bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of recombinant type II collagen: arthritogenicity and tolerogenicity in DBA/1 mice
Authors: MYERS  , BRAND  , YE  , CREMER  , ROSLONIEC  , BODO  , MYLLYHARJU  , HELAAKOSKI  , NOKELAINEN  , PIHLAJANIEMI  , KIVIRIKKO  , YANG  , ALA‐KOKKO  , PROCKOP  , NOTBOHM  , FIETZEK  , STUART  , KANG
Published: March 2003

Recombinant human type II collagen (rhCII) was produced using both the HT1080 mammalian cell expression system (rhCIIht) and a baculovirus expression system (rhCIIbac). The biosynthesis of CII requires extensive post‐translational modifications, such as the hydroxylation of prolyl and lysyl residues and glycosylation of hydroxylysyl residues. Amino acid analyses indicated that the rhCIIbac was adequately hydroxylated at prolyl residues but underhydroxylated at lysyl residues and underglycosylated compared with tissue‐derived hCII, while rhCIIht was hyperhydroxylated and hyperglycosylated at lysyl residues. When the murine collagen‐induced arthritis (CIA) model was used to investigate the immunological properties of the two forms of recombinant CII, each induced a high incidence of arthritis following immunization of susceptible mice when emulsified with complete Freund’s adjuvant (CFA). However, the severity of the arthritis, as assessed by the number of affected limbs, was significantly higher in mice immunized with rhCIIht than in mice immunized with rhCIIbac. These data indicate that the degree of hydroxylysine glycosylation may play a role in the induction of the arthritogenic response to CII. Each of the recombinant collagens was comparable to tissue‐derived hCII in their ability to induce tolerance and suppress arthritis when given as intravenous or oral tolerogens. Taken together, our data suggest that recombinant CII can be prepared in adequate amounts for therapeutic uses and that the material is immunologically comparable to tissue‐derived hCII when used to induce tolerance.
Enhancing the immunotherapeutic potential of mycobacteria by transfection with tumour necrosis factor‐α
Authors: HALEY  , YOUNG  , ALEXANDROFF  , JAMES  , JACKSON
Published: March 2003

In an attempt to enhance the anti‐tumour properties of mycobacteria we have developed recombinant forms of Mycobacterium smegmatis which express and secrete biologically active human tumour necrosis factor‐α (TNF‐α). This was achieved by transfecting M. smegmatis using shuttle plasmids incorporating the cDNA sequence for the human TNF‐α mature peptide. In vitro experiments on a panel of human bladder tumour cell lines (EJ18, MGH‐U1, RT4, RT112) indicate that our genetically modified mycobacteria are more effective than wild‐type at inducing or up‐regulating the expression of intracellular adhesion molecule‐1 and the secretion of an array of proinflammatory cytokines [interleukin‐1 (IL‐1), IL‐6, IL‐8, granulocyte–macrophage colony‐stimulating factor]. We have also demonstrated increased adhesion molecule and cytokine expression in response to mycobacteria transfected with vector containing no gene insert. However, this was not as pronounced as that observed following tumour cell stimulation by the TNF‐α‐transfected strain. In contrast, in three out of four tumour cell lines all M. smegmatis strains were found to down‐regulate the secretion of the anti‐inflammatory cytokine transforming growth factor‐β1. Our studies have also confirmed that M. smegmatis is a powerful inhibitor of bladder tumour cell growth and revealed that its antiproliferative potency is enhanced by transfecting with human TNF‐α and, to a lesser extent, with vector alone. All M. smegmatis strains were effective in the activation of peripheral blood leucocyte cultures. However, no differences were observed in the ability of the TNF‐α‐transfected, mock‐transfected and wild‐type mycobacteria to induce tumour cell killing activity. These results suggest that the immunomodulatory effects of M. smegmatis can be enhanced by transfection with vectors which allow the secretion of human TNF‐α, thus increasing mycobacterial immunotherapeutic potential.
Prognostic importance of antigen‐presenting dendritic cells during vaccine therapy in a murine hepatitis B virus carrier
Authors: AKBAR  , HORIIKE  , ONJI
Published: March 2003

As, the outcome of vaccine therapy was extremely heterogeneous in both human and murine hepatitis B virus (HBV)‐carriers, the experiments presented here were performed to find out a prognostic marker of vaccine therapy using an animal model of HBV‐carrier state, HBV‐transgenic mice (Tg). Neither the prevaccinated titres of viral markers, such as hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) or HBV DNA, nor the function of lymphocytes prior to vaccination, had significant influence on the outcome of vaccine therapy. Two independent, placebo‐controlled, trials of vaccine therapy for 12 months, one in 17 HBV‐Tg and the other in 26 HBV‐Tg (total, n=43) showed that the eight of 17 and 15 of 26 HBV‐Tg that had potent dendritic cell (DC) function at the start of vaccine therapy became completely negative for HBsAg, HBeAg and reduced HBV DNA, whereas all 19 HBV‐Tg that had poor DC function at the start of vaccine therapy became complete non‐responders, although, the prevaccinated titres of HBsAg, and HBeAg were similar in all 43 HBV‐Tg. Further study to find the mechanism underlying this revealed that there was up‐regulation of major histocompatibility complex (MHC) class II, CD86 antigens on DC and increased production of interleukin‐12 (IL‐12) by DC and of IL‐2, and tumour necrosis factor‐α (TNF‐α) in DC/T‐cell cultures when vaccine containing HBsAg was injected in HBV‐Tg with potent DC function but not in HBV‐Tg with poor DC function. This is the first report on the prognostic importance of DC during an immune therapy. Degree of activation of DC following vaccination would possibly help to predict the outcome of vaccine therapy in human HBV‐carriers. These data also provide the scientific and logical basis to up‐regulate the function of the DC before an immune therapy.
Selective protection by hsp 70 against cytotoxic drug‐, but not Fas‐induced T‐cell apoptosis
Authors: CREAGH  , COTTER
Published: March 2003

