bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of the induction of the inducible nitric oxide synthase in murine brain microglial cells by sodium salicylate
Authors: KIM  , LEE  , SHIN  , CHUNG  , AN
Published: March 2003

The induction of the inducible nitric oxide synthase (iNOS) has been proposed to play a role in a variety of inflammatory diseases. Sodium salicylate (NaSal) is the most commonly used anti‐inflammatory agent. We investigated whether NaSal can diminish the induction of iNOS in murine brain microglial cells. In primary cultures, interferon‐γ (IFN‐γ) or lipopolysaccharide (LPS) separately did not stimulate nitric oxide (NO) production, whereas IFN‐γ combined with LPS synergistically induced iNOS. NaSal inhibited both the production of NO and expression of iNOS in microglial cells. Synergy between IFN‐γ and LPS was mainly dependent on tumour necrosis factor‐α (TNF‐α) secretion as the increase of the induction of the iNOS by IFN‐γ plus LPS was associated with the increase of TNF‐α secretion and IFN‐γ plus LPS‐induced TNF‐α secretion by microglial cells was decreased by the treatment with NaSal. These results suggest a possible use of NaSal in managing inflammation of the central nervous system through inhibition of the iNOS induction.
Interleukin‐15 differentially enhances the expression of interferon‐γ and interleukin‐4 in activated human (CD4+) T lymphocytes
Authors: BORGER  , KAUFFMAN  , POSTMA  , ESSELINK  , VELLENGA
Published: March 2003

In this study interleukin (IL)‐15 was examined for its ability to modulate the expression of interferon‐γ (IFN‐γ) and IL‐4 in activated human T lymphocytes. The effect of IL‐15 was compared with IL‐2 and IL‐7, cytokines all known to use the IL‐2 receptor γC chain. The results demonstrate that the extent of upregulation of IFN‐γ and IL‐4 mRNA was dependent on the applied cytokine (IL‐2>IL‐15>IL‐7) and on the stimulatory signal. IFN‐γ and IL‐4 mRNAs were upregulated by IL‐15 in concanavalin A‐ (twofold) and anti‐CD3 plus anti‐CD28‐ (fivefold) stimulated T lymphocytes. IFN‐γ mRNA accumulation, but not IL‐4 mRNA, was additively upregulated by IL‐15 plus IL‐7 (ninefold) in anti‐CD3 stimulated T lymphocytes, and bypassed the requirement of CD28 signalling. Fluorescence‐activated cell sorting (FACS) experiments demonstrated that IFN‐γ mRNA was upregulated by IL‐15 in both CD4+ and CD8+ T lymphocytes, whereas IL‐4 mRNA accumulation predominantly occurred in CD4+ cells. Preincubation of highly purified CD4+ T lymphocytes during 7 days with IL‐15 and/or IL‐7, followed by activation, also showed enhanced IL‐4 protein secretion, but predominantly upregulated IFN‐γ protein. The net effect was a dramatically increased IFN‐γ/IL‐4 ratio. Taken together, IL‐15 and IL‐7 can act as costimulatory signals, which may favour a T helper 1 (Th1) immune response, particularly in the absence of sufficient CD28 costimulation.
B‐cell antigens within normal and activated human T cells
Authors: SANDILANDS  , PERRY  , WOOTTON  , HAIR  , MORE
Published: March 2003

In this study we compared cell surface staining for human peripheral blood lymphocyte (PBL) CD antigens by flow cytometry, with staining obtained following permeabilization of PBL using the Cytoperm method (Serotec). Six CD antigens (CD20, CD21, CD22, CD32, CD35 and major histocompatibility complex class II antigen) normally found on the surface of B cells, were also found to be expressed within T cells. We also showed, by immunoelectron microscopy, that these inappropriately expressed (‘occult’) CD antigens are located within cytoplasmic vesicles or within the rough endoplasmic reticulum. Following in vitro activation of T cells a distinct increase in expression of all of these cytoplasmic antigens was observed but staining at the cell surface was, by comparison, weak. We therefore propose that up‐regulation of various B‐cell CD antigens occurs within the cytoplasm of T cells following activation and that these antigens may be synthesized and released into the fluid‐phase as soluble immunoregulatory molecules.
The role of urocanic acid in UVB‐induced suppression of immunity to Trichinella spiralis infection in the rat
Authors: GARSSEN  , NORVAL  , CROSBY  , DORTANT  , VAN LOVEREN
Published: March 2003

The naturally occurring trans‐isomer of urocanic acid (trans‐UCA), found in the stratum corneum, absorbs ultraviolet light (UV) and isomerizes to the cis‐form. Cis‐UCA has been shown to impair some cellular immune responses, and has been proposed as an initiator of the suppression that follows UV irradiation. UVB exposure leads to an increase in cis‐UCA in the skin of rats from about 10% to 40% of the total UCA. Previously it has been demonstrated that UVB lowers immune responses to Trichinella spiralis after oral infection of rats with the parasitic worm. In the present study we investigated the role of cis‐UCA in the control of this parasitic infection. Rats were infected orally with T. spiralis and injected with different doses of cis‐ or trans‐UCA subcutaneously. Mitogenic responses and the mixed lymphocyte reaction were not affected by either isomer. In contrast, the number of T. spiralis larvae in muscle tissue of infected rats was increased significantly in the cis‐UCA‐treated animals compared with the trans‐UCA‐treated animals. In addition, delayed‐type hypersensitivity (DTH) to T. antigen in infected rats was significantly impaired by cis‐UCA but not by trans‐UCA. If rats were injected with a monoclonal antibody with specificity for cis‐UCA 2 hr prior to UVB exposure, the UVB‐induced suppression in DTH to T. spiralis and the increase in larvae counts were significantly inhibited compared with rats that were similarly injected with a control antibody. Thus cis‐UCA can inhibit the specific resistance to parasitic infections and acts as an important mediator of UVB‐induced suppression of immunity to T. spiralis in the rat.
Splenic but not thymic autoreactive T cells from New Zealand Black mice respond to a dominant erythrocyte Band 3 peptide
Authors: SHEN  , WRAITH  , ELSON
Published: March 2003

