bluuebunny/crossref_metadata_2025 huggingface dataset:
VS38 immunostaining in neuroendocrine tumours
Authors: BANERJEE S.S. , SHANKS J.H. , HASLETON P.S.
Published: November 2003

VS38 was originally described as a marker of neoplastic and non‐neoplastic plasma cells. We recently described its immunoreactivity with melanocytic lesions. The aim of the present study was to investigate the reactivity of VS38 with various neuroendocrine tumours. Using paraffin sections of routinely processed archival tissue we found that 72% of 50 neuroendocrine tumours tested showed immunohistochemical positivity for VS38. A wide spectrum of neuroendocrine tumours were positive. Use of a panel of markers and awareness of the lack of specificity of VS38 will avoid misdiagnosis, particularly when neuroendocrine tumours and other lesions which react immunohistochemically with this antibody are located at unusual sites and exhibit plasmacytoid features.
Immunohistochemical metallothionein expression in thymoma: correlation with histological types and cellular origin
Authors: KUO T. , LO S.K.
Published: November 2003

Metallothioneins (MTs) are low molecular weight and cysteine‐rich intracellular proteins involved in metal homeostasis and detoxication. They are found in certain normal tissues, and are overexpressed in various tumours with correlation to more aggressive behaviour in certain tumours. Since the histopathological types of thymoma have unpredictable invasive potential, MT over‐expression was investigated as a possible marker of the invasive potential of thymomas. We studied immunohistochemical MT expression in 27 non‐invasive thymomas, 20 micro‐invasive thymomas, and 23 macro‐invasive thymomas with a mouse monoclonal anti‐MT antibody E9 on formalin‐fixed paraffin‐embedded tissues. MT expression was significantly different among the three groups of thymomas (P = 0.02) with a stronger expression in invasive thymomas (P = 0.003). However, MT expression was not exclusively limited to invasive thymomas. Therefore, it could not be used as a marker of aggressive potential in individual thymomas. Analysis of MT expression according to the histological types of the thymomas revealed that eight of nine spindle cell thymomas, none of 10 small polygonal cell thymomas, four of 14 mixed thymomas, seven of 29 large polygonal cell thymomas, and seven of eight squamoid thymomas significantly expressed MT. There was a statistically significant difference in MT expression among different histological types of thymomas (P = 0.000). The strongest and most consistent expression was observed in spindle cell thymoma and squamoid thymoma. Since spindle cell thymoma was usually non‐invasive and squamoid thymoma was more aggressive, MT expression does not correlate with the invasive potential of different histological types of thymomas. But because medullary epithelial cells of the thymus were positive for MT, our results suggest that both spindle cell thymoma and squamoid thymoma might derive from the medullary compartment of the thymus.
G‐cell density in the antral mucosa: a feasibility study
Authors: PESCE C. , ROSSI R. , LENTI E. , TANZI R.
Published: November 2003

In this work a new method for gastrin‐secreting cell (G‐cell) density measurement was employed in order to test the hypothesis that G‐cell density of the antral mucosa can be estimated with an acceptable degree of error. The zone of antral mucosa where most G‐cells are located was demarcated, its area was measured and the G‐cells within this area were counted. The variation in the e error of the estimate according to normal distribution theory, and the size of the sampling expected to yield G‐cell density values with a given error were assessed. Our data indicate that a very large surface of the G‐cell zone should be explored in order to estimate G‐cell density with an acceptable degree of error. G‐cell density measurement appears not to be feasible on biopsy specimens or, for routine diagnosis, on gastrectomy specimens.
Histology of aberrant crypt foci in the human colon
Authors: Di Gregorio C. , Losi L. , Fante R. , Modica S. , Ghidoni M. , Pedroni M. , Tamassia M.G. , Gafà L. , PONZ de Leon M. , Roncucci L.
Published: November 2003