The phenomenon of heat‐shock (HS) protection to many cytotoxic insults has previously been described; however, the specific molecular mechanism underlying this HS‐mediated protection remains undefined. To gain insight into this protective mechanism, heat‐shocked Jurkat T cells were treated with a range of cytotoxic agents. Those against which HS conferred protection (camptothecin and actinomycin D) were compared with agents against which HS showed no protective effect (anti‐Fas monoclonal antibody (mAb)). Reactive oxygen species (ROS) production was found to be an event common to apoptosis induced by camptothecin and actinomycin D, whereas Fas‐mediated apoptosis was shown to occur via a ROS‐independent mechanism. The selective protection observed against these agents was found to be mimicked by pretreatment with antioxidant compounds. Furthermore, this antioxidant protection appears to be occurring downstream of ROS production. Experiments were extended using heat‐shock protein (hsp) 70 gene‐transfected Jurkat T cells to confirm that the protective effects observed were caused by hsp 70 synthesis rather than any other cellular response to HS. Bcl‐2 expression levels were also examined to determine whether any correlation existed between Bcl‐2‐ and hsp 70‐mediated protection.
Vectorial function of major histocompatibility complex class II in a human intestinal cell line
Authors: Lopes  , Hughson  , Anstee  , O’neil  , Katz  , Chain
Published: March 2003

This study explores the expression and the function of major histocompatibility complex class II in the intestinal epithelial cell line CaCo‐2, which has been widely used as a model for the human gastrointestinal epithelium. Human leucocyte antigen (HLA)‐DR expression on CaCo‐2 cells is induceable by interferon‐γ (IFN‐γ), but responsiveness to IFN‐γ is dependent on cell differentiation and IFN‐γ availability at the basolateral cell surface. HLA‐DR expression is concentrated in apical cytoplasmic vesicles and on the basolateral cell surface. Invariant chain is expressed in apical vesicles but is absent from the cell surface. Immunoprecipitation studies show a slow rate of dissociation of HLA‐DR from Ii. Double labelling shows some overlap between HLA‐DR expression and basolateral endosomal markers but no overlap with apical endosomal markers. Functional studies show processing and presentation of lysozyme endocytosed from the basolateral, but not apical surfaces. CaCo‐2 cells may provide a useful model with which to dissect the antigen‐processing pathways in polarized epithelial cells. The regulated access of antigens taken up from the gut lumen to the processing compartments may prevent overloading the immune system with antigens derived from normal gut contents.
Systemic mycobacterial infection inhibits antigen‐specific immunoglobulin E production, bronchial mucus production and eosinophilic inflammation induced by allergen
Authors: Yang  , Wang  , Fan  , Zhu
Published: March 2003

As the burden of infectious diseases becomes reduced in many countries, a remarkable increase in the incidence of allergies has occurred. The basis for the rise in atopic disorders as a correlate of the decline in infectious diseases has not been defined. In the present study, we tested experimentally whether prior systemic infection with Mycobacterium bovis bacillus Calmette Guérin (BCG) had any effect on ovalbumin (OVA) Al(OH)3 (alum)‐induced immunoglobulin E (IgE) production, airway mucus production and eosinophilic inflammation. The data showed that allergen‐specific IgE production and OVA‐induced eosinophilia and goblet cell development were significantly inhibited by prior infection with BCG. Correspondingly, following immunization with OVA alum, BCG‐infected mice exhibited significantly higher levels of allergen‐driven interferon‐γ (IFN‐γ) production than the mice without infection. The ratio of IFN‐γ: interleukin (IL)‐4 production was higher in OVA‐sensitized mice with prior BCG infection than in those without infection. The abrogation of OVA‐induced mucus production and pulmonary eosinophilia in BCG‐infected mice correlated with significantly decreased IL‐5 production and increased IFN‐γ and IL‐12 production. These data provide direct evidence that intracellular bacterial infection (i.e. BCG) can inhibit antigen‐specific IgE and airway reactivity induced by environmental allergen. Furthermore, the results suggest that changes in cytokine‐producing patterns of T lymphocytes and other cells may be the mechanism by which infections influence allergies.
Selective correlation of interferon‐γ, tumour necrosis factor‐α and granulocyte–macrophage colony‐stimulating factor with immunoglobulin G1 and immunoglobulin G3 subclass antibody in leprosy
Authors: Hussain  , Kifayet  , Dojki  , Dockrell
Published: March 2003

