003

Tat, the human immunodeficiency virus type 1 (HIV‐1) transactivating protein, binds through its RGD‐motif to human integrin receptors. Candida albicans, the commonest cause of mucosal candidiasis in subjects infected with HIV‐1, also possesses RGD‐binding capacity. The present study reveals that Tat binds to C. albicans but not to C. tropicalis. Tat binding was markedly reduced by laminin and to a lesser extent by a complement C3 peptide containing the RGD motif, but not by a control peptide. The outgrowth of C. albicans was accelerated following binding of Tat, but phagocytosis of opsonized C. albicans was also increased after Tat binding. Thus, Tat binding promotes fungal virulence by inducing hyphae but may also reduce it by augmenting phagocytosis. The net effect of Tat in vivo is difficult to judge but in view of the many disease‐promoting effects of Tat we propose that accelerating the formation of hyphae dominates over the augmentation of phagocytosis.
Expression of β‐defensin 1 and 2 mRNA by human monocytes, macrophages and dendritic cells
Authors: Duits Louise A. , Ravensbergen Bep , Rademaker Mirjam , Hiemstra Pieter S. , Nibbering Peter H.
Published: March 2003

Human β‐defensins are broad‐spectrum antimicrobial peptides known to be produced by epithelial cells. It was recently shown that β‐defensins also display chemotactic activity for dendritic cells (DC) and T cells, and thus may serve to link innate and adaptive immunity. The aim of the present study was to explore expression of mRNA for these peptides in mononuclear phagocytes and DC. The results revealed that monocytes, monocyte‐derived‐macrophages (MDM), and monocyte‐derived‐dendritic cells (DC) all express human‐β‐defensin‐1 (hBD‐1) mRNA. hBD‐1 mRNA expression by monocytes and MDM was increased after activation with interferon‐γ (IFN‐γ) and/or lipopolysaccharide (LPS) in a dose‐ and time‐dependent fashion. Alveolar macrophages showed an intense hBD‐1 expression, which could not be further increased. Expression of hBD‐1 mRNA by immature DC was low, and increased considerably after maturation. Monocytes, MDM, alveolar macrophages and DC showed a limited expression of human β‐defensin‐2 (hBD‐2) mRNA, which could only be increased in monocytes and alveolar macrophages by IFN‐γ and/or LPS in a dose‐ and time‐dependent fashion. Immunocytochemical stainings demonstrated the expression of hBD‐2 peptide by freshly isolated blood monocytes and alveolar macrophages in cytospin preparations.
Human small intestinal mucosa harbours a small population of cytolytically active CD8+ αβ T lymphocytes
Authors: Melgar Silvia , Bas Anna , Hammarström Sten , Hammarström Marie‐Louise
Published: March 2003

