bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear to cytoplasmic compartment shift of the p33ING1b tumour suppressor protein is associated with malignancy in melanocytic lesions
Authors: Nouman G S , Anderson J J , Mathers M E , Leonard N , Crosier S , Lunec J , Angus B
Published: March 2003

Nuclear to cytoplasmic compartment shift of the p33

ING1b

tumour suppressor protein is associated with malignancy in melanocytic lesions

Aims: Cutaneous malignant melanoma is an unpredictable neoplasm. Studies of cell cycle and proliferation‐associated proteins may help in the understanding of the genesis of melanomas. The tumour suppressor gene TP53 has been shown to be involved in melanomas. However, the incidence of TP53 malfunction in cutaneous melanoma is unclear, and other regulators of cell cycle control are likely to be involved in both the development and progression of melanocytic neoplasia. A candidate is the ING1 gene, which co‐operates with TP53 in growth suppression and apoptosis. Thus loss of ING1 function may have similar consequences to loss of TP53 function and may contribute to tumorigenesis. Therefore we have studied the expression of p33ING1b protein in cutaneous melanocytic neoplasia.

Methods and results: Sixty‐seven melanocytic lesions were studied by immunohistochemistry for the expression of p33ING1b. In our series there was loss of nuclear p33ING1b expression in invasive malignant melanoma compared with normal cutaneous melanocytes or the melanocytes of benign melanocytic naevi. This was associated with an enhancement of cytoplasmic p33ING1b expression which was particularly prominent in invasive malignant melanoma.

Conclusions: Cytoplasmic immunostaining for p33ING1b using MAb GN2 is strongly associated with `activated' melanocytic lesions; therefore it is possible that this MAb could be of value in diagnostic practice. Furthermore, the reduction in p33ING1b expression and perhaps translocation from the nucleus to the cytoplasm may play a central role in the development and progression of melanomas.
Differential Ki67 and bcl‐2 immunoexpression in solid–glandular and spindle cell components of biphasic synovial sarcoma: a double immunostaining assessment with cytokeratin and vimentin
Authors: Lopes J M , Nesland J M , Reis‐Filho J S , Holm R
Published: March 2003

Differential Ki67 and bcl‐2 immunoexpression in solid–glandular and spindle cell components of biphasic synovial sarcoma: a double immunostaining assessment with cytokeratin and vimentin

Aims: Synovial sarcoma is a malignant soft tissue of uncertain histogenesis that may show a biphasic (spindle and solid/glandular components) or a monophasic histological appearance. In previous studies, we demonstrated that the solid/glandular component possesses higher proliferation rates than the spindle cell component of biphasic synovial sarcomas and that the spindle cell component may exhibit a progressive transition from or to the solid‐glandular component in biphasic synovial sarcoma. To evaluate this hypothesis further, we designed a novel approach to correlate immunoexpression of Ki67, bcl‐2 and bax in the spindle cell and in the solid‐glandular component of biphasic synovial sarcomas. We also performed a double‐immunohistochemical assessment of the Ki67 proliferative indices and the immunoexpression of anti‐apoptotic protein bcl‐2 in neoplastic cells expressing either vimentin or cytokeratin.

Methods and results: Immunohistochemistry for vimentin (10 cases), bcl‐2 (10 cases), Ki67(10 cases), cytokeratin (10 cases), and bax (eight cases), and double‐immunostaining for vimentin/Ki67 (10 cases), vimentin/bcl‐2 (nine cases), cytokeratin/Ki67 (10 cases), and cytokeratin/bcl‐2 (10 cases) assays were performed in 10 cases of primary biphasic synovial sarcoma. Semiquantitative assessment was adopted for each case in both components. Statistical analysis was performed using Fisher's exact test or χ2 test. On conventional immunohistochemistry, the solid/glandular component revealed more expression of Ki67, bax and cytokeratin than the spindle cell component (P=0.0004, P=0.082, and P < 0.0001, respectively); on the other hand, the latter showed higher expression of bcl‐2 and vimentin than the former (P=0.0281 and P=0.059, respectively). Double immunohistochemistry analysis revealed higher co‐expression levels of cytokeratin/Ki67 and cytokeratin/bcl‐2 than the spindle cell component (P=0.015 and P < 0.0001, respectively); conversely, the latter presented higher co‐expression of vimentin/bcl‐2 than the former (P=0.0007). All cases showed no more than 10% of cells coexpressing cytokeratin/bcl‐2, cytokeratin/Ki67, and no case revealed cells coexpressing vimentin/Ki67.

Conclusion: Our findings indicate that in biphasic synovial sarcoma the acquisition of epithelial phenotype (solid/glandular component) is associated with a high expression of pro‐apoptotic proteins and a high proliferative differentiation status, and conversely, mesenchymal phenotype (spindle cell component) is associated with a high expression of apoptosis‐inhibitor bcl‐2 and a low proliferative terminal‐type differentiation status.
p53 expression related to the aggressive infiltrative histopathological feature of basal cell carcinoma
Authors: Auepemkiate S , Boonyaphiphat P , Thongsuksai P
Published: March 2003

p53 expression related to the aggressive infiltrative histopathological feature of basal cell carcinoma

Aims: To determine whether the p53 protein expression which is involved in the genesis and progression of various malignant tumours may relate to age, sites or the aggressive histopathological feature of the basal cell carcinoma.

Methods and results: One hundred and fifty‐eight basal cell carcinoma specimens from Songklanakarind Hospital, southern Thailand, collected from January 1992 to December 2000, were examined by immunohistochemistry using polyclonal anti‐p53‐CM1 (Novocastra Laboratories, Newcastle, UK; dilution 1:700). p53 protein expression was demonstrated in 48.7% of cases. The multivariate analysis showed that the aggressive infiltrative histopathological type was significantly associated with p53 expression (odds ratio 2.95, 95% confidence interval 1.10–7.90), whereas age, sun‐exposure site, cellular response and fibrosis were not.