Previous work from our laboratory suggested that erythrocyte Band 3 peptide 861–874 is the dominant epitope recognized by splenic T cells from adult New Zealand Black (NZB) mice that are developing autoimmune haemolytic anaemia (AIHA). Here, it is shown that splenic T cells from 6‐week‐old NZB mice mount a vigorous in vitro proliferative response to peptide 861–874 and some other selected Band 3 peptides. As the donors grow older, splenic T cells respond to an increasing number of Band 3 peptides and the magnitude of their response also becomes greater. Splenic T cells from 3‐week‐old NZB mice still responded vigorously to peptide 861–874 and Band 3. By contrast, neither thymocytes nor single‐positive CD4‐enriched thymus cells from NZB mice responded to peptide 861–874 or Band 3, although they responded to concanavalin A (Con A). However, thymocytes from mice expressing a transgenic T‐cell receptor (TCR)‐specific for myelin basic protein (MBP) peptide Ac 1–9 responded vigorously to Ac 1–9. It is considered that the T‐cell response of NZB mice to Band 3 is initially focused on peptide 861–874 and later spreads to other Band 3 peptides as the disease progresses and that peptide 861–874‐reactive T cells are primed in the periphery rather than the thymus.
B‐cell responses to a peptide epitope: mutations in heavy chain alone lead to maturation of antibody responses
Authors: TUTEJA
Published: March 2003

In the present study, the genetic mechanisms responsible for generation of antibodies recognizing the dominant epitope within a synthetic peptide PS1CT3 were examined. PS1CT3 is a peptide model antigen containing residues 28–42 of the large protein of the surface antigen of hepatitis B virus as B epitope (designated PS1), and the known T‐helper‐cell epitope derived from the circumsporozoite protein of the malaria parasite Plasmodium falciparum (designated CT3). To characterize the repertoire generated, the immunoglobulin heavy chain variable regions from IgM and IgG monoclonal antibodies against PS1CT3 were sequenced. Although all IgG monoclonal antibodies were directed against the immunodominant epitope, the genetic elements used were diverse. Comparison of the sequence of germ line precursor IgM to a mature IgG revealed that during maturation of the primary IgM response only the heavy chain fragment of the antibody molecule underwent somatic mutation.
Autoreactive cytotoxic T cells in mice are induced by immunization with a conserved mitochondrial enzyme in Freund’s complete adjuvant
Authors: Karanikas  , Rowley  , Mackay  , Loveland
Published: March 2003

Standard methods to generate autoimmune reactions in mice, by immunization with antigens emulsified with adjuvants, stimulate strong helper (CD4) T‐cell and antibody responses but are not reported to induce cytolytic CD8 T cells. The aim of this study was to assess whether specific autoreactive CD8 T cells could be readily generated after immunization with a ‘weak’ autoantigen in adjuvant. Mice were immunized intraperitoneally three times with the E3 subunit of the mitochondrial 2‐oxoacid dehydrogenase enzyme complexes (dihydrolipoamide dehydrogenase) emulsified with Freund’s complete adjuvant. Splenic and lymph node lymphocytes were harvested after 14 days for in vitro functional studies. T lymphocytes were tested for proliferative responses and cytotoxicity against antigen‐loaded isogeneic target cells. An autoreactive cytolytic T lymphocyte (CTL) response was detectable only after the in vitro restimulation of lymphocytes with E3 antigen‐loaded syngeneic splenocytes. These CTL were identified as H‐2‐restricted CD8+ T cells. A proliferative response to E3 was demonstrable against antigen‐pulsed syngeneic splenocytes. Immunized mice also generated strong antibody responses to E3. Liver histology showed portal infiltrates interpreted as a response of the liver to a non‐specific immunological stimulus. It is concluded that autoreactive cytolytic T cells can be generated experimentally upon appropriate stimulation of the immune system, and can be identified in vitro upon release from the controlling mechanisms that are likely to regulate them in vivo.
Establishment of long‐term CD154‐dependent porcine B‐cell cultures
Authors: Takamatsu  , Andersen  , Denyer  , Parkhouse
Published: March 2003

Cells of the B‐cell lineage play an essential part in the immune response, not only as the producers of antigen‐specific antibodies, but also as antigen‐presenting cells. Unlike T cells, however, the establishment of long‐term normal B‐cell lines has proved to be exceedingly difficult. In this paper we demonstrate that cell membrane‐expressed CD154 (CD40 ligand) is able to support the continual growth of porcine mesenteric lymph node B‐cell cultures for more than 4 months without the addition of exogenous cytokines, such as interleukin‐4 (IL‐4). Addition of IL‐4, but not interferon‐γ (IFN‐γ) or IL‐13, to these cultures enhanced proliferation, as, to a lesser extent, did addition of IL‐2. Interestingly, however, whilst IFN‐γ‐supplemented cultures largely consisted of immunoglobulin M (IgM)‐positive cells, cultures with IL‐13 or IL‐4 contained a significantly increased proportion of IgG‐positive cells.
Recombinant interleukin‐16 selectively modulates surface receptor expression and cytokine release in macrophages and dendritic cells
Authors: Hermann  , Darcissac  , Idziorek  , Capron  , Bahr
Published: March 2003