Dysregulation of both B‐ and T‐cell responses is observed in leprosy. Immunoglobulin G1 (IgG1) and IgG3 antibody subclasses are selectively elevated towards the lepromatous or disseminated form of the disease accompanied by a depression of T‐cell responses. T‐cell and macrophage cytokines influence antibody class switching, differentiation and proliferation of B cells. To understand the dynamic nature of the immune response in leprosy, we examined the relationship between circulating Mycobacterium leprae‐specific antibodies and secreted cytokines [interferon‐γ (IFN‐γ), interleukin‐2 (IL‐2), IL‐5, IL‐10, IL‐6, tumour necrosis factor‐α (TNF‐α) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF)] in leprosy patients (19 lepromatous patients; 25 tuberculoid patients) and their exposed household contacts (HC=14) in response to M. leprae antigens. Paired comparison revealed a highly significant negative correlation between IFN‐γ and IgG (P=0·016), IgG1 (P<0·001) and IgG3 (P=0·007) antibodies. No significant relationship was observed with other T‐cell cytokines (IL‐2, IL‐5 and IL‐10). These results strongly suggest that IFN‐γ may play a role in down‐regulating antigen‐specific IgG1 and IgG3 antibodies. Among the macrophage cytokines, TNF‐α and GM‐CSF which have not been shown to play a role in B‐cell activation were positively associated with IgG1 (TNF‐α, P=0·0005; GM‐CSF, P=0·001) and IgG3 (TNF‐α, P=0·001; GM‐CSF, P=0·021) antibodies. Since macrophages have high‐affinity Fc receptors for IgG1 and IgG3, it is possible that antigen uptake via these receptors may influence cytokine expression of TNF‐α, a key modulator of disease pathogenesis in mycobacterial diseases. We are currently investigating the role of Fc receptors on activated macrophages, in expression of pro‐inflammatory cytokines in mycobacterial diseases.
Granuloma formation is required to contain bacillus growth and delay mortality in mice chronically infected with Mycobacterium tuberculosis
Authors: Saunders  , Frank  , Orme
Published: March 2003

Previous studies in this laboratory have shown that mice with a gene disruption to the intracellular adhesion molecule‐1 (ICAM‐K/O) express normal cell‐mediated immunity but cannot mount delayed‐type hypersensitivity reactions following Mycobacterium tuberculosis infection. However, even in the absence of any appreciable granuloma formation, these mice control bacterial growth for at least 90 days. While not required to control the infection initially, we hypothesized that granuloma formation was required to control chronic infection, acting by surrounding infected cells to prevent bacterial dissemination. To test this, ICAM‐1 knockout mice were infected with a low dose aerosol of M. tuberculosis Erdman and were found to succumb to infection 136±30 days later, displaying highly elevated bacterial loads compared to wild‐type mice. Lung tissue from ICAM‐K/O mice displayed extensive cellular infiltration and widespread tissue necrosis, but no organized granulomatous lesions were evident, whereas the control mice displayed organized compact granulomas. These data demonstrate that while a granulomatous response is not required initially to control M. tuberculosis infection, absence of granulomas during chronic infection leads to increased bacterial growth and host death. Thus these data support the hypothesis that granuloma formation is required to control chronic infection, acting by surrounding and walling off sites of infection to prevent bacterial dissemination and maintain a state of chronic infection.
Up‐modulation of interferon‐γ mediates the enhancement of spontanous cytotoxicity in prolactin‐activated natural killer cells
Authors: Matera L. , Contarini M. , Bellone G. , Forno B. , Biglino A.
Published: March 2003

Prolactin (PRL) has been shown to participate in lymphocyte activation. In particular, the constitutive natural killer (NK) and the lymphokine‐activated killer (LAK) cytotoxicity of CD56+ CD16+ cells is increased by its physiological to supraphysiological concentrations. As PRL has been shown to up‐regulate the production of interferon‐γ (IFN‐γ) by peripheral blood mononuclear cells, we studied its effect on IFN‐γ production by NK cells as a possible mechanism of autocrine activation of cytotoxicity. Released and intracellular IFN‐γ, as well as IFN‐γ mRNA expression, were increased by pituitary and recombinant human PRL, which stimulated optimal NK and LAK cytotoxicity. Treatment with blocking anti‐IFN‐γ monoclonal antibody (mAb) selectively affected PRL‐increased killing of K562 targets, demonstrating that PRL‐mediated enhancement of spontaneous cytotoxicity depends, at least in part, on up‐regulation of IFN‐γ.
Regulation of epidermal Langerhans cell migration by lactoferrin
Authors: Cumberbatch M. , Dearman R. J. , Uribe‐luna S. , Headon D. R. , Ward P. P. , Conneely O. M. , Kimber I.
Published: March 2003

Lactoferrin (LF) is a member of the transferrin family of iron‐binding glycoproteins to which several anti‐inflammatory functions have been ascribed. LF has been shown to down‐regulate expression of the pro‐inflammatory cytokine tumour necrosis factor‐α (TNF‐α), although the possibility has been raised that the activity of LF in this regard was indirect and secondary to its ability to bind to and inactivate the bacterial lipopolysaccharide (LPS) used to induce cytokine production. However, the identification of putative membrane receptors for LF raises the possibility that the interaction of LF with its receptor may be one important route through which this protein exerts anti‐inflammatory activity. In the present investigations the biological properties of LF have been examined in a model of cutaneous immune function where the allergen‐induced migration of epidermal Langerhans cells (LC) from the skin and their subsequent accumulation as dendritic cells (DC) in skin‐draining lymph nodes are known to be dependent upon the de novo synthesis of TNF‐α, but independent of exogenous LPS. Consistent with the protein having direct anti‐inflammatory properties, it was found that the intradermal injection of recombinant murine LF (either iron‐saturated or iron‐depleted LF) inhibited significantly allergen (oxazolone) ‐induced LC migration and DC accumulation. That these inhibitory effects were secondary to the inhibition of local TNF‐α synthesis was suggested by the findings that first, LF was unable to inhibit LC migration induced by intradermal injection of TNF‐α itself, and second, that migration stimulated by local administration of another epidermal cytokine, interleukin 1β, which is also dependent upon TNF‐α production, was impaired significantly by prior treatment with LF. Finally, immunohistochemical analyses demonstrated the presence of LF in skin, associated primarily with keratinocytes. Collectively these data support the possession by LF of direct immunomodulatory and/or anti‐inflammatory activity, probably associated in this case with inhibition of cytokine production. Furthermore, the results suggest that as a constituent of normal skin, LF may play a role in homeostatic regulation of cutaneous immune function.
Lack of metallothionein‐I and ‐II exacerbates the immunosuppressive effect of ultraviolet B radiation and cis‐urocanic acid in mice
Authors: Reeve V. E. , Nishimura N. , Bosnic M. , Michalska A. E. , Choo K. H. A.
Published: March 2003