Aberrant crypt foci (ACF) have been identified in the methylene‐blue stained mucosa of the human colon. Some lines of evidence suggest that ACF may be precursors of colon cancer. The objective of the present study was to establish morphological criteria able to define and classify ACF in histological sections. Twenty‐four colectomy specimens were collected after operation for colorectal cancer and fixed in 10% formalin. Strips of grossly normal mucosa were stained in a 0.2% solution of methylene blue in saline for 5–10 min. The strips were measured, put on a glass slide and observed under a light microscope at ×25. One hundred and fourteen ACF identified by topology were sectioned parallel to the muscularis mucosae. Eighty‐four ACF were evident at histological examination and could be classified into three main groups: group A (61 ACF, 72.6%) including foci whose epithelial cells had regular nuclei, with only mild or focal crowding but no stratification, no mucin depletion and no dysplasia; group B (16 ACF, 19.1%), in which features of hyperplasia were evident; and group C (seven ACF, 8.3%) including foci with enlarged, crowded and stratified nuclei, mucin depletion, frequent mitoses, and evident dysplasia, diffuse or focal (mild in five cases, moderate in two) representing microadenomas. Finally, hyperplastic foci were significantly larger than foci of group A and C. Group B ACF were also more frequent in the rectum than in the colon. In conclusion, selected histological features allow the definition of groups of ACF, which may represent sequential steps in the development of human colorectal tumours.
Stem cell factor enhances immunoglobulin E‐dependent mediator release from cultured rat bone marrow‐derived mast cells: activation of previously unresponsive cells demonstrated by a novel ELISPOT assay
Authors: HILL P. B. , MACDONALD A. J. , THORNTON E. M. , NEWLANDS G. F. J. , GALLI S. J. , MILLER H. R. P.
Published: November 2003

Mucosal mast cells (MMC) are important effector cells in the immune response against gastrointestinal nematodes. We used cultured rat bone marrow‐derived mast cells (BMMC) as an in vitro model of MMC to study the effects of the multifunctional cytokine stem cell factor (SCF) on immunoglobulin E (IgE)‐dependent secretion of granule mediators. SCF (≤1000 ng/ml) was not a direct secretagogue for these cells, but it significantly enhanced IgE‐mediated secretion of the granule constituents rat mast cell protease‐II (RMCP‐II) and β‐hexosaminidase from mature BMMC in a dose‐dependent manner (>10 ng/ml). Maximum up‐regulation of secretion occurred after cells were pretreated with SCF (50 ng/ml) for 5 minutes before challenge with anti‐IgE, but the effect then declined and was absent in cells incubated with the cytokine for 3 to 24 h. In a novel ELISPOT assay developed to identify individual BMMC secreting RMCP‐II, the proportion of mature BMMC responding to anti‐IgE was significantly increased by treatment with SCF. To investigate this effect further, the percentage release of RMCP‐II and β‐hexosaminidase from populations of mature BMMC was directly compared to the proportion of individual cells releasing RMCP‐II as detected by ELISPOT. The release of both mediators was enhanced by SCF, and the increased percentage release reflected both an increased proportion of secreting cells, and enhanced mediator release from individual cells. These results suggest that SCF can enhance IgE‐dependent mediator release from BMMC not only by augmenting the secretory response from individual cells, but also by activating previously unresponsive cells.
Role of polymorphic residues of human leucocyte antigen‐DR molecules on the binding of human immunodeficiency virus peptides
Authors: JURCEVIC S. , PRAUD C. , COPPIN H. L. , BERTRAND A. , RICARD S. , THOMSEN M. , LAKHDAR‐GHAZAL F. , DE PREVAL C.
Published: November 2003