Intraepithelial lymphocytes (IEL) in normal human small intestine exhibit cytotoxicity. This study was undertaken to characterize the effector cells and their mode of action. Freshly isolated jejunal IEL and lamina propria lymphocytes (LPL), as well as IEL and LPL depleted of CD4+, CD8+ and T‐cell receptor (TCR)‐γδ+ cells were used as effector cells in anti‐CD3‐mediated redirected cytotoxicity against a murine FcγR‐expressing cell line. Effector cell frequencies were estimated by effector to target cell titration and limiting dilution. The capacity of IEL and LPL to kill a Fas‐expressing human T‐cell line was also analysed. T‐cell subsets were analysed for perforin, granzyme B, Fas‐ligand (FasL), tumour necrosis factor‐α (TNF‐α) and TNF‐related apoptosis inducing ligand (TRAIL) mRNA expression by reverse transcription–polymerase chain reaction (RT‐PCR). Frequencies of IEL expressing the perforin and FasL proteins were determined by immunomorphometry. Both IEL and LPL exhibited significant Ca2+‐dependent, anti‐CD3‐mediated cytotoxicity, ≈ 30% specific lysis at the effector to target cell ratio 100. The cytotoxic cells constituted, however, only a small fraction of IEL and LPL (≈ 0·01%). CD8+ TCR‐αβ+ cells accounted for virtually all the cytotoxicity and expressed mRNA for all five cytotoxic proteins. The frequency of granzyme B‐expressing samples was higher in CD8+ cells than in CD4+ cells (P<0·05 and <0·01 for IEL and LPL, respectively). In addition, both IEL and LPL exhibited significant spontaneous anti‐CD3‐independent cytotoxicity against Fas‐expressing human T cells. This killing was mediated by Fas–FasL interaction. On average, 2–3% of the IEL expressed perforin and FasL. We speculate that CD8+ memory cells accumulate in the jejunal mucosa and that the CD8+ TCR‐αβ+ lymphocytes executing TCR/CD3‐mediated, Ca2+‐dependent cytotoxicity are classical cytotoxic T lymphocytes ‘caught in the act’ of eliminating infected epithelial cells through perforin/granzyme exocytosis. The observed Fas/FasL‐mediated cytotoxicity may be a reflection of ongoing down‐regulation of local immune responses by ‘activation‐induced cell death’.
Mechanism of induction of complement susceptibility of erythrocytes by spider and bacterial sphingomyelinases
Authors: Tambourgi Denise V. , De Sousa Da Silva Marcelo , Billington Stephen J. , Gonçalves De Andrade Rute M. , Magnoli Fábio C. , Songer J. Glenn , Van Den Berg Carmen W.
Published: March 2003

We have recently shown that the sphingomyelinase toxins P1 and P2 from the venom of the spider Loxosceles intermedia induce complement (C)‐dependent lysis of autologous erythrocytes by induction of the cleavage of cell surface glycophorins through activation of an endogenous metalloproteinase facilitating the activation of the alternative pathway of C. Phospholipase D (PLD) from Corynebacterium pseudotuberculosis shows some degree of homology with the spider sphingomyelinases and can induce similar clinical symptoms to those observed after spider envenomation. The aim of this study was to investigate if the bacterial PLD‐induced haemolysis of human erythrocytes was C dependent and if cleavage of glycophorins occurred. We show here that haemolysis of both PLD‐ and P1‐treated human erythrocytes was C dependent, but while PLD‐mediated haemolysis was dependent on activation of the classical pathway of C, P1 induced lysis via both the classical and alternative pathways. P1, but not PLD, induced cleavage of glycophorins and no change in expression of complement regulators was induced by either of the toxins. In both cases, annexin V binding sites were exposed, suggesting that the membrane asymmetry had been disturbed causing exposure of phosphatidylserine to the cell surface. Our results suggest that C susceptibility induced by L. intermedia and C. pseudotuberculosis PLD is a result of exposure of phosphatidylserine, and the higher potency of P1 toxin can be explained by its additional effect of cleavage of glycophorins.
Life and death within germinal centres: a double‐edged sword
Authors: Guzman‐Rojas Liliana , Sims‐Mourtada Jennifer C. , Rangel Roberto , Martinez‐Valdez Hector
Published: March 2003

Within germinal centres, B lymphocytes are destined to die by apoptosis via Fas signalling, unless they are positively rescued by antigen and by signals initiated by CD40–CD154 interactions. Thus, while the germinal centre microenvironment can become a virtual graveyard for most B lymphocytes that fail to bind antigen with high affinity, it concomitantly provides the necessary stimuli for the survival of cells that successfully accomplish affinity maturation. Such dichotomy in the physiology of germinal centre reaction that results in survival of the functional B‐cell repertoire and the elimination of abnormal cells, dictates the fate towards B‐cell homeostasis or disease. Consequently, the death and survival‐signalling arms within germinal centres predominantly reside on the timely and controlled expression of Fas and its ligand (FasL), and CD40 and CD154, respectively. In keeping with this notion, lymphoproliferation or deficient immunity are documented landmarks of inactivation of either the Fas/FasL or CD40/CD154 signalling pathways. The present review considers two different scenarios in the control of B‐cell survival and death within germinal centres. The first is an idealistic scenario, in which a discriminatory and co‐ordinate signalling initiated by the CD40/CD154 and Fas/FasL pairs, respectively, leads the rescue of the functional B‐cell repertoire and the elimination of the abnormal phenotype. The second is a gloomy scenario in which both the lack and the hyperexpression of either receptor/ligand pairs, are seen as equally deleterious.
Inducible nitric oxide synthase deficiency in mice increases resistance to chronic infection with Echinococcus multilocularis
Authors: Dai Wen J. , Waldvogel Andreas , Jungi Thomas , Stettler Marianne , Gottstein Bruno
Published: March 2003