The effect of a null mutation for the metallothionein (MT)‐I and ‐II isoforms in mice on the immunosuppressive action of ultraviolet B (UVB; 280–320 nm) radiation has been examined. Mice were exposed to a series of increasing daily UVB doses, each dose administered to the dorsum on 3 consecutive days. Erythema was assessed, and measured as its oedema component by the post‐irradiation dorsal skinfold thickness, but there was no effect of the null mutation (MT–/–) observed after 3 × 3·4 kJ/m2 of UVB radiation. Immune function was assessed by the contact hypersensitivity (CHS) response, which was initiated by sensitization on unirradiated abdominal skin, and thus demonstrated the systemic effects of dorsal treatments. In comparison with the wild‐type MT+/+ mouse, the MT–/– mouse was significantly more immunosuppressed by moderate daily UVB doses (1·75–5·9 kJ/m2). When topically applied cis‐urocanic acid (cis‐UCA) replaced UVB radiation as the immunosuppressive agent, contact hypersensitivity in MT–/– mice was again markedly more suppressed than in MT+/+ mice, in a dose‐responsive manner. The results infer that MT, which was shown immunohistochemically to be strongly induced in the epidermis of MT+/+ mice, but to be absent in MT–/– epidermis, has the potential to protect from photoimmunosuppression, and that the mechanism of action may be via the inactivation of the epidermal UVB‐photoproduct, cis‐UCA.
Repeat bacterial challenge in a subcutaneous chamber model results in augmented tumour necrosis factor‐α and interferon‐γ response, and suppression of interleukin‐10
Authors: Houri‐haddad Y. , Soskolne W. A. , Halabi A. , Barak V. , Shapira L.
Published: March 2003

The present study compared the effect of a single or a repeat challenge with the Gram‐negative pathogen Porphyromonas gingivalis on the local inflammatory response within subcutaneous chamber model in mice. Subcutaneous chambers were implanted 2 weeks prior to the final challenge. The repeat‐challenge (REP) group received two intrachamber bacterial injections 14 days apart, while the single‐injection group (SIN) received only a single bacterial challenge. Injection of saline was used as the control. The cellular contents of the chamber exudates were used for differential cell counts, and the supernatants were analysed for tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), and interleukin (IL)‐10 levels. Immunoglobulin G1 (IgG1) and IgG2a levels to P. gingivalis in the exudates were also determined. The results showed that the leucocyte counts increased significantly post‐challenge, and the REP group showed the highest number of lymphocytes and neutrophils. Both P. gingivalis‐challenged groups exhibited significant increase in TNF‐α and IL‐10 levels at day 1 post‐challenge. TNF‐α levels in the chamber exudate were threefold higher in the REP group compared with the SIN group on day 1 post‐challenge (P < 0·05). In contrast, IL‐10 levels were significantly lower in the REP group 1 day post‐challenge compared with the SIN group. The REP group had significantly higher levels of IFN‐γ at baseline, and this difference remained significant 1 day post‐challenge. Analysis of antibody levels to P. gingivalis showed that while the control and the SIN groups had no anti‐P. gingivalis IgG in the chamber exudate during the 7‐day study period, the REP group showed high anti‐P. gingivalis IgG levels. In addition, the titres of IgG2a were fivefold higher than the IgG1 titres. The results showed that a repeat local challenge with P. gingivalis augmented the proinflammatory cytokines TNF‐α and IFN‐γ, while inhibiting the accumulation of the anti‐inflammatory cytokine IL‐10. This shift towards a T helper 1 (Th1)‐dominant response was reflected in the relatively high anti‐P. gingivalis IgG2a titres in the local inflammatory environment 7 days post‐challenge.
Nerve growth factor‐β induces mast‐cell marker expression during in vitro culture of human umbilical cord blood cells
Authors: Welker P. , Grabbe J. , Gibbs B. , Zuberbier T. , Henz B. M.
Published: March 2003

Nerve growth factor‐β (NGF) is known as a growth factor for human basophils and murine mast cells and has recently been shown to also up‐regulate mast cell characteristics in human leukaemic mast cells. We have examined here the effect of NGF on the differentiation of normal human mast cells from cord blood progenitors during culture with stem cell factor (SCF), NGF alone or in combination, or fibroblast supernatants. All these supplements induced mast cell immunoreactivity against tryptase, c‐Kit and FcεRIα, but none of the cells reacted against the basophil specific antibody 2D7 before or during culture. Intracellular tryptase activity increased as well, with maximal levels on combined culture with SCF and NGF. On reverse transcription–polymerase chain reaction (RT–PCR), cells lacked tryptase and chymase and expressed low levels of FcεRI and c‐Kit mRNA prior to culture, with marked up‐regulation of FcεRI and c‐Kit, and with de novo expression of mast‐cell specific α‐ and β‐tryptase by week 3, and of chymase by week 5. Only the TrkA and not the p75 NGF receptor was detected at m‐RNA and protein level, and only the TrkA NGF receptor was up‐regulated during NGF‐driven culture. These findings show therefore that, like SCF, NGF is another growth factor that can induce and regulate human mast‐cell development and differentiation.
Expression and functional activity of the very late activation antigen‐4 molecule on human natural killer cells in different states of activation
Authors: Macías C. , Ballester J. M. , Hernández P.
Published: March 2003