A study was made of the binding properties of 96 human immunodeficiency virus peptides to human leucocyte antigen (HLA)‐DR1 and HLA‐DR103 molecules, which differ by three amino acids at positions 67, 70 and 71 in the β chains. The affinity of the peptides was characterized by their inhibitory concentrations in competitive binding assays which displace half of the labelled influenza haemagglutinin peptide HA306‐318 (IC50). Among the high‐affinity peptides (IC50 ≤ 1 μm), seven bound to DR1, three to DR103 and five equally well to both alleles (promiscuous peptides). Thirty‐two other peptides showed medium or low affinity for DR molecules. The role of polymorphic residues was analysed using six mutated DR molecules, intermediates between DR1 and DR103 and differing by one or two substitutions at positions 67, 70 or 71. We reached the same conclusions when using DR1‐specific or DR103‐specific peptides: modification of residue 70 had no effect on peptide affinity, but single substitution at positions 67 or 71 decreased the allele specificity of the peptides while double substitution at 67 and 71 completely reversed the peptide specificity. In functional assays, DR‐binding peptides are able to outcompete specific T‐cell proliferation. Furthermore, modification at position 67 or 70 significantly affects the T‐cell response and mutation at position 71 abolishes completely the T‐cell proliferation. Thus, the polymorphic positions 67 and 71 contributed to the peptide binding with direct effects on T‐cell receptor (TCR) recognition while position 70 seems to be mostly engaged in TCR interactions. Furthermore, our results suggest that polymorphic residues may select allele‐specific peptides and also influence the conformation of promiscuous peptides.
Regulation of T‐cell activation in the lung: isolated lung T cells exhibit surface phenotypic characteristics of recent activation including down‐modulated T‐cell receptors, but are locked into the G0/G1 phase of the cell cycle
Authors: STRICKLAND D. , KEES U. R. , HOLT P. G.
Published: November 2003

Peripheral lung tissue contains large numbers of T cells, strategically located for immune surveillance at the blood–air interface. Given the intensity of antigenic exposure at this site, it is clear that local T‐cell activation events require strict control, in order to maintain tissue homeostasis. How this control is achieved in this unique tissue microenvironment is unknown, and the present study sought to elucidate the process via detailed analysis of the surface phenotypic characteristics of freshly isolated lung T cells. We report below that these cells display typical characteristic of ‘postactivation’, notably elevated basal Ca2+ concentrations, down‐modulated T‐cell receptors, expression of Ia and ‘late’ activation antigens and concomitant CD4/CD8. However, levels of interleukin‐2 receptor and CD2 expression were below those expected of ‘activated’ T‐cell populations, and virtually all of the cells were found to be in the G0/G1 phases of the cell cycle. These properties bear a remarkable similarity to those of T cells activated in the presence of endogenous tissue (alveolar) macrophages from the lung (see accompanying paper). We hypothesize that they reflect the in vivo operation of an endogenous macrophage‐mediated T‐cell anergy‐induction process, the function of which is to limit the local clonal expansion of T cells in peripheral lung tissue after in situ activation.
Multiple B‐ and T‐cell epitopes on a major allergen of Kentucky Bluegrass pollen
Authors: ZHANG L. , YANG M. , CHONG P. , MOHAPATRA S. S.
Published: November 2003

The B‐ and T‐cell epitopes of a recombinant grass allergen, rKBG60, were delineated using a set of overlapping synthetic peptides. Direct binding by enzyme‐linked immunosorbent assay (ELISA) utilizing serum pools led to the identification of 13 murine immunoglobulin‐, and nine to 13 human IgG‐ and five to seven human IgE‐reactive overlapping peptides. Of the peptides which bound to human IgE antibodies, all but three peptides bound to human and/or murine IgG antibodies. Furthermore, eight out of 12 synthetic peptides induced antigen‐specific antibodies in mice, suggesting that these peptides contained epitopes that recognized and/or induced T cells. These results, in conjunction with cross‐recognition of different peptides at the C‐terminus of rKBG60 by antibodies to neighbouring or non‐overlapping peptides suggest that the C‐terminus of this antigen represents a dominant antigenic and allergenic site. Peripheral blood mononuclear cell (PBMC) proliferation studies using these synthetic peptides for 13 grass allergic individuals indicated that seven potential human T‐cell epitopes exist on this allergen. Taken together, the results demonstrate that multiple B‐ and T‐cell epitopes exist on this major group of grass allergens, the majority of which are localized at the C‐terminus of this antigen.
Expression of RANTES in human airway epithelial cells: effect of corticosteroids and interleukin‐4, ‐10 and ‐13
Authors: BERKMAN N. , ROBICHAUD A. , KRISHNAN V. L. , ROESEMS G. , ROBBINS R. , JOSE P. J. , BARNES P. J. , CHUNG K. F.
Published: November 2003