Interleukin‐16 (IL‐16), a natural ligand for the CD4 receptor, has been found to modulate T‐lymphocyte function and to inhibit human immunodeficiency virus type 1 (HIV‐1) replication. Antigen‐presenting cells (APC), including macrophages and dendritic cells, are known to express functional surface CD4 molecules, to be susceptible to HIV‐1 infection and to play a critical role in different immune processes. Therefore, we evaluated the ability of recombinant IL‐16 (rIL‐16) to regulate receptor expression and cytokine release in monocyte‐derived macrophages (MDM) and monocyte‐derived dendritic cells (MDDC). Recombinant IL‐16 was found to up‐regulate CD25 and CD80 but to down‐regulate CD4 and CD86 surface expression in MDM cultures. However, no change could be observed on the level of CD4, CD80 and CD86 expression in IL‐16‐stimulated MDDC, although a significant up‐regulation of CD25 and CD83 was consistently detected. Furthermore, the level of gene expression of the chemokine receptors CCR5 and CXCR4 was significantly reduced in rIL‐16‐treated MDM and costimulation with IL‐2 did not modify the activity of the recombinant cytokine. The effects on chemokine receptor gene expression were less evident in MDDC and only a transient down‐regulation of weak intensity could be detected following stimulation with rIL‐16. Analysis of supernatants from rIL‐16‐stimulatedcultures revealed a different profile of released cytokines/chemokines among the two cell populations studied. These findings establish an important role for IL‐16 in modulating the activity of APC and may have relevance regarding the protection of reservoir cells against HIV‐1 infection.
Lipopolysaccharide enhances FcγR‐dependent functions in vivo through CD11b/CD18 up‐regulation
Authors: Rubel  , Miliani De Marval  , Vermeulen  , Isturiz  , Palermo
Published: March 2003

Fc receptors for immunoglobulin G (IgG) (FcγR) mediate several defence mechanisms in the course of inflammatory and infectious diseases. In Gram‐negative infections, cellular wall lipopolysaccharides (LPS) modulate different immune responses. We have recently demonstrated that murine LPS in vivo treatment significantly increases FcγR‐dependent clearance of immune complexes (IC). In addition, we and others have reported the induction of adhesion molecules on macrophages and neutrophils by LPS in vivo and by tumour necrosis factor‐α (TNF‐α) in vitro. The aim of this paper was to investigate CD11b/CD18 participation in LPS enhancing effects on Fcγ‐dependent functionality of tissue macrophages. Our results have demonstrated that LPS can enhance antibody‐dependent cellular cytotoxicity (ADCC) and IC‐triggered cytotoxicity (IC‐Ctx), two reactions which involve the Fcγ‐receptor but different lytic mechanisms. In vitro incubation of splenocytes from LPS‐treated mice with anti‐CD11b/CD18 abrogated ADCC and IC‐Ctx enhancement, without affecting FcγR expression. Similar results were obtained with physiological concentrations of fibrinogen. In this way cytotoxic values of LPS‐splenocytes decreased to the basal levels of control mice. Time and temperature requirements for such inhibition strongly suggested that anti‐CD11b/CD18 could modulate intracellular signals leading to downregulation of FcγR functionality. Data presented herein support the hypothesis that functional and/or physical associations between integrins and FcγR could be critical for the modulation of effector functions during an inflammatory response.
Human parathymic lymph node: morphological and functional significance
Authors: Tanegashima  , Yamashita  , Yamamoto  , Fukunaga
Published: March 2003

Parathymic lymph nodes (PTLNs) have been identified in several species, but in humans they have been noted only once before in a study 90 years ago using fetal material. We now report their occurrence in children. Human PTLNs are small but distinctive lymphatic organs located on the surface of the thymus (or sometimes between the upper and lower lobes of the thymus) and covered with the thymic capsule. Histologically, the medullary cords of these lymph nodes were found to be thin, with only small numbers of plasma cells. In addition, they had a well‐developed paracortical area rich with high endothelial venules (HEV), but a thin cortex, including only a few undeveloped follicles. Flow cytometric analysis of PTLNs revealed that the ratios of T:B cells (14·6±9·3) and of CD4+:CD8+ T cells (4·9±1·4) in PTLNs were much higher than in other peripheral lymphoid tissues and in peripheral blood. Because of these characteristics of the human PTLNs, we propose that the human PTLNs might influence the functional differentiation of T cells.
Regulation of major histocompatibility complex class II antigens on human alveolar macrophages by granulocyte–macrophage colony‐stimulating factor in the presence of glucocorticoids
Authors: Caulfield  , Fernandez  , Sousa  , Lane  , Lee  , Hawrylowicz
Published: March 2003

Alveolar macrophages (AM) present antigen poorly to CD4+ T cells and respond weakly to interferon‐γ (IFN‐γ) for up‐regulation of major histocompatibility complex (MHC) class II and costimulatory molecule expression. In atopic asthma, however, AM exhibit enhanced antigen‐presenting cell (APC) activity. Since granulocyte–macrophage colony‐stimulating factor (GM‐CSF) is increased in the airways of asthmatic patients, we have investigated its role in modulating the APC function of AM. The effects of glucocorticoids were also studied since earlier studies showed optimal induction of MHC antigens on monocytes by GM‐CSF in their presence. GM‐CSF in the presence, but not the absence, of dexamethasone enhanced the expression of HLA‐DR, ‐DP and ‐DQ antigens by AM. However AM and monocytes differed in the optimal concentration of steroid required to mediate this effect (10−10 m and 10−7 m, respectively). Induction of MHC antigens was glucocorticoid specific and independent of IFN‐γ. These studies suggest the existence of an IFN‐γ‐independent pathway of macrophage activation, which may be important in regulating APC function within the lung.
Low dose of orally administered antigen down‐regulates the T helper type 2‐response in a murine model of dust mite hypersensitivity
Authors: Sato  , Carvalho  , Silva  , Maciel Jr  , Fusaro  , Duarte
Published: March 2003