In the present study we describe the expression and functional activity of the α4β1 heterodimer molecule on human natural killer (NK) cells. Flow cytometric analyses showed that fresh and activated NK cells expressed high levels of very late activation antigen‐4 (VLA‐4) molecules. These cells bound to fibronectin (FN) and to its 38 000‐MW proteolytic fragment through the VLA‐4 integrin that was blocked with HP2/1 anti‐α4 monoclonal antibodies (mAbs) and with the FN peptide fragment CS1. No inhibitory effects were observed in the presence of anti‐α5 mAb, FN peptide fragment CS2 or other irrelevant mAb. Fresh NK cells were unable to aggregate, despite their expression of VLA‐4, and only activated (cultured and lymphocyte‐activated killer cells) NK cells showed homotypic aggregation with HP1/7 and HP2/4 anti‐α4 mAb related to cellular activation. These results underline new evidence of how NK cells in different states of activation maintain different constitutive levels of α4β1 integrin activity, and highlight the possibility of a different functional regulation by the cells bearing VLA‐4, in the expression of these epitopes and their ability to interact with their ligands.
Tamoxifen alleviates disease severity and decreases double negative T cells in autoimmune MRL‐lpr/lpr mice
Authors: Wu W.‐M. , Suen J.‐L. , Lin B.‐F. , Chiang B.‐L.
Published: March 2003

Previous study suggested that MRL‐lpr/lpr mice treated with tamoxifen (TAM) had less severe proteinuria, reduced serum titre of anti‐dsDNA autoantibodies and an increased survival rate. To investigate further the regulatory mechanisms of TAM on MRL‐lpr/lpr female mice, a total dose of 200 µg per mice (5·5 mg/kg) was given every 2 weeks subcutaneously, while the control mice were injected with oil only. After being treated with TAM four times, the mice were killed and cellular functions were evaluated. The TAM‐treated groups had smaller sized spleen and lymph nodes. Flow cytometric analysis of splenocytes had a significantly lower percentage of cell number of T cells and double negative T cells (CD4– CD8– T cells). There was no difference in cytokine production (interleukin (IL)‐2, IL‐4, IL‐5, IL‐10 and interferon‐γ (IFN‐γ)) from splenocytes stimulated with concanavalin A (Con A) or cytokines (IL‐6) secreted by peritoneal exudate cells when stimulated with lipopolysaccharide (LPS). However, IL‐2 from lymph node cells was significantly higher on TAM‐treated mice. Finally, splenocytes or purified T cells stimulated with anti‐CD3 antibody plus cross‐linking immunoglobulin G (IgG) of the TAM‐treated group had higher 3H‐incorporation of proliferation assay compared with that of control groups. In vitro study further demonstrated that IL‐2‐activated proliferation of lymph node double negative (DN) T cells can be inhibited by TAM treatment in a dose‐dependent manner. Our finding demonstrated that TAM may potentially influence T cells and modulate the immune function, which offers a novel approach to explore the feasibility of hormone therapy for autoimmune diseases.
Clonal anergy induced in a CD8+ hapten‐specific cytotoxic T‐cell clone by an altered hapten‐peptide ligand
Authors: Preckel T. , Hellwig S , Pflugfelder U , Lappin M. B. , Weltzien H. U.
Published: March 2003

Clonal T‐cell anergy has been proposed as a mechanism to ensure peripheral tolerance in vivo. Anergy has been reported to result from T cell activation with inappropriate antigen‐presenting cells (APC) or, in the case of CD4+ T cells, also by altered peptide ligands. This study reveals that altered hapten ligands can also induce anergy in CD8+ T cells. The Kb‐restricted, trinitrophenyl (TNP) specific cytotoxic T lymphocyte (CTL) clone E6 was found to lyse target cells presenting the TNP‐modified peptides M4L‐TNP (derived from mouse serum albumin) or O4TNP (derived from chicken ovalbumin), but not the corresponding dinitrophenol (DNP)‐modified peptides. However, whereas M4L‐DNP was found totally unreactive, O4DNP antagonistically inhibited M4L‐TNP‐mediated kill if expressed on the same target cell. Moreover, when presented alone on APC, O4DNP, but not M4L‐DNP, induced anergy in clone E6 by preventing its subsequent proliferative response to M4L‐TNP. The anergic state did not affect agonist‐specific cytolysis or T‐cell receptor (TCR) down‐modulation by the anergized CTL, and proliferative responses were regained upon addition of interleukin (IL)‐2 or IL‐12 plus IL‐18. These findings substantiate the similarity between hapten‐and peptide‐recognition by T cells. The induction as well as the reversal of anergy in CD8+ CTL may thus be of relevance not only in autoimmunity or tumour rejection, but also in contact hypersensitivity reactions to haptens.
The human macrophage cell line U937 as an in vitro model for selective evaluation of mycobacterial antigen‐specific cytotoxic T‐cell function
Authors: Passmore J. S. , Lukey P. T. , Ress S. R.
Published: March 2003

Despite strong evidence for CD8+ T‐cell function in murine mycobacterial infections, their corresponding role in human tuberculosis has proven more difficult to demonstrate. We have evaluated the human macrophage (Mφ) cell line U937 as an in vitro model for human leucocyte antigen (HLA) class I‐restricted presentation of mycobacterial antigens, as HLA class I is constitutively expressed at high levels by U937 cells in the absence of detectable HLA class II or CD1 molecules. U937 cells were evaluated for their ability to phagocytose Mycobacterium tuberculosis and for their ability to present mycobacterial antigens to human HLA class I‐matched cytotoxic T lymphocytes (CTLs). Differentiated U937 cells were capable of efficient phagocytosis of M. tuberculosis but did not generate a subsequent respiratory burst response, and were permissive for intracellular growth of both bacillus Calmette–Guérin (BCG) and the virulent M. tuberculosis H37Rv strain. CTL activity was restricted to live mycobacterial organisms and was shown to be mediated by M. tuberculosis‐specific, HLA class I‐matched, purified CD8+ CTL lines and CD8+ T‐cell clones. Furthermore, M. tuberculosis‐infected U937 targets were more rapidly and strongly lysed by CD8+ CTLs than were infected autologous Mφ. Finally, M. tuberculosis‐infected U937 cells simultaneously provided a sensitive indicator for detection of mycobacterial‐specific, HLA‐unrestricted γδ+ CTL activity.
Primary human alveolar epithelial cells can elicit the transendothelial migration of CD14+ monocytes and CD3+ lymphocytes
Authors: Eghtesad M. , Jackson H. E. , Cunningham A. C.
Published: March 2003