Conclusions: The p53 expression is found to be related to the aggressive histopathological feature, which may be of predictive value for the behaviour of basal cell carcinoma. However, this result does not support the relation between sun exposure inducing basal cell carcinoma and p53 protein expression.
Ulcerative terminal ileitis after BCG therapy for bladder carcinoma
Authors: Satgé D , Pommepuy I , Hassan T , Goburdhun J , Flejou J‐F
Published: March 2003

Aim:  We evaluated the aetiology of ulcerative ileitis in a 77‐year‐old patient receiving BCG therapy for recurring bladder carcinoma.

Methods and results:  Histological examination, laboratory data and the clinical history allowed us to exclude a classical cause such as Crohn's disease, tuberculosis or ischaemia. The important locoregional reaction suggests a BCG ileitis of the terminal ileum. Intestinal BCG ileitis has been exceptionally described after BCG vaccination.

Conclusion:  Our observation of ulcerative ileitis indicates that a digestive tract location is possible as a reaction to BCG therapy.
Malignant mesothelioma: PAS–diastase positivity and inversion of polarity in intravascular tumour
Authors: Adams S A , Sherwood A J , Smith M E F
Published: March 2003

Aims:  Periodic acid–Schiff (PAS)–diastase‐positive material was identified within pseudoglandular structures within the small intravascular component of two pleural malignant mesotheliomas. The aim of this study was to ascertain the nature of this material and to asses the polarity of the cells forming the pseudoglandular structure.

Methods and results:  Immunohistochemistry was performed using antibodies to laminin and type IV collagen and the antibody HBME‐1. These demonstrated the material to be basement membrane rather than mucin. The apical polarity marker HBME‐1 was not related to the internal pseudoglandular structure but stained the periphery of intravascular tumour clumps.

Conclusions:  Pseudoluminal PAS–diastase‐positive material in malignant mesothelioma may easily be mistaken for epithelial mucin, leading to an erroneous diagnosis of adenocarcinoma. The presence of basement membrane material in pseudolumina, as defined by the presence of laminin and type IV collagen, surrounded by tumour cells whose external surface expresses the apical polarity marker HBME‐1 implies inversion of polarity of tumour cells within vascular spaces.
Hashimoto's thyroiditis shares features with early papillary thyroid carcinoma
Authors: Arif S , Blanes A , Diaz‐Cano S J
Published: March 2003

Neoplastic transformation is a multistep process that results in a continuous spectrum from the normal (physiological) state to a fully established neoplasm. The gold standard for diagnosis of papillary thyroid carcinoma is conventional histology, the essential element being the characteristic nuclear features, regardless of whether papillary structures are present or not. However, other criteria are being used increasingly in the diagnosis of neoplasms, including immunohistochemical staining and molecular profile. The RET/PTC gene rearrangement is highly specific for papillary thyroid carcinoma and is associated with the characteristic nuclear features seen in papillary thyroid carcinoma. There is an overlap in the morphological features, immunohistochemical staining pattern, and most importantly, molecular profile between papillary thyroid carcinoma and Hashimoto's thyroiditis. Although considered a ‘benign’ condition, Hashimoto's thyroiditis almost always harbours a genetic rearrangement that is strongly associated with and is highly specific for papillary thyroid carcinoma. Submicroscopic foci of papillary thyroid carcinoma must be present in Hashimoto's thyroiditis, although the clinical behaviour is still benign. Further studies are required to predict which foci will progress to papillary thyroid carcinoma.
Small epithelial cells in human liver cirrhosis exhibit features of hepatic stem‐like cells: immunohistochemical, electron microscopic and immunoelectron microscopic findings
Authors: Xiao J‐C , Ruck P , Adam A , Wang T‐X , Kaiserling E
Published: March 2003

Aims:  To investigate whether cells with features similar to those of the oval cells of rodents and the small epithelial cells (SEC) recently described in certain human liver diseases, i.e. hepatic progenitor cells, also occur in human liver cirrhosis.

Methods and results:  Surgical specimens from 35 cases of hepatitis B virus‐positive cirrhosis (30 cases containing hepatocellular carcinoma) were investigated by immunohistochemical staining for cytokeratin 7 and albumin. Electron microscopic investigations, and immunoelectron microscopic investigations using the same antibodies and a double‐labelling technique were performed in 15 and seven cases, respectively. SEC were observed in proliferated bile ductules, at the margins of regenerating nodules and in the fibrous septa in all cases of cirrhosis. The SEC were morphologically similar to the SEC described previously, and to the oval cells seen in experimental hepatocarcinogenesis. They were characterized by their small size, oval shape, scanty electron‐dense or electron‐lucent cytoplasm, a high nucleo–cytoplasmic ratio, tonofilaments and intercellular junctions. Immunoelectron microscopy revealed that the SEC co‐expressed cytokeratin 7 and albumin. Both relatively undifferentiated SEC and SEC with morphological and immunophenotypical signs of differentiation towards biliary epithelial cells and hepatocytes were found.