One of the main goals of immunotherapy of allergic diseases is the down‐regulation of the type I hypersensitivity reaction. We investigated in this study the effect of oral administration of varying doses (0·25, 1·0, 4·0 and 10 mg) of dust mite extract (Dermatophagoides pteronyssinus, Dp) in sensitized A/Sn mice. A marked decrease of the allergen‐specific immunoglobulin E (IgE) response was observed with all antigen doses. The mice orally tolerized with low Dp dose (0·25 mg) had a significant decrease in the total serum IgE and in the immunoglobulin G1 (IgG1), IgG2a and IgG2b antibody levels. The higher Dp dose (10·0 mg), however, enhanced the IgG1 antibody response, suggesting the stimulation of a pre‐existing immune response of the sensitized animals. Animals fed with the low Dp dose had a significant decrease in the frequency of interleukin‐4 (IL‐4) secreting cells. These animals also showed a significant decrease in the frequency of Dp‐specific IgE‐ and IgG1‐positive plasma cells. Our data suggest that feeding dust mite extract to Dp‐sensitized mice down‐regulates the development of type I hypersensitivity, by inhibition of the T helper 2 response.
Regulation of VH gene repertoire and somatic mutation in germinal centre B cells by passively administered antibody
Authors: Song  , Nie  , Basu  , Singh  , Cerny
Published: March 2003

Immunization with T‐dependent antigens induces a rapid differentiation of B cells to plasmacytes that produce the primary immunoglobulin M (IgM) and IgG antibodies with low affinities for the immunogen. It is proposed that the IgG antibody forms immune complexes with the residual antigen which provide an important stimulus for the formation of germinal centres (GC) and the activation of somatic mutation. This hypothesis was tested by passive administration of hapten‐specific antibody into mice shortly after the immunization with nitrophenyl (NP) coupled to chicken gamma globulin (NP‐CGG) in an environment of limited T‐cell help. Athymic mice that received normal T helper cells at 72 hr after the administration of antigen produced low levels of anti‐NP antibody and the splenic GC formation was delayed until day 12 after the antigen administration. The analysis of VDJ segments from NP‐reactive GC B cells showed very few mutations in the VH genes. Passive injection of anti‐NP IgG1 monoclonal antibody – but, not IgM – stimulated the GC formation up to normal levels and the somatic mutation activity in the GC B cells was fully restored. In addition, GC B cells in the recipients of IgG1 antibody demonstrated a change in the usage of germline‐encoded VH genes which was not apparent among the primary antibody‐forming cells. These results suggest the existence of a specific feedback mechanism whereby the IgG antibody regulates the GC formation, clonotypic repertoire and somatic mutation in GC B cells.
Tolerance and bystander suppression, with involvment of CD25‐positive cells, is induced in rats receiving serum from ovalbumin‐fed donors
Authors: Karlsson M. R. , Kahu H. , Hanson L. Å. , Telemo E. , Dahlgren U. I.
Published: March 2003

In the present study we have investigated if transfer of serum from rats fed ovalbumin (OVA) leads to specific tolerance and bystander suppression in recipient animals. Rats that received serum from OVA‐fed donors had a lower delayed‐type hypersensitivity reaction (DTH) both against OVA and the bystander antigen, human serum albumin (HSA), compared with recipients given serum from control‐fed animals. The in vitro proliferation of OVA‐ and HSA‐stimulated spleen cells and the serum immunoglobulin G (IgG) antibody levels against OVA and HSA were also lower in the animals that received serum from OVA‐fed animals compared with the controls. There was no reduction of the immune response to HSA if the recipient animals, given serum from OVA‐fed donors were immunized with OVA and HSA at separate sites. Depletion of CD25‐positive cells from spleen suspensions from rats receiving serum from OVA‐fed animals, resulted in a significant increase in proliferation of OVA‐stimulated cells in vitro compared with the controls. Tolerogenic activity could be demonstrated, both in a fraction from serum containing structures smaller than 100 000 MW and a fraction with components larger than 100 000 MW, compared with size‐related serum fractions obtained from control‐fed animals. This implies that the tolerogenic activity could be mediated by more than one serum component. The tolerogenic activity was most prominent in animals receiving the larger size fraction with a more pronounced suppression of the DTH reaction and lower levels of IgG anti‐OVA antibodies in serum compared with controls. A novel finding in the present study was that the transfer of serum, collected from rats fed OVA, led to a reduction of the immune response to a bystander antigen in the recipients. This suggests that the induced tolerance is at least partly due to suppression. The suppression could have been mediated by CD25‐positive cells since removal of these cells resulted in an increased in vitro proliferation against OVA.
Biological function of CD40 on human endothelial cells: costimulation with CD40 ligand and interleukin‐4 selectively induces expression of vascular cell adhesion molecule‐1 and P‐selectin resulting in preferential adhesion of lymphocytes
Authors: Kotowicz K. , Dixon G. L. J. , Klein N. J. , Peters M. J. , Callard R. E.
Published: March 2003