RANTES is a C–C chemokine with strong chemoattractant and activating properties for eosinophils, basophils and T lymphocytes. We investigated the expression of RANTES in human airway epithelial cells and its modulation. Epithelial cells obtained from tracheas of donor lungs were stimulated with interleukin‐1β (IL‐1β), tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ) or with a mixture of the three cytokines (‘cytomix’). Levels of mRNA and protein were assayed by Northern blot and radioimmunoassay, respectively. Each individual cytokine produced a small increase in RANTES protein: IL‐1β 24 ± 1 pM, TNF‐α 13 ± 7 pM and IFN‐γ 29 ± 7 pM, but cytomix increased protein to 236 ± 22 pM (P < 0.002) and mRNA expression by >20‐fold (P < 0.002). Both RANTES protein and mRNA expression were inhibited by dexamethasone (10−6 m) (38 ± 5%, P < 0.05 and 55 ± 8%, P < 0.007, respectively), and by IL‐4 (42 ± 7%, P < 0.03 and 19 ± 1%, not significant, respectively). No inhibitory effect was observed with IL‐10 or IL‐13. We also demonstrated in vivo expression of RANTES protein by epithelial cells in human airways using immunohistochemistry. Our data show that human airway epithelial cells can be stimulated to express and release RANTES, an effect that is inhibited by corticosteroids and IL‐4, but not by IL‐10 and IL‐13.
Interaction of natural killer cells with MHC class II: reversal of HLA‐DR1‐mediated protection of K562 transfectant from natural killer cell‐mediated cytolysis by brefeldin‐A
Authors: JIANG Y. Z. , COURIEL D. , MAVROUDIS D. A. , LEWALLE P. , MALKOVSKA V. , HENSEL N. F. , DERMIME S. , MOLLDREM J. , BARRETT A. J.
Published: November 2003

Major histocompatibility complex (MHC) class I antigens on tumour cell surfaces have been shown to modulate target susceptibility to natural killer (NK) cell‐mediated lysis in some, although not all, systems investigated. MHC class II expression may also affect NK cell function, but the mechanism by which MHC class II antigen regulates NK cell activity has not been fully examined. In this study we induced HLA‐DR1 expression by gene transfection into the classic NK‐sensitive K562 cell line to study the interaction of NK cells with MHC class II molecules and the effect of brefeldin‐A (BFA), an endogenous antigen‐processing pathway blocker, on NK–target cell interaction. We demonstrated that the expression of HLA‐DR1 on the cell surface reduced K562 cell susceptibility to NK lysis by peripheral blood monuclear cells and a NK cell line. The effect was demonstrable in prolonged (8 hr) cytotoxicity assays and was blocked by pretreatment of target cells with anti‐HLA‐DR antibody. Treatment of K562 DR transfectant with BFA abrogated the resistance of K562 transfectant to NK‐mediated cytolysis. These findings indicate that HLA class II molecules regulate NK cell function and target recognition, and suggest that endogenous peptides presented through MHC molecules are responsible for regulating NK cytolysis.
Th1/Th2 cell dichotomy in acquired immunity to Bordetella pertussis: variables in the in vivo priming and in vitro cytokine detection techniques affect the classification of T‐cell subsets as Th1, Th2 or Th0
Authors: BARNARD A. , MAHON B. P. , WATKINS J. , REDHEAD K. , MILLS K. H. G.
Published: November 2003

In studies of the mechanism of immunity to Bordetella pertussis in a murine respiratory infection model, we have previously demonstrated that natural infection of immunization with a whole cell vaccine induces a potent protective immune response, which is mediated by T‐helper type‐1 (Th1) cells. In contrast an acellular vaccine generates Th2 cells and is associated with delayed bacterial clearance following respiratory challenge. In the present study we have investigated the apparent Th1/Th2 cell dichotomy in acquired immunity and have examined the factors that affect their induction or detection. The cytokine profiles of B. pertussis‐specific T cells in immune animals were determined using antigen‐stimulated ex vivo spleen cells or CD4+ T‐cell lines and clones established in the presence of interleukin‐2 (IL‐2) or IL‐4. Antigen‐specific T cells derived from mice immunized with the acellular vaccine were almost exclusively of the Th2 cell type. In contrast, T‐cell lines and clones established following respiratory infection or immunization with the whole cell vaccine were predominantly of the Th1 type. However, a proportion of T cells from convalescent mice, especially when cultured in the presence of IL‐4, secreted IL‐4 and IL‐5 with or without detectable IL‐2 and interferon‐γ (IFN‐γ), suggesting that Th0 or Th2 cells were also primed during natural infection in vivo. Furthermore, when mice were assessed 6 months after infection, spleen cells produced significant levels of IL‐4 and IL‐5, which were not evident at 6 weeks. The route of immunization and the genetic background of the mice were also found to influence the preferential priming of Th1 cells, and this was directly related to the level of protection against respiratory or intracerebral (i.c.) challenge. Our findings underline the critical role of CD4+ Th1 cells in immunity to B. pertussis, but also demonstrate that a number of factors in the in vivo priming and in vitro restimulation can skew the apparent dominance of one Th cell type over another.
The method of deriving human T‐cell clones alters the proportion of IL‐10‐producing cells
Authors: COHEN S. B. A. , WEBB L. M. C. , FELDMANN M.
Published: November 2003