The production of nitric oxide (NO) by intraperitoneal macrophages of mice during secondary infection with Echinococcus multilocularis mediates immunosuppression at early and late stages of infection. We addressed the role of NO in host resistance against this extracellular metazoan parasite by infecting inducible nitric oxide synthase knockout ((iNOS‐KO) mice (of the C57BL/6 background) with 100 metacestode vesicles. The parasite weight was significantly lower in iNOS‐KO mice when compared with wild‐type (WT) mice at 4 months postinfection (late stage), thus demonstrating that iNOS deficiency confers a certain degree of resistance against persistent chronic infection. However, histological analysis of periparasitic tissue showed no differences between WT and iNOS‐KO mice, as both exhibited granuloma formation and the presence of giant cells. Together with histology, the production of a high level of interferon‐γ (IFN‐γ) in infected iNOS‐KO mice upon stimulation with concanavalin A (Con A) and VF‐antigen indicated normal T‐cell signalling in these animals. As expected, peritoneal exudate cells (PEC) from infected iNOS‐KO mice produced no detectable NO, while the PEC from infected WT mice produced high levels of NO after stimulation with lipopolysaccharide (LPS) and parasite protein or carbohydrate antigen, or even without in vitro stimulation. Consequently, the high level of NO production observed during chronic infection in WT mice appears to contribute more to immunosuppression than to limitation of parasite growth. This is also reflected by the fact that splenocyte proliferation was significantly higher and parasite masses lower in iNOS‐KO mice (at 1 and 4 months postinfection) than in WT mice.
Phenotypic profile and functional characterization of rat lymph node‐derived γδ T cells: implication in the immune response to cytomegalovirus
Authors: Dyugovskaya Larissa , Hirsh Mark , Ginsburg Haim
Published: March 2003

γδ T cells are unique, and their localization at sites of infection is considered critical in immune defence. We demonstrate the accumulation of γδ T cells in rat regional popliteal lymph nodes (PLNi) starting 2 days after inoculation of cytomegalovirus (CMV) into the footpad. Early‐appearance PLNi γδ T cells significantly inhibited plaque development and the spread of CMV infection. These γδ T cells were negative for CD4 and CD8beta receptors, proliferated in response to interleukin‐2 (IL‐2) and contained high levels of interferon‐γ (IFN‐γ), the appearance of which correlated with the curing of fibroblasts from virus infection. The addition of anti‐IFN‐γ abolished the ability of fibroblast monolayers to be cured from CMV infection. In contrast, this protection was not abolished by the addition of anti‐rat IL‐2 or anti‐rat TNF‐α, or by the depletion of NKR‐P1‐bearing cells within γδ T cells. In addition, the present study shows that while γδ T cells derived from naive and CMV‐infected rats are able to kill both YAC‐1 targets and CMV‐infected syngeneic fibroblasts in vitro, only the latter are able to clear CMV‐infected fibroblast monolayers. Finally, our data suggest that the expression of NKR‐P1 by γδ T cells is critical for cytotoxicity, but its contribution to the curing from CMV infection was limited.
The role of intermediaries in electronic marketplaces: developing a contingency model
Authors: Giaglis George M. , Klein Stefan , O'Keefe Robert M.
Published: March 2003