The expression of adhesion molecules on vascular endothelial cells determines the pattern of migration and extravasation of leucocytes in inflammation and immunity. Here we show that costimulation with CD40 ligand (CD40L) and interleukin (IL)‐4 (or IL‐13) gives rise to a unique pattern of adhesion molecule expression by human umbilical vein endothelial cells (HUVEC). CD40 ligation alone enhanced expression of vascular cell adhesion molecule‐1 (VCAM‐1), intracellular adhesion molecule‐1 (ICAM‐1) and E‐selectin whereas IL‐4 and IL‐13 increased expression of VCAM‐1 and P‐selectin but not ICAM‐1 or E‐selectin. When IL‐4 and CD40L were combined there was an additional increase of both VCAM‐1 and P‐selectin, but ICAM‐1 and E‐selectin were both inhibited. The combined effects of IL‐4 and CD40L signalling were not the result of altered response kinetics, enhanced sensitivity of the endothelium, or increased expression of CD40 or the IL‐4 receptor. The rise in VCAM‐1 expression induced by combined IL‐4 and CD40L stimulation was slower and more sustained than with tumour necrosis factor‐α (TNF‐α) and occurred only on a subset (75–80%) of the endothelial cell population compared to 100% with TNF‐α. Costimulation with IL‐4 and CD40L increased adhesion of T cells and B cells above levels obtained with either signal alone, but decreased adhesion of neutrophils. Furthermore, CD40 and IL‐4 synergistically increased IL‐6 but decreased IL‐8 production by HUVEC. These results show that interactions between IL‐4 and CD40 on endothelial cells give rise to specific patterns of adhesion molecule expression and cytokine production that may have important implications for lymphocyte and neutrophil migration and function at sites of inflammation.
Xenogeneic islet re‐transplantation in mice triggers an accelerated, species‐specific rejection
Authors: Triponez F. , Oberholzer J. , Morel P. , Toso C. , Yu D. , Cretin N. , Buhler L. , Majno P. , Mentha G. , Lou J.
Published: March 2003

Xenogeneic islets could provide an unlimited source of tissue for the treatment of diabetes, and could in theory be transplanted repeatedly in a recipient. However, little is known on the consequences of islet re‐transplantation in a recipient who has rejected a first graft. In this study, we investigated the functional consequence of xeno islet re‐transplantation in mice sensitized with islets from different species. Sprague–Dawley (SD)‐rat islets transplanted in sensitized C57/Bl6 mice that rejected either SD‐ or Lewis‐rat islets underwent accelerated rejection. However, accelerated rejection was not found in mice sensitized with pig or human islets, suggesting that accelerated rejection was species specific. Immunohistochemistry showed increased binding of antibodies and accelerated leucocyte infiltration on re‐grafted islets in sensitized mice. In situ apoptosis detection indicated that islet cell apoptosis was correlated with the time of leucocyte infiltration, but not with the time of antibody binding. In vitro experiments with cultured islet cells showed that although antibody binding was increased after incubation with sensitized mouse serum, islet cell cytotoxicity was not increased, suggesting that humoral immunity did not play a direct role in islet destruction. These results indicate that there is a cell‐mediated, species‐specific accelerated rejection after re‐transplantation of xenogeneic islets.
In vivo rapid reduction of alloantigen‐activated CD8+ mature cytotoxic T cells by inhibitors of acidification of intracellular organelles, prodigiosin 25‐C and concanamycin B
Authors: Lee M.‐H. , Kataoka T. , Honjo N. , Magae J. , Nagai K.
Published: March 2003

Prodigiosin (PrG) 25‐C and concanamycin B (CMB) are immunosuppressants that specifically inhibit the induction of cytotoxic T cells (CTL) without affecting the function of B cells and helper T cells in vivo. Both compounds inhibit acidification of intracellular organelles and induce destruction of cytotoxic granules and degradation of perforin in vitro. Here we show that a single intraperitoneal (i.p.) injection of PrG 25‐C, and of CMB, into mice eliminates cytotoxic activity 7 days after alloantigen stimulation (when mature CTL activity has been detected in control mice), with minimal effect on the alloantigen‐specific antibody titre in serum. FK506 did not suppress the cytotoxic activity with this administration schedule. Suppression was accompanied by a decrease in the CD8+ population and in perforin expression of spleen cells induced by alloantigen stimulation. The suppression of CTL activity and decrease in CD8+ cell number was detected as early as 7 hr after the injection of compounds. These results suggest that inhibitors of acidification of intracellular organelles suppress CTL activity in vivo by reducing the number of mature CD8+ CTL.
Rewiring of CD40 is necessary for delivery of rescue signals to B cells in germinal centres and subsequent entry into the memory pool
Authors: Siepmann K. , Skok J. , Van Essen D. , Harnett M. , Gray D.
Published: March 2003

Memory B‐cell development is impaired by in vivo blockade of the CD40–CD40 ligand (CD40L) interaction using human Fc immunoglobulin G1 (IgG1)‐mouse CD40 fusion protein (CD40‐Ig); however, germinal centre (GC) formation is not. We show here that the block in B‐cell differentiation in these mice is at the stage of rescue from apoptosis and exit from the GC. Thus, GC from CD40‐Ig‐treated mice contain a three‐ to fourfold higher level of apoptotic cells than found in control mice injected with human IgG1 alone. This increase in apoptosis is not caused by a blockade of the CD40L‐mediated rescue signal but is the result of an intrinsic defect of GC B cells in CD40‐Ig‐treated mice to receive rescue signals via CD40. While anti‐CD40 stimulation maintained the viability in culture of GC B cells from control mice, it did not rescue GC B cells from CD40‐Ig‐treated mice. This data is consistent with the notion that a ‘rewiring’ of the CD40 molecule is induced by CD40 ligation early in the response and is necessary to allow B‐cell rescue from apoptosis when they subsequently enter the GC.
Short‐term immunoglobulin A B‐cell memory resides in intestinal lymphoid tissues but not in bone marrow of gnotobiotic pigs inoculated with Wa human rotavirus
Authors: Yuan Lijuan , Geyer Annelize , Saif Linda J.
Published: March 2003