It is now well documented that the mode of primary stimulation in the mouse determines the ratio of Th1‐, Th0‐ or Th2‐type T cells obtained. In this paper we determine whether driving and cloning human peripheral blood mononuclear cells (PBMC) non‐specifically with interleukin‐2 (IL‐2) and/or IL‐4 and mitogen, will differentially select T cells for IL‐10 production. The presence of IL‐2 during culture (with or without IL‐4) yielded predominantly Th1‐type clones (81% of clones with IL‐2 alone and 77% with IL‐2 + IL‐4) and a low frequency of Th0‐type clones (19% of clones with IL‐2 and 10% of clones with IL‐2 + IL‐4), whereas IL‐4 alone increased the yield of the IL‐4 producing Th0 (35%) clones. The proportions of IL‐2‐producing clones did not alter with treatment; however, the combination of IL‐2 + IL‐4 altered the proportions of IL‐10‐producing clones. 47% of IL‐2‐cultured cells and 51% of IL‐4‐cultured cells produced IL‐10 respectively, whereas only 22% of clones driven with IL‐2 + IL‐4 produced detectable levels of IL‐10. Thus in the preliminary experiments described here we have demonstrated that the cytokines used to initially drive out human T cells and maintain the T‐cell growth can skew the Th1/Th2/Th0 cytokine profiles of the clones obtained from mitogen‐stimulated cultures. Moreover and unexpectedly the proportion of IL‐10‐producing cells is altered. These results are important in interpreting analysis of the cytokine profiles of human T cells and raise questions concerning how we should derive T‐cell clones for a cytokine analysis that truly reflects the in vivo situation.
Altered patterns of migration of cytokine‐producing T lymphocytes in skin‐grafted naive or immune mice following in vivo administration of anti‐VCAM‐1 or ‐ICAM‐1
Authors: GORCZYNSKI R. M. , CHUNG S. , HOANG Y. , SULLIVAN B. , CHEN Z.
Published: November 2003

Naive or preimmunized (to B10.BR or BALB.k) C3H/HeJ mice received skin grafts from multiple minor histoincompatible B10.BR or BALB.k mice following antigen‐specific portal venous (p.v.) pretransplant transfusion, a protocol known to produce prolongation of graft survival in naive animals. In addition, groups of mice received intravenous (i.v.) infusion following transplantation with a mixture of monoclonal antibodies (mAb) to vascular adhesion molecule‐1L : very late activation antigen‐4 (VCAM‐1 : VLA‐4) or intracellular adhesion molecule‐1 : lymphocyte function‐associated antigen‐3 (ICAM‐1 : LFA‐1). Cells were harvested from different tissues of the grafted mice at various times post grafting. RNA was extracted and analysed, using polymerase chain reaction, for expression of different cytokines potentially involved in the regulation of graft rejection [interleukin‐2 (IL‐2), IL‐4, IL‐6, IL‐10, tumour necrosis factor‐α, interferon‐γ and transforming growth factor‐β]. In addition, using limiting dilution analysis, we investigated the frequency of allo‐specific and third‐party reactive cells producing IL‐2 and IL‐4 in vitro in different tissues of grafted mice following these treatments. The mAb treatment protocol which produced optimum increases in graft survival in naive versus immune mice was different, with anti‐LFA‐1 : ICAM‐1 superior for naive mice compared with anti‐VLA‐4 : VCAM‐1, and vice versa for immune animals. However, in each case, increased survival was associated with increases local to the graft in the frequency of occurrence of antigen‐specific type‐2 cytokine‐producing cells.
Involvement of LFA‐1/ICAM‐2 adhesive interactions and PKC in activation‐induced cell death following SEB rechallenge
Authors: TCHILIAN E. Z. , ANDERSON G. , MOORE N. C. , OWEN J. J. T. , JENKINSON E. J.
Published: November 2003