Conclusions:  SEC that exhibit morphological and immunophenotypical features of the SEC seen in certain other liver diseases are found in cirrhosis. These findings further support the hypothesis that a bipotent hepatic stem cell that may give rise to biliary epithelial cells and hepatocytes exists in the human liver.
The orf virus OV20.0L gene product is involved in interferon resistance and inhibits an interferon‐inducible, double‐stranded RNA‐dependent kinase
Authors: HAIG  , McINNES  , THOMSON  , WOOD  , BUNYAN  , MERCER
Published: March 2003

The parapoxvirus orf virus was resistant to type 1 (IFN‐α) and type 2 (IFN‐γ) interferons in cultures of ovine cells. The recently identified orf virus OV20.0L gene exhibits 31% predicted amino acid identity to the vaccinia virus E3L interferon‐resistance gene, and is referred to as the (putative) orf virus interferon‐resistance gene (OVIFNR). The objective of this study was to determine whether OVIFNR was involved in interferon resistance. Recombinant OVIFNR as a thioredoxin fusion protein (OVIFNR–Tx) inhibited the activation (by autophosphorylation) of an interferon‐inducible, double‐stranded (ds) RNA‐dependent kinase (PKR) of sheep, which was shown to bind dsRNA (poly I:C). PKR in other species is involved in the inhibition of protein synthesis as part of the antiviral state in infected cells. Virus‐infected cell lysates, but not control lysates, from cells grown in the presence of cytosine arabinoside also contained PKR inhibitory activity, which indicated that the inhibitory activity was associated with early viral gene expression. Significantly, the OVIFNR gene expressed in interferon‐treated ovine fibroblasts protected the unrelated Semliki Forest virus from the antiviral effect of both type 1 and type 2 interferons. Taken together, the results indicate that the OVIFNR gene functions as an interferon‐resistance gene, the product of which inhibits PKR in a similar way to the vaccinia virus E3L gene product.
Non‐responsiveness of antigen‐experienced CD4 T cells reflects more stringent co‐stimulatory requirements
Authors: HAMEL  , NOTEBOOM  , KRUISBEEK
Published: March 2003

We recently reported that previously activated T cells, irrespective of the nature of the first stimulus they encountered, are unable to respond to Staphylococcal enterotoxin B (SEB), nor to soluble anti‐CD3 monoclonal antibody (mAb) presented by splenic antigen‐presenting cells (APC). Such previously activated T cells are, however, fully capable of responding to plate‐bound anti‐CD3 plus splenic APC. These data suggest differential integration of the T‐cell receptor (TCR) and co‐stimulatory signalling pathways in naive versus antigen‐experienced T cells. Consistent with this hypothesis, anti‐CD28 mAb restores the proliferative capacity of resting ex vivo CD45RBlo CD4+ T cells (representing previously activated T cells) to both soluble anti‐CD3 mAb and SEB. Interestingly, mAb‐mediated engagement of cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) completely negates the rescue effects mediated by anti‐CD28 mAb in CD45RBlo cells. Nevertheless, the non‐responsiveness of CD45RBlo CD4+ T cells cannot be reversed by anti‐CTLA‐4 Fab fragments, indicating that it is not related to negative regulatory effects of CTLA‐4 engagement itself. Interestingly, the addition of interleukin‐2 (IL‐2) restores the proliferative capacity of CD45RBlo CD4+ T cells to SEB and soluble anti‐CD3 mAb. Moreover, when rescued by IL‐2, the cells are less susceptible to the negative regulatory effects of CTLA‐4 engagement. Together, these findings suggest that the non‐responsiveness of CD45RBlo CD4+ T cells to certain stimuli may be related to inadequate TCR signalling, primarily affecting IL‐2 production.
The transmembrane region of CD2‐associated signal‐transducing proteins is crucial for the outcome of CD2‐mediated T‐cell activation
Authors: VON BONIN  , EHRLICH  , FLEISCHER
Published: March 2003

Signalling through the CD2 molecule was shown previously to employ similar signalling molecules as the T‐cell receptor (TCR). Here, we show that CD2‐mediated signalling is strongly influenced by the expressed transmembrane region of the employed signal‐transducing molecule. We used TCR‐negative cells expressing chimeric fusion proteins that consist of human interleukin‐2 (IL‐2) receptor α‐chain‐derived sequences (hCD25) fused to mouse‐specific ζ‐chain segments (hCD25‐ζ). One set of TCR‐negative cell lines expressed the hCD25‐derived extracellular part fused to mouse‐specific transmembrane and cytoplasmic ζ‐protein sequences (‘TZZ’). The second type of cell lines expressed the hCD25‐derived extracellular and transmembrane portions fused to the mouse‐specific ζ‐chain cytoplasmic segment (‘TTZ’). After cross‐linking the hCD25‐ζ molecules with specific monoclonal antibodies (mAb), all TCR‐negative cell lines produced similar amounts of IL‐2. Cross‐linking with stimulating pairs of CD2‐specific mAb, however, led to IL‐2 production only in cell lines expressing the ζ‐chain‐specific transmembrane segment. Co‐cross‐linking of CD25 and CD2 molecules resulted in an effective stimulation of both TZZ‐ and TTZ‐expressing cell lines. Moreover, TTZ‐ and TZZ‐expressing cell lines differed in their pattern of tyrosine‐phosphorylated proteins after stimulation with hCD25‐specific mAb. Thus, although CD2 and TCR molecules share signalling components and pathways, the fine tuning of CD2 co‐receptor function appears to be regulated in part by transmembrane regions of signal‐transducing molecules like the TCR‐associated ζ‐chain.
Wild isolates of murine cytomegalovirus induce myocarditis and antibodies that cross‐react with virus and cardiac myosin
Authors: Fairweather D. , Lawson C. M. , Chapman A. J. , Brown C. M. S. , Booth T. W. M. , Papadimitriou J. M. , Shellam G. R.
Published: March 2003