Immunological memory is important for protecting the host from reinfection. To investigate the development and sites of residence of intestinal memory B cells, and their role in protective immunity to reinfection with an enteric virus, we assessed the association between memory B cell and antibody‐secreting cell (ASC) responses and protection using a gnotobiotic pig model for human rotavirus (HRV) infection and diarrhoea. The isotypes, quantities and tissue distribution of rotavirus‐specific memory B cells and ASC were evaluated prechallenge (28 and 83 postinoculation days [PID]) and postchallenge (7 postchallenge days [PCD]), using enzyme‐linked immunospot (ELISPOT) assay, in gnotobiotic pigs inoculated once with virulent or three times with attenuated HRV and challenged at PID 28 with the corresponding virulent HRV. Complete protection against HRV shedding and diarrhoea was associated with significantly higher numbers of immunoglobulin A (IgA) and immunoglobulin G (IgG) memory B cells and ASC in the ileum of virulent HRV‐inoculated pigs at challenge. In contrast, pigs inoculated with attenuated HRV had lower numbers of IgA and IgG memory B cells and ASC in intestinal lymphoid tissues, but higher numbers in the spleen. The bone marrow had the lowest mean numbers of IgA and IgG memory B cells and ASC prechallenge in both groups of HRV‐inoculated pigs. Therefore, bone marrow was not a site for IgA and IgG rotavirus‐specific antibody production or for memory B cells after inoculation with live rotavirus, from 28 PID up to at least 83 PID. The effect of in vitro antigen dose was examined and it was determined to play an important role in the development of ASC from memory B cells for the different tissues examined.
Rat mast cell protease‐I enhances immunoglobulin E production by mouse B cells stimulated with interleukin‐4
Authors: Yoshikawa Tsutomu , Imada Teruaki , Nakakubo Hiroshi , Nakamura Norifumi , Naito Koji
Published: March 2003

Mast cell chymase plays important roles in inflammation and tissue remodeling. Here we show that mast cell chymase also functions as an enhancer of immunoglobulin production. In the culture of murine spleen cells stimulated with lipopolysaccharide and interleukin‐4, purified rat chymase (rat mast cell protease‐I; RMCP‐I), at physiological concentrations, enhanced immunoglobulin E (IgE) and IgG1 syntheses but not IgG3 synthesis. The enhancement was also evident when spleen cells depleted of T cells and macrophages were employed as responding cells. Enzymatic activity of RMCP‐I was required to enhance IgE and IgG1, because two inhibitors for chymotryptic enzymes, chymostatin and Y‐40613, a novel chymase inhibitor, suppressed the enhanced immunoglobulin production, and phenylmethylsulphonyl fluoride, an irreversible inhibitor for serine proteases, totally abolished the enhancing effect. Furthermore, a specific inhibitor for Zn2+‐dependent metalloproteases, GI 129471, could also completely inhibit the production of IgE and IgG1 that was enhanced by RMCP‐I, suggesting that a metalloprotease also played an essential role in the immunoglobulin production. Our results together with others show that proteases from mast cell granules have important function not only in the efferent phase but also in the afferent phase of immune responses.
Adrenaline suppression of the macrophage nitric oxide response to lipopolysaccharide is associated with differential regulation of tumour necrosis factor‐α and interleukin‐10
Authors: Zinyama Rutendo B. , Bancroft Gregory J. , Sigola Lynette B.
Published: March 2003

Adrenaline is a catecholamine hormone secreted by the adrenal medulla in response to acute stress. Previous studies have shown that adrenaline suppresses the nitric oxide (NO) response of murine macrophages (Mφs) stimulated in vitro with lipopolysaccharide (LPS). We have now extended these studies to examine the effects of adrenaline on the production of tumour necrosis factor alpha (TNF‐α) and interleukin‐10 (IL‐10). Our results showed that NO, TNF‐α and IL‐10 were concurrently produced following in vitro LPS (10 µg/ml) stimulation of murine peritoneal Mφs. Adrenaline suppressed both NO and TNF‐α with concomitant up‐regulation of the IL‐10 response above that seen with LPS alone. In this in vitro model of LPS stimulation we demonstrated that TNF‐α was required for NO production, as the TNF‐α neutralizing monoclonal antibody, TN3.19.12, abolished the response; in contrast, IL‐10 suppressed NO. In order to determine any functional consequence of adrenaline‐mediated IL‐10 augmentation on NO production, Mφs were stimulated with LPS and specific neutralizing anti‐IL‐10 antibodies were added to the cultures. The LPS NO response was suppressed to 43% of the control value by adrenaline (10−8 m) and an irrelevant control antibody had no effect on the adrenaline‐mediated inhibition of NO, but anti‐IL‐10 treatment restored the NO response to levels similar to those observed with LPS alone. Furthermore, we demonstrated that exogenous TNF‐α, at a dose range of 1·9–50 ng per ml, also restored the nitrite response to LPS in the presence of adrenaline. Together, the observations that neutralization of IL‐10 and addition of TNF‐α abrogate adrenaline's inhibition of NO, suggest that this hormone suppresses NO partly through up‐regulation of IL‐10 which, in turn, may suppress TNF‐α that is required for NO production. Finally, we also observed that the Mφ‐activating cytokine, interferon‐γ (IFN‐γ), attenuated the inhibitory effect of adrenaline on the LPS NO response.
Studies on the association between immunoglobulin E autoreactivity and immunoglobulin E‐dependent histamine‐releasing factors
Authors: Budde Ilona Kleine , De Heer Pleuni G. , Natter Susanne , Mahler Vera , Van Der Zee Jaring S. , Valenta Rudolf , Aalberse Rob C.
Published: March 2003