Ligation of T‐cell receptor (TCR) causes mature T cells to proliferate or, on re‐exposure to antigen, can cause them to die by activation‐induced cell death (AICD). In proliferative responses, costimulatory and adhesive interactions are required and activation of protein kinase C (PKC) has been shown to be essential. Whether or not interactions involving costimulatory signals and PKC have a role in facilitating AICD remains unclear. Here we have examined the role of CD28/B7 and leucocyte function associated antigen‐1 (LFA‐1)/intracellular adhesion molecule (ICAM) mediated interactions in AICD triggered by staphylococcal enterotoxin B (SEB) in murine lymph node T cells. We show that, after a primary proliferative response to SEB, LFA‐1/ICAM‐2 adhesive interactions can play a part in AICD following SEB rechallenge, while B7 and ICAM‐1 mediated interactions are not essential for this process. In addition, using a higly selective PKC inhibitor, Ro31.8425, we show that PKC activation is essential for the regulation of AICD by SEB rechallenge.
Role of leukotriene B4 in the interleukin‐4‐induced human mononuclear phagocyte activation
Authors: DUGAS N. , DUGAS B. , KOLB J.‐P. , YAMAOKA K. , DELFRAISS J. F. , DAMAIS C.
Published: November 2003

Interleukin‐4 (IL‐4) induced a time‐ and dose‐dependent production of leukotriene B4 (LTB4) by human resting monocytes indicating that IL‐4 induced the activation of the 5‐lipoxygenase pathway in resting human monocytes. Maximal effect was observed in the presence of 10 ng/ml IL‐4, and in kinetics experiments LTB4 production plateaued 40 min after the onset of stimulation. When stimulated for 48 hr with IL‐4, resting human monocytes expressed and released the low‐affinity receptor for IgE (CD23) and were partially inhibited in the presence of a highly non‐redox 5‐lipoxygenase inhibitor (BW B70C), suggesting that the production of LTB4 partially contributed to the IL‐4‐induced CD23 expression and release. This hypothesis was strengthened by the fact that exogenous LTB4 (10 nm) was found to increase the effect of a suboptimal dose of IL‐4 (1 ng/ml). In addition to these phenotypical changes, IL‐4 primed the phorbol‐12‐myristate‐13‐acetate (PMA)‐induced luminol‐dependent chemiluminescence response (LDCL) by normal human monocytes, this priming effect being abrogated in the presence of BW B70C. Taken together, these data indicated that IL‐4 induced the production of LTB4 by activation of the 5‐lipoxygenase pathway in human monocytes, and that the activation of this pathway could upregulate the expression and release of CD23 and the respiratory burst of these cells.
Reciprocal action of interferon‐γ and interleukin‐4 promotes granulomatous inflammation induced by Rhodococcus aurantiacus in mice
Authors: ASANO M. , KOHANAWA M. , MINAGAWA T. , NAKANE A.
Published: November 2003

An intravenous injection of Rhodococcus aurantiacus to mice causes granulomatous inflammation dependent on endogenous interferon‐γ (IFN‐γ). The present study examined the role of endogenous interleukin‐4 (IL‐4) on granulomatous inflammation. Endogenous IL‐4 in the spleen extracts was not detected during the phase of granuloma formation by enzyme‐linked immunosorbent assay (ELISA). However, IL‐4 protein level was elevated during the phase of granuloma regression. IL‐4 mRNA expression in the livers and spleens was also elevated during the phase of granuloma regression. In addition, IL‐4 levels during the phase of granuloma formation were increased by treatment with anti‐IFN‐γ monoclonal antibody (mAb), suggesting that endogenous IFN‐γ might inhibit IL‐4 production during the phase of granuloma formation. Administration of anti‐IL‐4 mAb on weeks 3 and 4 after the inoculation inhibited the regression of granulomas and augumented IFN‐γ level at 5 weeks. Endogenous IFN‐γ was produced by CD4+ T cells during the phase of granuloma regression and endogenous IL‐4 was produced by both CD4+ and CD8+ T cells. These findings suggest that during the phase of granuloma formation endogenous IL‐4 might be inhibited by IFN‐γ, while during the phase of granuloma regression endogenous IL‐4 might play a crucial role in the reduction of granulomas and IFN‐γ production.
Primary cytotoxic T‐cell responses to bovine respiratory syncytial virus in calves
Authors: GADDUM R. M. , COOK R. S. , THOMAS L. H. , TAYLOR G.
Published: November 2003