The laboratory‐adapted K181 strain of murine cytomegalovirus (MCMV) induces both acute and chronic myocarditis, associated with autoantibodies to cardiac myosin, in susceptible BALB/c mice. However, the K181 MCMV strain has been maintained in the laboratory for many years and may not resemble naturally occurring strains of MCMV in its ability to induce myocarditis. Accordingly, six different isolates of MCMV from wild Mus domesticus were compared with K181 MCMV for their ability to induce myocarditis and autoantibodies to cardiac myosin in BALB/c mice. These isolates were shown to induce acute myocarditis similar to K181 MCMV, with associated focal and diffuse myocardial inflammation. However, the levels of myocarditis induced by the wild isolates during the chronic phase of the disease (days 32–56 post‐infection) were low in contrast to the K181 strain. Interestingly, 30% of wild‐trapped mice showed histological evidence of myocarditis and all were sero‐positive to MCMV. Sera from BALB/c mice infected with wild MCMV isolates and from wild‐trapped mice contained antibodies that cross‐reacted with MCMV and cardiac myosin (S2 region). The cross‐reactive region of MCMV was found to be a 50 000–55 000 MW viral polypeptide. These findings suggest that molecular mimicry may be involved in the pathogenesis of autoimmune myocarditis following infection with both laboratory and wild MCMV strains.

EDTA, ethylenediaminetetraacetic acid ELISA, enzyme‐linked immunosorbent assay H&E, haematoxylin and eosin HMM, heavy meromyosin i.p., intraperitoneal LMM, light meromyosin  mAb, monoclonal antibodies MCMV, murine cytomegalovirus MEF, mouse embryo fibroblasts MOBS, mouse osmolarity buffered saline MW, molecular weight OD, optical density PBS, phosphate buffered saline PFU, plaque‐forming units p.i., post‐infection PMSF, phenylmethylsulphonyl fluoride PVDF, polyvinylidene difluoride SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis.
Modulation of antigen‐specific T‐cell activation in vitro by taurine chloramine
Authors: MARCINKIEWICZ J. , GRABOWSKA A. , CHAIN B. M.
Published: March 2003

Taurine chloramine (TauCl) is produced during inflammation by reaction of hypochlorous acid (HOCl) with taurine, the most abundant free amino acid in neutrophils. We previously reported that TauCl inhibits the generation of macrophage inflammatory mediators such as nitric oxide, prostaglandin E2 (PGE2),tumour necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6). In this study, the activity of TauCl in modulating T‐cell activation was investigated. Treatment of T cells with TauCl (0·1–0·3 mm), prior to activation, was found to inhibit interleukin‐2 (IL‐2) release in response to both mitogen and antigen stimulation. Similarly, pretreatment of A‐20 antigen presenting cells (APCs), at low cell numbers, was found to inhibit their ability to process and present ovalbumin (OVA) to a specific T‐cell hybridoma. In contrast, pretreatment of higher numbers of A‐20 cells with TauCl in the presence of OVA enhanced subsequent presentation of OVA. Finally, OVA modified with TauCl was processed and presented more efficiently than native VA. Thus, TauCl is able to modulate induction of a specific adaptive immune response at several independent points of the overall antigen‐presenting pathway.
Different roles are played by αβ and γδ T cells in acquired immunity to Chlamydia trachomatis pulmonary infection
Authors: YANG  , HAYGLASS  , BRUNHAM
Published: March 2003

Using gene knockout and wild‐type C57BL/6 mice, we examined the role of αβ and γδ T cells in the resolution of Chlamydia trachomatis mouse pneumonitis (MoPn) biovar pulmonary infection. The results show that αβ T‐cell‐deficient (α−/−) mice, when compared with wild‐type control mice, have dramatically increased mortality rate and greater in vivo growth of MoPn. The αβ T‐cell‐deficient mice were as susceptible to MoPn infection as T‐ and B‐lymphocyte‐deficient (RAG‐1−/−) mice. Moreover, both αβ T‐cell‐deficient and RAG‐1 mutant mice failed to mount delayed‐type hypersensitivity (DTH) responses to organism‐specific challenge and showed undetectable interferon‐γ (IFN‐γ) production by spleen cells upon in vitro organism‐specific restimulation. In contrast, γδ T‐cell‐deficient mice exhibited intact DTH responses and their mortality rate and in vivo chlamydial growth were comparable to those in wild‐type controls. More interestingly, γδ T‐cell‐deficient mice showed significantly higher levels of IFN‐γ production than did wild‐type mice. The data indicate that the αβ T cell is the major T‐cell population for acquired immunity to chlamydial infection and that γδ T cells may play an ancillary role in regulating the magnitude of αβ T‐cell responses.
Paradoxical early immune activation during acceptance of liver allografts compared with rejection of skin grafts in a rat model of transplantation
Authors: ROKAHR  , SHARLAND  , SUN  , WANG  , SHEIL  , YAN  , MCCAUGHAN  , BISHOP
Published: March 2003

Liver allografts in many animal models are often spontaneously accepted across a complete histocompatibility barrier without requirement for immunosuppression. In contrast, skin allografts are usually rejected, even across minor histocompatibility barriers. To identify the mechanism of liver allograft acceptance we have compared skin rejection with liver acceptance in DA rat strain recipients of PVG donors, a major histocompatibility complex (MHC) class I plus II mismatch. In spite of the established role of draining lymph nodes (LN) in induction of rejection of skin allografts, there was much greater involvement of LN after liver than after skin transplantation. Few donor cells migrated to these organs from transplanted skin but many cells migrated from transplanted liver. There was also a paradoxical increase in interleukin‐2 (IL‐2) and interferon‐γ (IFN‐γ) mRNA in LN and spleen of liver allograft recipients that greatly exceeded their expression in skin allograft recipients. For example, there were 2·7±1·6×104 molecules of IFN‐γ per 106 molecules of β‐actin mRNA in the LN draining liver allografts 1 day after transplantation compared with 2·0±0·3×103 molecules/106β‐actin in LN draining skin allografts and 8·1±1·8×102 molecules/106β‐actin in LN draining skin isografts. Examination of the graft showed that infiltration and cytokine mRNA up‐regulation occurred more slowly in the transplanted skin than in liver but progressed inexorably in skin grafts until rejection. These results show that liver acceptance is associated with a paradoxical marked early activation then subsequent decline of the immune response.
Genetics of graft‐versus‐host disease, I. A locus on Chromosome 1 influences development of acute graft‐versus‐host disease in a major histocompatibility complex mismatched murine model
Authors: ALLEN  , DOBKINS  , HARPER  , SLAYBACK
Published: March 2003