The ability of freshly isolated primary human alveolar epithelial cells (type II pneumocytes) to induce leucocyte migration across an endothelial monolayer was investigated. Three‐way factorial analysis of variance (anova) demonstrated that resting alveolar endothelial cells (AEC) could produce detectable quantities of monocyte chemoattractant protein 1 (MCP‐1), which was upregulated in response to tumour necrosis factor‐α (TNF‐α) in a dose‐ and time‐dependent fashion. Interferon‐γ (IFN‐γ) had no significant effect on this process. TNF‐α and IFN‐γ both induced AEC to provoke migration of CD14+ monocytes and CD3+ lymphocytes across endothelium. IFN‐γ and TNF‐α synergized in their ability to induce production of T lymphocyte, but not monocyte, chemoattractants from AEC. Leucocyte transendothelial migration was inhibited by anti‐MCP‐1 neutralizing antibody and by heparin, a polyanionic glycosaminoglycan (GAG). These data suggest that human AEC play a role in the multiple mechanisms that facilitate monocyte and T lymphocyte migration into the alveolar compartment of the lung under homeostasis and inflammatory conditions. One of these mechanisms is mediated via constitutive MCP‐1 production by alveolar epithelial cells, which is upregulated by TNF‐α.
Interferon‐γ is crucial for surviving a Brucella abortus infection in both resistant C57BL/6 and susceptible BALB/c mice
Authors: Murphy Erin A. , Sathiyaseelan Janaki , Parent Michelle A. , Zou Baixiang , Baldwin Cynthia L.
Published: March 2003

Brucella abortus is an intracellular bacterial pathogen that causes chronic infections in humans and a number of agriculturally important species of animals. It has been shown that BALB/c mice are more susceptible to infections with virulent strains of Brucella abortus than C57BL/6 or C57BL/10 strains. In experiments described here, gene knock‐out mice were utilized to elucidate some of the salient components of resistance. Resistant C57BL/6 mice with gene deletions or disruptions in the interferon‐γ (IFN‐γ), perforin or β2‐microglobulin genes had decreased abilities to control intracellular infections with B. abortus strain 2308 during the first week after infection. However, only the IFN‐γ knock‐out mice had a sustained inability to control infections and this resulted in death of the mice at approximately 6 weeks post‐infection. These mice had a continual increase in the number of bacterial colony‐forming units (CFU) in their spleens until death. When BALB/c mice with the disrupted IFN‐γ gene were infected they had more splenic CFU at one week post‐infection than control mice but the increase was not statistically significant and by 3 weeks they did not have more CFU than control mice. Moreover, the number of splenic bacteria did not increase in the BALB/c IFN‐γ knock‐out mice between 6 and 10·5 weeks, although they died at 10·5 weeks, the time by which normal BALB/c mice were clearing the infection. Death in both strains of IFN‐γ gene disrupted mice coincided with symptoms of cachexia and macrophages comprised ≥ 75% of the splenic leucocytes.
Changes in gene expression in macrophages infected with Mycobacterium tuberculosis: a combined transcriptomic and proteomic approach
Authors: Ragno Silvia , Romano Maria , Howell Steven , Pappin Darryl J. C. , Jenner Peter J. , Colston Michael J.
Published: March 2003

We investigated the changes which occur in gene expression in the human macrophage cell line, THP1, at 1, 6 and 12 hr following infection with Mycobacterium tuberculosis. The analysis was carried out at the transcriptome level, using microarrays consisting of 375 human genes generally thought to be involved in immunoregulation, and at the proteomic level, using two‐dimensional gel electrophoresis and mass spectrometry. The analysis of the transcriptome using microarrays revealed that many genes were up‐regulated at 6 and 12 hr. Most of these genes encoded proteins involved in cell migration and homing, including the chemokines interleukin (IL)‐8, osteopontin, monocyte chemotactic protein‐1 (MCP‐1), macrophage inflammatory protein‐1α (MIP‐1α), regulated on activation, normal, T‐cell expressed and secreted (RANTES), MIP‐1β, MIP‐3α, myeloid progenitor inhibitory factor‐1 (MPIF‐1), pulmonary and activation regulated chemokine (PARC), growth regulated gene‐β (GRO‐β), GRO‐γ, MCP‐2, I‐309, and the T helper 2 (Th2) and eosinophil‐attracting chemokine, eotaxin. Other genes involved in cell migration which were up‐regulated included the matrix metalloproteinase MMP‐9, vascular endothelial growth factor (VEGF) and its receptor Flk‐1, the chemokine receptor CCR3, and the cell adhesion molecules vesicular cell adhesion molecule‐1 (VCAM‐1) and integrin a3. In addition to the chemokine response, genes encoding the proinflammatory cytokines IL‐1β (showing a 433‐fold induction), IL‐2 and tumour necrosis factor‐α (TNF‐α), were also found to be induced at 6 and/or 12 hr. It was more difficult to detect changes using the proteomic approach. Nevertheless, IL‐1β was again shown to be strongly up‐regulated. The enzyme manganese superoxide dismutase was also found to be strongly up‐regulated; this enzyme was found to be macrophage‐, rather than M. tuberculosis, derived. The heat‐shock protein hsp27 was found to be down‐regulated following infection. We also identified a mycobacterial protein, the product of the atpD gene (thought to be involved in the regulation of cytoplasmic pH) in the infected macrophage extracts.
Lipopolysaccharide triggers invasive streptococcal disease in mice through a tumour necrosis factor‐α‐dependent mechanism
Authors: Diao Hongyan , Kohanawa Masashi , Yimin  , Nakajima Hirofumi , Sato Yuichiro , Minagawa Tomonori , Nakane Akio
Published: March 2003