Bovine respiratory syncytial virus (RSV) is a major cause of respiratory disease in young calves. Recent studies in calves, in which different T‐cell subsets were depleted, have shown that CD8+ T cells play a central role in recovery from RSV infection.1 The present study demonstrates that RSV‐specific, major histocompatibility complex‐restricted cytotoxic T cells appear in the peripheral blood of gnotobiotic calves 7–10 days after infection with bovine RSV and were also detected in the lungs 10 days after infection. The cytotoxic T lymphocytes (CTL) recognized antigenically distinct strains of bovine RSV. There was no correlation between either the level of CTL activity in the lung or the development of CTL in the peripheral blood and the extent of pneumonic consolidation. The demonstration of CD8+ CTL in the lungs at a time when bovine RSV has been cleared confirms the importance of these cells in recovery from infection.
Coexpression of H2‐Mb and H2‐Ab genes during fetal and postnatal development
Authors: GEORGIADES P. , KIESZKIEWICZ J. , ROZYCKA M. , BRICKELL P. M. , LUND T.
Published: November 2003

The major histocompatibility complex (MHC) class II‐like molecules, H2‐M, have an essential role in processing and presentation of antigens by the MHC class II molecules, because functional inactivation of these genes lead to surface expression of MHC class II molecules devoid of associated foreign peptides. We have used in situ hybridization to examine the expression of MHC class II and H2‐Mb genes in embryonic and neonatal mice and show here that expression of H2‐Ab and H2‐Mb mRNA is absent in 13.5‐day‐old mice. However, mRNA for both genes could be detected in the thymus of 14.5‐ and 15.5‐day‐old embryos, and the patterns of hybridization suggested that the two genes were expressed in the same cells. In spleen and thymus of neonatal mice the H2‐Ab and H2‐Mb genes were also coexpressed, with expression being localized to the white pulp of the spleen and to the thymic medulla, which are rich in antigen‐presenting cells. The steady‐state levels of H2‐Ab mRNA appeared to be approximately 10 to 14 times greater that the level of H2‐Mb mRNA molecules, irrespectively of the tissue and age, reflecting the different functions of the two molecules.
Induced expression of the new cytokine, activin A, in human monocytes: inhibition by glucocorticoids and retinoic acid
Authors: YU J. , SHAO L.‐E. , FRIGON, JR. N. L. , LOFGREN J. , SCHWALL R.
Published: November 2003

The capacity of recombinant human granulocyte–macrophage colony‐stimulating factor (GM‐CSF), glucocorticoids or all‐trans‐retinoic acid to modulate production of activin A by human monocytes was studied. It was shown that GM‐CSF stimulated monocytes to accumulate activin A RNA after as few as 4 hr of incubation, reaching a peak of stimulation at approximately 16 hr of incubation. The activin A transcripts accumulated in the monocytes after stimulation with only 5 U/ml of GM‐CSF and reached a maximum plateau level of expression between 25 and 50 U/ml of GM‐CSF. Biologically active activin A molecules were detected in the conditioned media by a bioassay, performed both in the absence and presence of a neutralizing antiserum for activin A. Accumulation of bioactive activin A in conditioned medium of monocyte cultures was detected after 24 hr of incubation with GM‐CSF and high levels of activin A were maintained for 72 hr. The production of the dimeric βAβA in these monocytes was further confirmed by a sandwich enzyme‐linked immunosorbent assay (ELISA) specific for activin A. In contrast to the stimulatory effect of GM‐CSF, hydrocortisone, dexamethasone or all‐trans‐retinoic acid at 1 × 10−7 to 1 × 10−5 m inhibited the constitutive expression of activin A and greatly suppressed the GM‐CSF‐stimulated production. Thus, the expression of activin A is modulated in monocytes by different agents. These observations may imply new roles for activin A at sites of inflammation where monocytes accumulate.
Definition of cotton rat immunoglobulins: sigmodon species differ in expression of IgG isotypes and production of respiratory syncytial virus antibody
Authors: COE J. E. , PRINCE G. A.
Published: November 2003