Graft‐versus‐host disease (GVHD) is the major complication occurring after bone marrow transplantation. The severity of GVHD varies widely, with this variation generally being attributed to variation in the degree of disparity between host and donor for minor histocompatibility antigens. However, it is also possible that other forms of polymorphism, such as polymorphisms in immune effector molecules, might play a significant role in determining GVHD severity. In order to investigate this hypothesis, we are studying the genetic factors that influence GVHD development in a murine model. We here report the first results of this analysis, which demonstrate that a locus on Chromosome 1 of the mouse, and possibly also a locus on Chromosome 4, exert considerable influence over the development of one aspect of acute GVHD – splenomegaly – in a parent→F1 murine model. These results demonstrate that non‐MHC genes can exert quite significant effects on the development of GVHD‐associated pathology and that gene mapping can be used as a tool to identify these loci. Further analysis of such loci will allow identification of the mechanism whereby they influence GVHD and may lead in the future to improved selection of donors for human bone marrow transplantation.
A phage‐displayed mimotope inhibits tumour necrosis factor‐α‐induced cytotoxicity more effectively than the free mimotope
Authors: CHIRINOS‐ROJAS  , STEWARD  , PARTIDOS
Published: March 2003

A phage‐displayed peptide library was screened by direct interaction with human tumour necrosis factor‐α (TNF‐α) to identify novel antagonistic molecules of its biological activities. After several rounds of affinity selection, a phage displaying a mimotope sequence was shown to strongly inhibit, in a dose‐dependent fashion, both mouse and human TNF‐α‐mediated cytotoxicity in L929 cells. The identified mimotope did not bear any sequence homology to the primary structures of the extracellular domains of either the 55 000 MW or the 75 000 MW TNF‐α receptors, suggesting that it represents or mimics a conformational epitope involved with binding to TNF‐α. The free 15‐mer mimotope weakly inhibited TNF‐α‐induced cytotoxicity in vitro, and it did not bind to TNF‐α as assessed by surface plasmon resonance, demonstrating the importance of mimotope presentation for its biological activities. In conclusion, this study highlights the potential of random combinatorial peptide libraries for the identification of novel inhibitors, which may serve as important tools in research that could lead to the development of TNF‐α antagonists with therapeutic potential.
Characterization of immune responses following intramuscular DNA immunization with the MOMP gene of Chlamydia trachomatis mouse pneumonitis strain
Authors: ZHANG  , YANG  , SHEN  , BRUNHAM
Published: March 2003

Studies were carried out to characterize the cellular and humoral immune responses evoked by intramuscular DNA vaccination with the major outer membrane protein (MOMP) gene of Chlamydia trachomatis mouse pneumonitis strain. The data demonstrate that DNA vaccinated mice develop antigen‐specific delayed‐type hypersensitivity, lymphocyte proliferation and interferon‐γ (IFN‐γ) production. Serum antibody responses (mainly immunoglobulin G2a; IgG2a) were evoked in two‐thirds of the mice. We conclude that intramuscular DNA immunization with the MOMP gene evokes cellular and humoral immune responses suggestive of a T helper 1 (Th1) bias.
Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti‐tumoral cell therapy
Authors: MASURIER  , PIOCHE‐DURIEU  , COLOMBO  , LACAVE  , LEMOINE  , KLATZMANN  , GUIGON
Published: March 2003

Dendritic cells (DC) are professional antigen‐presenting cells that can be used as immune adjuvant for anti‐tumoural therapies. This approach requires the generation of large quantities of DC that are fully characterized on the immunophenotypical and functional levels. In a murine model, we analysed the in vitro effects of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) alone or combined with interleukin‐4 (IL‐4) or Flt3 ligand (Flt3‐L) on the number, immunophenotype and functions of bone marrow‐derived DC. In GM‐CSF cultures, we have identified two populations based on their level of expression of major histocompatibility complex (MHC) class II molecules: MHC‐IIhi cells, exhibiting the typical morphology and immunophenotype of myeloid DC (CD11c+ 33D1+ DEC‐205+ F4/80+), and MHC‐IIlo cells, heterogeneous for DC markers (30% CD11c+; 50% 33D1+; DEC‐205−; F4/80+). The addition of Flt3‐L to GM‐CSF induced a twofold increase in MHC‐IIhi DC number; besides, the MHC‐IIlo cells lost all DC markers. In contrast, after addition of IL‐4 to GM‐CSF, the two populations displayed a very similar phenotype (CD11c+ 33D1− DEC‐205+ F4/80−), differing only in their expression levels of MHC class II and costimulatory molecules, and showed similar stimulatory activity in mixed leucocyte reaction. We next analysed the migration of these cultured cells after fluorescent labelling. Twenty‐four hours after injection into the footpads of mice, fluorescent cells were detected in the draining popliteal lymph nodes, with an enhanced migration when cells were cultured with GM‐CSF+Flt3‐L. Finally, we showed that MHC‐IIhi were more efficient than MHC‐IIlo cells in an anti‐tumoral vaccination protocol. Altogether, our data highlight the importance of characterizing in vitro‐generated DC before use in immunotherapy.
Evaluation of C3a receptor expression on human leucocytes by the use of novel monoclonal antibodies
Authors: ZWIRNER  , GÖTZE  , BEGEMANN  , KAPP  , KIRCHHOFF  , WERFEL
Published: March 2003