It has been reported that serum immunoglobulin E (IgE) from certain atopic patients can sensitize basophils to release histamine in response to IgE‐dependent histamine‐releasing factors (HRFs). It has also been shown that patients suffering from severe forms of atopy may contain IgE autoantibodies. It was investigated whether HRF‐responsive sera contained IgE autoantibodies and if there was an association between IgE autoreactivity and IgE‐dependent responsiveness to HRF. The presence of HRF‐responsive IgE (IgE+) in serum of patients with respiratory atopy was determined by stimulating stripped human basophils sensitized by serum with peripheral blood mononuclear cell (PBMC)‐derived HRF, and measuring the release of histamine. In parallel, these sera were screened for the presence of IgE autoantibodies to nitrocellulose‐blotted human cellular extracts. The capacity of IgE autoantigen‐containing preparations to induce histamine release was tested in the stripped basophil assay. Eleven out of 52 sera contained IgE autoantibodies to blotted cellular extracts of human PBMCs or of the human epithelial cell line A431. No significant association was found between IgE autoreactivity and IgE‐dependent responsiveness to HRF: 7/26 IgE+ sera contained IgE to human cellular extracts, and 4/26 of the sera without IgE+ did also. IgE autoantigen‐containing extracts did not induce histamine release of appropriately sensitized basophils. By size‐exclusion chromatography it was shown that a 32 000 MW autoantigen eluted in the >55 000 MW fraction, which indicates that this protein forms polymers or complexes with other macromolecules. This might explain the discrepancy between binding and histamine‐releasing activity. A 20 000 MW IgE‐defined autoantigen cross‐reacted with a shrimp allergen. Our results indicate that IgE‐reactivity to immunoblotted human protein and IgE‐dependent HRF activity are distinct entities that may co‐occur in atopic patients.
Mechanism of antigen presentation after hypertonic loading of soluble antigens
Authors: Enders Georg A.
Published: March 2003

Hypertonic loading of proteins into cells has been used to introduce soluble proteins into the major histocompatibility complex class I pathway of antigen presentation followed by cytotoxic T‐lymphocyte (CTL) induction. The precise mechanism for this pathway is not completely understood. The antigen is either processed and presented by/on the same cell or by professional antigen‐presenting cells (APC) after taking up the antigen from damaged or apoptotic cells. After loading labelled ovalbumin (OVA), it could be co‐precipitated with the proteasome complex, supporting the role of this pathway for antigen processing. The processing speed however, appeared to be slow since intact OVA could be detected inside the cells even after 18 hr. This corresponded well with the processing of OVA by isolated proteasomes. On the other hand, enough peptides for recognition of target cells by CTLs were generated in this reaction. One reason for the low level of processing might be that hypertonic loading may damage the cells and inhibit direct processing. In fact, at least 50% of the cells became positive for Annexin V binding after hypertonic loading which indicates severe membrane alterations usually associated with the progress of apoptosis. Annexin V binds to phosphatidylserine residues which also serve as ligand for CD36 expressed on monocytes and some immature dendritic cells. This may direct the phagocytic pathway to hypertonically loaded cells and thus enable professional APCs to present OVA‐peptides. Therefore, in addition to the direct processing of OVA, CTLs can be primed by professional APC after uptake of apoptotic, OVA‐loaded cells.
Induction of immunoglobulin G1, interleukin‐6 and interleukin‐10 by Taenia crassiceps metacestode carbohydrates
Authors: Dissanayake Senarath , Khan Nasir , Shahin Allen , Wijesinghe Shanaka , Lukic Miodrag
Published: March 2003

T helper type 2 (Th2) ‐polarized immune responses are characteristically dominant in helminth infections. Two murine models that show a Th1 to Th2 polarization with infection progression are those of Schistosoma mansoni and Taenia crassiceps. In both, an early Th1 response is replaced by a late Th2 response. We report that the nucleic acid‐, protein‐ and lipid‐free carbohydrate fraction of T. crassiceps metacestodes (denoted T‐CHO) possesses Th2‐like immunomodulatory activity. Immunization of two strains of rats (Dark Agouti and Albino Oxford) and BALB/c mice with chicken albumin in the presence of T‐CHO resulted in selective enhancement of immunoglobulin G1 (IgG1) antibodies, considered to be associated with Th2 responses in both rats and mice. Interleukin‐6 (IL‐6) followed by IL‐10 were the dominant cytokines detected in in vitro cultures of mouse spleen cells stimulated with T‐CHO. IL‐4 and IL‐5 were not detected in these culture supernates. Furthermore, Taenia carbohydrates were mitogenic to spleen cells, activated serine phosphorylation of proteins and up‐regulated the expression of the anti‐apoptotic protein, Bcl‐2. When mouse spleen cells were cultured in the presence of Taenia carbohydrates, a concentration‐dependent down‐regulation of IL‐2 and an overlapping up‐regulation of IL‐6 secretion were seen.
Distribution of Toll‐like receptor 1 and Toll‐like receptor 2 in human lymphoid tissue
Authors: Ochoa Maria‐Teresa , Legaspi Annaliza J. , Hatziris Zena , Godowski Paul J. , Modlin Robert L. , Sieling Peter A.
Published: March 2003

To determine how distinct receptors of the immune system can contribute to innate immunity, we investigated the pattern of Toll‐like receptor 1 (TLR1) and TLR2 expression in human lymphoid tissue. We found that TLR1 and TLR2 were co‐expressed on cells of the innate immune system, including macrophages and dendritic cells. In addition, TLR1 and TLR2 were expressed in mucosa‐associated lymphoid tissue on tonsillar crypt epithelium. Of the lymphoid tissue examined, spleen expressed the highest levels of TLR2. Although TLR1‐ and TLR2‐positive cells were in close proximity to T lymphocytes in vivo, lymphocytes themselves were devoid of TLR1 and TLR2 expression. The co‐expression of TLR1 and TLR2 on myeloid cells in lymphoid tissue provides the host with the ability to respond to a variety of microbial ligands at sites conducive to the generation of an immune response.
Breast milk macrophages spontaneously produce granulocyte–macrophage colony‐stimulating factor and differentiate into dendritic cells in the presence of exogenous interleukin‐4 alone
Authors: Ichikawa Masao , Sugita Masahiko , Takahashi Megumi , Satomi Misao , Takeshita Toshiyuki , Araki Tsutomu , Takahashi Hidemi
Published: March 2003