Streptococcus pyogenes sometimes induces invasive streptococcal infection, including streptococcal toxic shock syndrome (STSS). Muscular necrosis is one of the peculiar symptoms of invasive streptococcal infection and STSS. We inoculated S. pyogenes into the muscles of mice. To do so, 5 × 108 bacteria in 0·2 ml phosphate‐buffered saline were injected into the right hind thigh. None of the mice injected with the bacteria showed muscular necrosis and none died. Tumour necrosis factor‐α (TNF‐α) and infiltration of leucocytes were detected in the muscles of infected sites, although the condition of the infected mice did not deteriorate after anti‐TNF‐α monoclonal antibody treatment. The infected mice treated intraperitoneally with Escherichia coli lipopolysaccharide (LPS) showed augmentation of bacterial growth, muscular necrosis and death. TNF‐α was detected in the sera of the infected mice treated with LPS, but not in the muscles of the infected sites. Infiltration of leucocytes into the infected muscle was not observed in the infected mice treated with LPS. Anti‐TNF‐α monoclonal antibody treatment decreased mortality in the infected mice treated with LPS. Moreover, the infected mice treated with recombinant TNF‐α showed augmentation of muscular necrosis and death. These results suggest that systemic production of TNF‐α induced by stimulation with LPS inhibits infiltration of leucocytes into the infected site and exacerbates muscular infection, and that TNF‐α produced in streptococcal infection is not a defence factor for the host. Invasive streptococcal infection and STSS appear to be induced by both S. pyogenes and the host's immune system.
Local T‐cell activation after segmental allergen challenge  in the lungs of allergic dogs
Authors: Out Theo A. , Wang Shan‐Ze , Rudolph Karin , Bice David E.
Published: March 2003

Dogs with immunoglobulin E (IgE) allergy for ragweed that are sensitized by intrapulmonary exposure to ragweed can be used to study the pulmonary immune response that is important in allergic asthma. Using this model, we tested the hypothesis that T lymphocytes are activated locally in the airways shortly after allergen exposure of the lungs. The airways of six allergic dogs and three non‐allergic dogs were exposed to ragweed by segmental allergen challenge (SAC). T‐cell subsets and T‐cell activation in blood and bronchoalveolar lavage (BAL) fluid were measured by flow cytometry before SAC and at 4, 24 and 72 hr thereafter. SAC caused a statistically significant increase in the percentage of major histocompatibility complex (MHC) class II‐positive CD4 and CD8 T cells in BAL fluid and a significant increase in the mean fluorescent activity of MHC class II from 4 hr after SAC onward. This activation was significantly different from that found in cells from lung lobes challenged with saline, or from lung lobes in non‐allergic dogs challenged with ragweed. The percentage of CD45RAbright CD8 cells increased significantly in allergic dogs after both ragweed and saline challenges. This was significantly higher than in non‐allergic dogs. We conclude that T‐cell activation in the airways of dogs can be measured after in vivo activation of the cells by measuring MHC class II and CD45RA expression in BAL fluid T cells. Furthermore, in allergic dogs, T cells are activated locally in the lungs within 4 hr after exposure to ragweed allergen. These results suggest a role for T lymphocytes in the development of late‐phase allergic reactions in the airways.
Developmentally regulated interactions of human thymocytes with different laminin isoforms
Authors: Kutleša SnjeŽana , Siler Ulrich , Speiser Angelika , Wessels Johannes T. , Virtanen Ismo , Rousselle Patricia , Sorokin Lydia M. , Müller Claudia A. , Klein Gerd
Published: March 2003

The gene family of heterotrimeric laminin molecules consists of at least 15 naturally occurring isoforms which are formed by five different α, three β and three γ subunits. The expression pattern of the individual laminin chains in the human thymus was comprehensively analysed in the present study. Whereas laminin isoforms containing the laminin α1 chain (e.g. LN‐1) were not present in the human thymus, laminin isoforms containing the α2 chain (LN‐2/4) or the α5 chain (LN‐10/11) were expressed in the subcapsular epithelium and in thymic blood vessels. Expression of the laminin α4 chain seemed to be restricted to endothelial cells of the thymus, whereas the LN‐5 isoform containing the α3 chain could be detected on medullary thymic epithelial cells and weakly in the subcapsular epithelium. As revealed by cell attachment assays, early CD4− CD8− thymocytes which are localized in the thymus beneath the subcapsular epithelium adhered strongly to LN‐10/11, but not to LN‐1, LN‐2/4 or LN‐5. Adhesion of these thymocytes to LN‐10/11 was mediated by the integrin α6β1. During further development, the cortically localized CD4+ CD8+ thymocytes have lost the capacity to adhere to laminin‐10/11. Neither do these cells adhere to any other laminin isoform tested. However, the more differentiated single positive CD8+ thymocytes which were mainly found in the medulla were able to bind to LN‐5 which is expressed by medullary epithelial cells. Interactions of CD8+ thymocytes with LN‐5 were integrin α6β4‐dependent. These results show that interactions of developing human thymocytes with different laminin isoforms are spatially and developmentally regulated.
Antigen binding to GM1 ganglioside results in delayed presentation: minimal effects of GM1 on presentation of antigens internalized via other pathways
Authors: Nashar Toufic O. , Betteridge Zoe E. , Mitchell Richard N.
Published: March 2003