Varying results have been published in the past regarding the reactivity of different leucocyte subpopulations, including neutrophils, monocytes and B lymphocytes, to the anaphylatoxin C3a and its degradation product C3a(desArg). To better characterize the cellular distribution of C3a receptor (C3aR) expression, monoclonal antibodies against two different epitopes on the third extracellular domain of the human C3aR were generated. Quantification of C3aR as compared with C5aR densities was performed on peripheral blood leucocytes by quantitative indirect immunofluorescence. Eosinophils and basophils expressed similar numbers of C3aR and C5aR molecules/cell. On eosinophils 10 700±4500 (mean±SD) C3aR and 14 700±4100 C5aR were found, whereas basophils carried 8100±2100 C3aR and 13 500±3800 C5aR. Monocytes expressed approximately six times more C5aR than C3aR molecules on their surface (6000±2500 C3aR versus 34 100±9300 C5aR molecules) whereas on neutrophils, the expression of C5aR was more than 20 times higher than the expression of C3aR (3100±1000 C3aR versus 63 500±12 200 C5aR). No C3aR expression was detectable on peripheral blood‐derived B lymphocytes and on tonsillar B cells before and after stimulation with interleukin‐2/Staphylococcus aureus Cowan strain I. Our findings correspond well with the paucity of data on C3a‐induced functional activities in monocytes and neutrophils and suggest that eosinophilic and basophilic granulocytes represent the primary effector cells in the peripheral blood which can be stimulated by C3a.
Both kinetic data and epitope mapping provide clues for understanding the anti‐coagulant effect of five murine monoclonal antibodies to human β2‐glycoprotein I
Authors: Regnault  , Arvieux  , Vallar  , Lecompte
Published: March 2003

The interaction between five murine monoclonal antibodies (mAb) and β2‐glycoprotein I (β2GPI) in the absence of phospholipids was studied using surface plasmon resonance‐based biosensor technology. Two separate epitope regions were confirmed for the five mAb but epitopes of two mAb were shown to be overlapping but not identical. The characteristics of binding on both immobilized β2GPI, using different chemistries of coupling to a dextran matrix and antibody surfaces prepared by two strategies of immobilization, were compared. Binding was strongly influenced by the orientation of the immobilized partner, and the five mAb showed heterogeneity in their binding to immobilized and soluble β2GPI. The observed stoichiometries of mAb‐β2GPI complexes and the detailed analysis of the kinetics of the association and dissociation phases of the interactions with soluble and immobilized β2GPI revealed differences in the dissociation rate constants, resulting in a 10‐fold higher affinity for immobilized β2GPI compared to soluble β2GPI for four out of five mAb. This suggests bivalent binding of these mAb to immobilized β2GPI. In addition, the kinetic data helped explain the differing anti‐coagulant properties of these mAb.
Interleukin‐12 can directly induce T‐helper 1 responses in interferon‐γ (IFN‐γ) receptor‐deficient mice, but requires IFN‐γ signalling to downregulate T‐helper 2 responses
Authors: Mountford  , Coulson  , Cheever  , Sher A. , Wilson R. A. , Wynn
Published: March 2003

An in vivo model of pulmonary granuloma formation around embolized schistosome eggs was investigated as an environment in which to analyse a role for interleukin‐12 (IL‐12) in the differentiation of T‐helper 1 (Th1) and Th2 subsets. Specifically, mice deficient for the interferon‐γ receptor (IFN‐γR−/–) were used to determine the role for IL‐12 in the absence of IFN‐γ‐mediated signalling. We show that recombinant IL‐12 administered to IFN‐γR−/– mice caused the up‐regulation of mRNA for IFN‐γ in lung tissue, and the secretion of abundant IFN‐γ by in vitro‐cultured lymph node cells in response to egg antigens. This indicates that IL‐12 can act independently of IFN‐γ to induce the development of Th1 cells. Administration of rIL‐12 to wild‐type mice markedly reduced the secretion of Th2‐associated cytokines, IL‐4 and IL‐5. However, these cytokines were not dramatically reduced in IFN‐γR−/– mice treated with IL‐12. We conclude that inhibition of these cytokines by IL‐12 is primarily dependent upon effective IFN‐γ signalling, although abrogation of T‐cell derived IL‐10 appeared to be dependent upon IL‐12. We also show that increases in mRNA for the β2 subunit of the IL‐12 receptor and the p40 subunit of IL‐12 after rIL‐12 treatment were lower in IFN‐γR−/– mice, compared to wild‐type mice, indicating that their expression was primarily dependent upon IFN‐γ with only a minor role for IL‐12.
Different effect of granulocyte colony‐stimulating factor or bacterial infection on bone‐marrow cells of cyclophosphamide‐treated or irradiated mice
Authors: Buisman  , Van Zwet TH. L. , Langermans  , Geertsma M. F. , Leenen  , Van Furth
Published: March 2003