Peripheral blood monocytes extravasate and differentiate into tissue macrophages to mediate effective local defence, but how tissue‐specific stimuli and environments may influence their functions remains unknown. Here, we found that peripheral blood monocytes gained the ability to produce granulocyte–macrophage colony‐stimulating factor (GM‐CSF) upon exposure to breast milk and differentiated into CD1+ dendritic cells (DCs) in the presence of exogenous interleukin‐4 (IL‐4) alone. This in vitro observation appeared physiologically relevant since macrophages that were freshly isolated from breast milk were also found to produce GM‐CSF spontaneously. Furthermore, in contrast to peripheral blood monocytes that differentiated into DCs only in the presence of both exogenous GM‐CSF and IL‐4, differentiation of breast milk macrophages into DCs was induced by incubation with exogenous IL‐4 alone. These IL‐4‐stimulated breast milk macrophages were efficient in stimulating T cells, suggesting their potential role in mediating T‐cell‐dependent immune responses in situ. On the other hand, unexpected expression of DC‐SIGN, a DC‐specific receptor for human immunodeficiency virus (HIV), even in unstimulated breast milk macrophages, may favour HIV infection, resulting in an increased risk of mother‐to‐infant vertical transmission of the virus via breast milk. Thus, tissue‐specific development of macrophages is often linked to effective local immunity, but may potentially provide an opportunity for a pathogen to spread and transmit.
Contextualized access to knowledge: theoretical perspectives and a process‐tracing study
Authors: Mao Ji‐Ye , Benbasat Izak
Published: March 2003

This paper investigates the theoretical foundations and empirical evidence concerning contextualized access to task domain knowledge enabled by hypertext‐style links. It examines several relevant theoretical perspectives, including theories of discourse comprehension, contextualized learning and the production paradox, and reports on an exploratory study in the knowledge‐based systems (KBS) domain. Process‐tracing data was collected using a ‘thinking‐aloud’ procedure, and data analysis focused on some highly illustrative verbal protocols. Results indicate that contextualized access to domain knowledge can be critical for understanding KBS output, and that lack of it can cause comprehension difficulties. Contextualized access is highly effective for resolving comprehension difficulties arising from the users' lack of task domain knowledge and for reducing the motivational ‘cost’ of learning. We conclude that it has the potential for substantially increasing the effectiveness of information systems.
The Brave New World of development in the internetwork computing architecture (InterNCA): or how distributed computing platforms will change systems development
Authors: Lyytinen Kalle , Rose Gregory , Welke Richard
Published: March 2003

This essay is a speculation of the impact of the next generation technological platform — the internetwork computing architecture (InterNCA) — on systems development. The impact will be deep and pervasive and more substantial than when computing migrated from closed computer rooms to ubiquitous personal computers and flexible client‐server solutions. Initially, by drawing upon the notion of a technological frame, the InterNCA, and how it differs from earlier technological frames, is examined. Thereafter, a number of hypotheses are postulated with regard to how the architecture will affect systems development content, scope, organization and processes. Finally, some suggestions for where the information systems research community should focus its efforts (if the call for relevance is not to be taken lightly) are proposed.
The use of critical systems thinking in designing a system for a university information systems support service
Authors: Warren Lorraine , Adman Peter
Published: March 2003

Abstract. This paper reports the practical application of critical systems thinking (CST) to an information systems design (ISD)‐related problem. The potential benefits of using CST for ISD are explored, followed by a description of an intervention to derive a design for part of a UK university’s IS user support service. More specifically, total systems intervention (TSI) was used as a meta‐methodology to operationalize CST and to guide the course of the intervention. The intervention serves as a vehicle to gain insight into the potential value of CST to ISD‐related problems, and concludes with reflections on the actual benefits realized and suggestions for further research.
A report on the use of action research to evaluate a manufacturing information systems development methodology in a company
Authors: Grant Delvin , Ngwenyama Ojelanki
Published: March 2003

Abstract. The paper reports on an action research study that evaluated the usefulness of a Manufacturing Information Systems (MIS) development methodology at a manufacturing technology company. The evaluation process is based upon a five‐stage action research method. The ISD methodology, in conjunction with the action research method, was used to solve five technical and organizational problems identified in the Engineering Release Function of the company. Results of the study include reduction in cycle time, work‐in‐process and rework.
Relative abundance, isolation and structure of phlebotomine microsatellites
Authors: Day J. C. , Ready P. D.
Published: March 2003

Popular classes of microsatellites are not always abundant in insects or easily isolated from them. Dot‐blot hybridizations demonstrated much variation in the relative abundance of four repeat classes in four phlebotomine sandfly species. Only AAT‐class repeats were specifically isolated from a phagemid library of Lutzomyia whitmani, even though other microsatellites had similar abundances. An enrichment step would have targeted classes but was omitted because relatively long flanking sequences were sought. All fourteen sandfly loci had a non‐coding structure, and a minority of dipteran AAT‐class repeats found in DNA databases and the literature were from exons. Therefore, this class should often provide neutral alleles for population studies. Perfect, not imperfect, AAT‐class repeats were polymorphic in wild L. whitmani.