Plasma membrane rafts are sphingolipid‐ and cholesterol‐rich patches that function as membrane trafficking and surface signalling regions. Ganglioside GM1 is an integral component of these microdomains, and Escherichia coli enterotoxin B subunit (EtxB) is a pentamer that binds with high affinity to GM1 resulting in GM1 cross‐linking. We previously demonstrated that antigen coupled directly to EtxB resulted in enhanced presentation relative to antigen taken up by fluid‐phase endocytosis. Here we demonstrate a new role for GM1 in antigen presentation by examining the effects of cross‐linking GM1 on the kinetics of presentation and processing of antigen by the B‐cell receptor (BCR), fluid‐phase endocytosis and GM1‐targeted antigen. EtxB bound to B cells does not augment the subsequent kinetics or magnitude of presentation of either BCR‐internalized antigen or soluble antigen. Moreover, presentation of GM1‐bound antigen is significantly slower than antigen presentation following BCR‐mediated uptake. In contrast to the rapid internalization of BCR‐bound antigen (which has a half life of 60 min), the majority of EtxB‐bound antigen forms a plasma membrane depot detectable for many hours after initial incubation (and with a half life of 12 hr). We conclude that cross‐linking of GM1 by EtxB minimally affects the processing and presentation of antigens internalized via other pathways. Nevertheless, binding of antigens to GM1 results in delayed presentation that has important implications for in vivo immunization using GM1‐targeted adjuvants.
Murine NFX.1: isolation and characterization of its messenger RNA, mapping of its chromosomal location and assessment of its developmental expression
Authors: Arlotta Paola , Miyazaki Dai , Copeland Neal G. , Gilbert Debra J. , Jenkins Nancy A. , Ono Santa J.
Published: March 2003

We have previously isolated (by expression cloning) a human cDNA, termed NFX.1, encoding a nucleic acid‐binding protein that interacts with the conserved X1 box cis‐element first discovered in class II major histocompatibility complex (MHC) genes. Functional studies involving expression of NFX.1 and assessment of expression from class II reporter constructs and endogenous class II MHC genes indicated that the factor could repress transcription of class II MHC genes. Subsequent studies have extended the biological significance of the factor, indicating that it plays an important role in neuronal development. Indeed, the reiterated RING finger motifs in the central domain of the polypeptide strongly suggest that NF‐XI is a probable E3 ubiquitin protein ligase, indicating that the protein may have multiple activities. Here we report the cloning of the mouse homologue of the human NfX.1 cDNA: m‐Nfx.1. Comparison of the deduced primary sequence of mouse and human NFX.1 proteins shows very high homology and confirms that m‐NFX.1 contains the conserved cysteine‐rich DNA‐binding motif first described in human NFX.1 (95% homology). Expression of MHC class II genes is substantially reduced following expression of m‐NFX.1, which confirms that we have isolated the functional murine homologue of human NfX.1 cDNA. Further evidence comes from the mapping of m‐Nfx.1 gene to the proximal region of mouse chromosome 4, a region syntenic to the location of human Nfx.1 (short arm of chromosome 9). Expression profiling shows that m‐NFX.1 is expressed ubiquitously in both adult tissues and during development, supporting the hypothesis that it may have yet‐undescribed roles in distinct biological processes.
Regulation of HLA‐DR and co‐stimulatory molecule expression on natural killer T cells by granulocyte–macrophage colony‐stimulating factor
Authors: Saikh Kamal U. , Kissner Teri , Ulrich Robert G.
Published: March 2003

A subset of mononuclear cells present in most tissues coexpresses receptors of both natural killer (NK) and T cells. Although linked to antiviral immunity, the function of these putative NKT cells is uncertain. We present evidence that human CD56+ DR− NKT cells exhibit hybrid adaptive and innate immune functions. These cells spontaneously lysed tumour cell targets and upon engagement of T‐cell antigen receptors secreted the cytokines interferon‐γ and granulocyte–macrophage colony‐stimulating factor (GM‐CSF). Conversely, GM‐CSF treatment transformed the NKT cells into dendritic cells, inducing rapid expression of HLA‐DR and the co‐stimulatory molecules CD80 and CD86. The ability to stimulate tetanus toxoid‐specific responses from naïve T cells was acquired within 3 days of activating CD56+ NKT cells with GM‐CSF. These results suggest a potential role for NKT cells in the initiation and control of primary immunity during the acute phase of infection.
Regulation of thymocyte homeostasis by Fliz1
Authors: Hwang Eun Sook , Ho I‐Cheng
Published: March 2003

Fliz1 (fetal liver zinc finger protein 1) is a novel zinc finger protein preferentially expressed in fetal liver hematopoietic progenitors and in several adult organs, including the thymus. To investigate the in vivo function of Fliz1 in regulating the development of T cell lineage, we generated and studied transgenic mice overexpressing human Fliz1 in a T‐cell specific and inducible manner. We found that overexpression of human Fliz1 in adult animals resulted in a substantial decrease in total number of thymocytes due to enhanced apoptosis, whereas maturation of thymocytes remained undisturbed. The Fliz1‐induced apoptosis is dependent on the N‐terminus of Fliz1, which contains an acidic and a serine‐rich domains, and might be due to augmented expression of bad, a pro‐apoptotic gene. Taken together, our data suggest that Fliz1 plays a role in regulating thymocyte homeostasis.