In the present study, the effect of treatment with granulocyte colony‐stimulating factor (G‐CSF) on cellular composition of the bone marrow and the number of circulating leucocytes of granulocytopenic mice, whether or not infected with Staphylococcus aureus, was assessed. With two monoclonal antibodies, six morphologically distinct cell populations in the bone marrow could be characterised and quantitated by two‐dimensional flow cytometry. Granulocytopenia was induced by cyclophosphamide or sublethal irradiation. Cyclophosphamide predominantly affected the later stages of dividing cells in the bone marrow resulting in a decrease in number of granulocytic cells, monocytic cells, lymphoid cells and myeloid blasts. G‐CSF administration to cyclophosphamide‐treated mice increased the number of early blasts, myeloid blasts and granulocytic cells in the bone marrow, which indicates that this growth factor stimulates the proliferation of these cells in the bone marrow. During infection in cyclophosphamide‐treated mice the number of myeloid blasts increased. However, when an infection was induced in cyclophosphamide and G‐CSF‐treated mice, the proliferation of bone‐marrow cells was not changed compared to that in noninfected similarly treated mice. Sublethal irradiation affected all bone‐marrow cell populations, including the early blasts. G‐CSF‐treatment of irradiated mice increased only the number of myeloid blasts slightly, whereas an infection in irradiated mice, whether or not treated with G‐CSF, did not affect the number of bone‐marrow cells. Together, these studies demonstrated that irradiation affects the early blasts and myeloid blasts in the bone marrow more severely than treatment with cyclophosphamide. Irradiation probably depletes the bone marrow from G‐CSF‐responsive cells, while cyclophosphamide spared G‐CSF responsive cells, thus enabling the enhanced G‐CSF‐mediated recovery after cyclophosphamide treatment. Only in these mice, bone marrow recovery is followed by a strong mobilisation of mature granulocytes and their band forms from the bone marrow into the circulation during a bacterial infection.
Functional expression of chemokine receptor CXCR4 on human epithelial cells
Authors: Murdoch  , Monk  , Finn
Published: March 2003

Chemokines and their receptors play an important role in the process of leucocyte recruitment at sites of inflammation. However, recent evidence suggests that these proteins can also regulate non‐leucocyte cell functions such as angiogenesis, migration and proliferation. We have investigated the expression of the CXC chemokine receptor 4 (CXCR4) on primary cultures of type II alveolar epithelial cells, their transformed counterpart, the A549 cell line and also on other epithelial cell lines from various tissues. We found that all epithelial cell types tested express mRNA for CXCR4. Flow cytometric analysis and immunocytochemical staining shows that CXCR4 chemokine receptor is abundantly expressed on the surface of A549 epithelial cells. Furthermore, A549 cells responded to the CXCR4 ligand, stromal‐derived factor‐1α (SDF‐1α) with a rapid and robust calcium mobilization and not to other CXC chemokines, suggesting that CXCR4 is functionally active and is able to couple to G‐protein signalling mechanisms. A549 cells did not proliferate in response to either SDF‐1α or interleukin‐8 (IL‐8) CXC chemokines. These findings may have important implications for epithelial physiology and pathology.
Sulfasalazine prevents T‐helper 1 immune response by suppressing interleukin‐12 production in macrophages
Authors: Kang  , Chung  , Im  , Choe  , Kim
Published: March 2003

Interleukin‐12 (IL‐12) plays a pivotal role in the development of T‐helper 1 (Th1) immune response, which may be involved in the pathogenesis of chronic inflammatory autoimmune disorders. In this study we investigated the effects of sulfasalazine, a drug for treating inflammatory bowel disease and rheumatoid arthritis, on the production of IL‐12 from mouse macrophages stimulated with lipopolysaccharide (LPS). Sulfasalazine potently inhibited the production of IL‐12 in a dose‐dependent manner, in part through the down‐regulation of nuclear factor κB (NFκB) activation in IL‐12 p40 gene. Activation of macrophages by LPS resulted in markedly enhanced binding activities to the κB site, which significantly decreased upon addition of sulfasalazine as demonstrated by an electrophoretic gel shift assay. Importantly, macrophages pretreated with sulfasalazine either in vitro or in vivo reduced their ability to induce interferon‐γ (IFN‐γ) and increased the ability to induce IL‐4 in antigen‐primed CD4+ T cells. From these results, sulfasalazine may induce the Th2 cytokine profile in CD4+ T cells by suppressing IL‐12 production in macrophages, and sulfasalazine‐induced inhibition of IL‐12 production in macrophages may explain some of the known biological effects of sulfasalazine.
Lipopolysaccharide‐dependent down‐regulation of CD27 expression on T cells activated with superantigen
Authors: Kai  , Rikiishi  , Sugawara  , Takahashi  , Takada  , Kumagai
Published: March 2003

To investigate the mechanisms underlying T‐cell responses during superantigen (SAg) stimulation, we analysed the effects of SAg on CD27 expression with or without lipopolysaccharide (LPS) as a novel regulator of T‐cell function. CD27 is expressed on the majority of resting peripheral blood T cells (CD27low). Activation of T cells by SAg induces high levels of CD27 surface expression (CD27high) accompanied with simultaneous CD30 receptor expression. After prolonged activation in vitro, the level of CD27 expression became intermediate. The effects of LPS on down‐regulation of CD27high expression on CD30+ T cells were dose‐dependent. Separating LPS‐stimulated monocytes from T cells by mechanical dispersion abolished its inhibitory effect, indicating the requirement for direct interactions between monocytes and T cells. We also found that SAg up‐regulated CD80 expression on CD14+ monocytes and LPS inhibited SAg‐induced CD80 expression after 24 hr of stimulation. Up‐regulation of CD152 (CTLA‐4) was selective, since it was found to be preferentially expressed on the CD30+ population. Competitive experiments using soluble blocking peptides showed that addition of CD28 or CD80 peptide recovered LPS‐induced down‐regulation of CD27high expression on CD30+ T cells. These observations suggested that the presence of low levels of CD80 on monocytes may partially inhibit CD27 expression due to inefficient delivery of positive signals via CD28/CD80 interaction, and that the increased levels of CD80 enhance the inhibition through interactions with CD152 which is expressed at the highest levels after 48 hr of activation.
Some Bence–Jones proteins enter cultured renal tubular cells, reach nuclei and induce cell death
Authors: Matsuura K. , Ikoma S. , Watanabe M. , Togawa A. , Sinohara H.
Published: March 2003