Abstract.Early research in electronic markets seemed to suggest that e‐ commerce transactions would result in decreased costs for buyers and sellers alike, and would therefore ultimately lead to the elimination of intermediaries from electronic value chains. However, a careful analysis of the structure and functions of electronic marketplaces reveals a different picture. Intermediaries provide many value‐adding functions that cannot be easily substituted or ‘internalized’ through direct supplier–buyer dealings, and hence mediating parties may continue to play a significant role in the e‐commerce world. In this paper we provide an analysis of the potential roles of intermediaries in electronic markets and we articulate a number of hypotheses for the future of intermediation in such markets. Three main scenarios are discussed: the disintermediation scenario, in which market dynamics will favour direct buyer–seller transactions; the reintermediation scenario, in which traditional intermediaries will be forced to differentiate themselves and re‐emerge in the electronic marketplace; and the cybermediation scenario, in which wholly new markets for intermediaries will be created. The analysis suggests that the likelihood of each scenario dominating a given market is primarily dependent on the exact functions that intermediaries play in each case. A detailed discussion of such functions is presented in the paper, together with an analysis of likely outcomes in the form of a contingency model for intermediation in electronic markets.
Assessing and managing the benefits of enterprise systems: the business manager's perspective
Authors: Shang Shari , Seddon Peter B
Published: March 2003

This paper focuses on the benefits that organizations may achieve from their investment in enterprise systems (ES). It proposes an ES benefit framework for summarizing benefits in the years after ES implementation. Based on an analysis of the features of enterprise systems, on the literature on information technology (IT) value, on data from 233 enterprise systems vendor‐reported stories published on the Web and on interviews with managers of 34 organizations using ES, the framework provides a detailed list of benefits that have reportedly been acquired through ES implementation. This list of benefits is consolidated into five benefits dimensions: operational, managerial, strategic, IT infrastructure and organizational, and illustrated using perceived net benefit flow (PNBF) graphs. In a detailed example, the paper shows how the framework has been applied to the identification of benefits in a longitudinal case study of four organizations.
Cultivation and engineering of a software metrics program
Authors: Iversen Jakob , Mathiassen Lars
Published: March 2003

Abstract. This paper reports from a case study of an organization that implements a software metrics program to measure the effects of its improvement efforts. The program measures key indicators of all completed projects and summarizes progress information in a quarterly management report. The implementation turns out to be long and complex, as the organization is confronted with dilemmas based on contradictory demands and value conflicts. The process is interpreted as a combination of a rational engineering process in which a metrics program is constructed and put into use, and an evolutionary cultivation process in which basic values of the software organization are confronted and transformed. The analysis exemplifies the difficulties and challenges that software organizations face when bringing known principles for software metrics programs into practical use. The article discusses the insights gained from the case in six lessons that may be used by Software Process Improvement managers in implementing a successful metrics program.
Molecular cloning and chromosomal localization of a prophenoloxidase cDNA from the malaria vector Anopheles gambiae
Authors: Lee W.‐J. , Ahmed A. , Della Torre A. , Kobayashi A. , Ashida M. , Brey P. T.
Published: March 2003

A cDNA clone for prophenoloxidase was isolated from the most important human malaria vector, Anopheles gambiae. The clone encoded a polypeptide of 79341 Da that contains the two copper binding domains common to all invertebrate prophenoloxidases and haemocyanins. Expression of the prophenoloxidase gene was detected throughout all life stages from egg to imago in two strains of A. gambiae; however, the strongest expression was observed in developing embryos in eggs. The prophenoloxidase gene was mapped to the inversion rich region of the right arm of chromosome‐2 in region 13B.
Patterns of mitochondrial versus nuclear DNA sequence divergence among nymphalid butterflies: the utility of wingless as a source of characters for phylogenetic inference
Authors: Brower A. V. Z. , DeSalle R.
Published: March 2003

To investigate the utility of a region of wingless (wg) as a marker for molecular systematics, we compared wg sequences to mitochondrial COII sequences from twenty‐two nymphalid butterfly taxa and one outgroup. Compositional characteristics of the two gene regions are compared, and their contributions to a cladogram inferred from the combined data set are assessed. Primarily due to its uniform base composition, wg appears to become saturated more slowly than mtDNA, although the two genes appear to be evolving at quite similar rates. We suggest that wg will be a useful source of characters for phylogenetic studies of butterflies, and perhaps other insect taxa, with divergence times up to 60 million years ago.