Eighteen monoclonal Bence–Jones proteins (BJPs) were examined for their effects on cultured LLC‐PK1 (porcine kidney proximal tubule) cells as well as for their amidase and DNase activities. Five proteins were found to enter the cell and to gain access to the nucleus without degradation of epitopes. Intranuclear BJPs ultimately induced DNA fragmentation and cell death. BJPs with relatively high amidase activity were cytotoxic. On the other hand, three of four BJPs with DNase activity had a cytocidal effect on cultured cells; the remaining BJP, which had a relatively high DNase activity but a very low amidase activity, failed to enter the cell and was not cytotoxic in vitro. These results suggest that catalytic and cytotoxic activities of some BJPs may make a significant contribution, in a substantial proportion of myeloma patients, to the development and/or deterioration of the disease.
Human antigen‐presenting cell/tumour cell hybrids stimulate strong allogeneic responses and present tumour‐associated antigens to cytotoxic T cells in vitro
Authors: Dunnion D. J. , Cywinski A. L. , Tucker V. C. , Murray A. K. , Rickinson A. B. , Coulie P. , Browning M. J.
Published: March 2003

Most tumours do not stimulate effective antitumour immune responses in vivo. In order to enhance the immunogenicity of human tumour cells, we fused a variety of tumour cell lines with an Epstein–Barr virus transformed B‐lymphoblastoid cell line (EBV B‐LCL) in vitro, to produce stable hybrid cells. Hybrid cell lines showed a marked increase in their ability to stimulate primary allogeneic T‐cell responses in vitro, as compared with the parent tumour cells. The hybrid cells induced proliferation of naive (CD45RA+) as well as memory (CD45RO+) T lymphocytes, and both CD4+ and CD8+ subpopulations of T cells were directly stimulated. The stimulatory hybrids expressed human leucocyte antigen (HLA) class I and II, and a wide range of surface accessory molecules, including the T‐cell co‐stimulatory ligand molecules CD40, CD80 (B7.1) and CD86 (B7.2), the expression of which was required for optimal stimulation of T‐cell responses. Fusion of the EBVB‐LCL with a melanoma cell line (518.A2) yielded hybrid cells that expressed the melanoma‐associated antigens MAGE‐1 and MAGE‐3, and presented these antigens to antigen‐specific, HLA class I‐restricted cytotoxic T‐lymphocyte clones with greater efficiency than the parent melanoma cell line. These findings suggest that the generation of human antigen‐presenting cell/tumour cell hybrids offers promise as an approach to cancer immunotherapy.
Systematic characterization of porcine ileal Peyer's patch, I. Apoptosis‐sensitive immature B cells are the predominant cell type
Authors: Andersen J. K. , Takamatsu H. , Oura C. A. L. , Brookes S. M. , Pullen L. , Parkhouse R. E. M.
Published: March 2003

It is now apparent that the Peyer's patches of some species exhibit structural, functional and developmental heterogeneity. In sheep, for example, the ileal Peyer's patch (IPP) is the primary, antigen‐independent site for the generation of the primary immunoglobulin repertoire and consequent production of the systemic B‐cell pool. The pig has three distinct Peyer's patches, including an IPP, but the functional status of this organ, as primary or secondary lymphoid tissue, is not clear. Here, we have systematically characterized pig IPP follicular lymphocytes and show that about 90% B cells that are positive for surface immunoglobulin G (sIgM+) and express an immature phenotype characterized by expression of myeloid marker sWC3 (74‐22‐15) and two molecules recognized by IPP B‐cell‐specific monoclonal antibodies (F10/4, F12/35). Extensive apoptosisin vivoandin vitrowas demonstrated by electron microscopy, immunohistology with TdT‐mediated dUTP nick end labelling, DNA analysis and fluorescence‐activated cell sorter analysis. Thus, when isolated IPP follicular cells were incubated at 37°in vitro, the majority of them became apoptotic. The few that survived, however, had lost their expression of sWC3, F10/4, F12/35, but showed an increased expression of sIgM and major histocompatibility complex class II indicating that such surviving cells were of a more mature phenotype. Although more T cells were observed in porcine IPP follicles than in sheep IPP, CD3+cells comprised less than 5% of the IPP follicular lymphocytes. Thus, the results clearly indicate that pig IPP is equivalent to sheep IPP.
Systematic characterization of porcine ileal Peyer’s patch, II. A role for CD154 on T cells in the positive selection of immature porcine ileal Peyer’s patch B cells
Authors: Andersen J. K. , Takamatsu H. , Pullen L. , Parkhouse R. M. E.
Published: March 2003

We previously demonstrated that the majority (≥ 90%) of porcine ileal Peyer’s patch (IPP) follicular cells are immature B cells destined to die by apoptosis, when incubated at 37°. In this paper we approached the mechanisms responsible for positive selection of porcine IPP follicular immature B‐cell selection, by screening for various cell types, cytokines and polyclonal and monoclonal antibodies for promoting the survival of IPP B cells. Of these reagents, only CD3 cross‐linked purified T cells from mesenteric lymph nodes were able to rescue IPP follicular B cells from apoptosis, although polyclonal anti‐IPP lymphocyte antibodies delayed apoptosis. This survival effect could be reproduced simply by incubating IPP follicular B cells with soluble and cell membrane‐expressed CD154, an observation consistent with the demonstrated presence of CD40 and CD154 on porcine IPP follicular B cells and activated T cells, respectively. The IPP follicular B cells rescued in this manner expressed a more mature surface marker phenotype. Immunohistology and fluorescence‐activated cell sorter analysis demonstrated that subpopulations of IPP follicular T cells (less than 0·5%) express CD154. Thus, perhaps unexpectedly, CD154 on T cells may play a role in the positive selection of immature B cells in the porcine IPP. The origin and control of the activated T cells identified within the porcine IPP remains to be investigated.