The cotton rat (Sigmodon species) is the preferred animal model for experiments with a number of human pathogens, especially the respiratory viruses. The cotton rat is classified in the family Cricetidae (with hamsters and gerbils) and is a distant cousin of the common laboratory rat (Rattus) classified in the family Muridae (with the common laboratory mouse, Mus). Antibody reagents that are specific for cotton rat immunoglobulins have not been described. To enhance the usefulness of this model, four immunoglobulins in Sigmodon serum were characterized (IgG1, IgG2, IgA, IgM) and antisera specific for each immunoglobulin were made. Sera from three different species of Sigmodon were examined, S. hispidus (SH), S. arizoni (SA) and S. fulviventer (SF). Although IgA and IgM appeared similar in all three species, the IgG were expressed differently because normal serum levels of IgG2 were deficient in SH when compared with SA and SF and to other rodents. Similarly, IgG2 antibody response to purified protein antigen was deficient in SH although the IgG1 antibody response was superior to that in SF and SA. The three cotton rat species were infected with respiratory syncytial virus, and the kinetics of the antibody response was measured. Neutralizing antibody developed faster and to a higher titre in SH than in SA and SF. The enhanced immunoresponsiveness in SH may compensate for the IgG2 deficiency in SH and these changes appear to be relatively recent events in the evolution of this most populous species of Sigmodon
Limited heterogeneity of biased T‐cell receptor Vβ gene usage in lung but not blood T cells in active pulmonary sarcoidosis
Authors: SILVER R. F. , CRYSTAL R. G. , MOLLER D. R.
Published: November 2003

Sarcoidosis is a multisystem disorder characterized by non‐caseating granulomas and the accumulation of CD4+ T cells in involved tissues such as the lung. To evaluate the diversity of the CD4+ T‐cell repertoire in this disorder, a detailed clonal analysis was performed in five individuals with active sarcoidosis who demonstrated preferential accumulation of T cells expressing the T‐cell receptor variable gene family Vβ8 in either the lung or blood. In three individuals, analysis of unselected samples of nucleotide sequences derived from Vβ8+ lung T cells demonstrated degrees of clonality ranging from 11% to 46%, indicating the expansion of limited numbers of Vβ8+ T‐cell clones in the lung. Analysis of the corresponding deduced amino acid sequences demonstrated common VDJ junctional amino acid residues in the dominant Vβ8+ T‐cell clones derived from two oligoclonal Vβ8+ lung T‐cell populations, consistent with an antigen‐specific T‐cell response. In contrast, analysis of Vβ8+ CD4+ T cells from the blood of an individual with a marked bias for peripheral blood Vβ8+ T cells demonstrated no evidence of oligoclonality, suggesting that the stimulus for circulating biased Vβ‐specific T cells in sarcoidosis may derive from a different, perhaps superantigenic, origin. Clinical improvement in the disease either in response to treatment with corticosteroids or as a result of spontaneous resolution was associated with a decrease in the proportion of Vβ8‐specific T cells in the biased lung and/or blood T‐cell compartments. Together, these observations are consistent with a role for this T‐cell subset in the clinical manifestations of active granulomatous disease.
Immune responses in congenic mice to multiple antigen peptides based on defined epitopes from the malaria antigen Pf332
Authors: AHLBORG N. , ANDERSSON R. , PERLMANN P. , BERZINS K.
Published: November 2003

Repeat sequences from the Plasmodium falciparum blood stage antigen Pf332 frequently comprise the pentapeptide VTEEI, an epitope recognized by certain parasite neutralizing antibodies. This B‐cell epitope was assembled in an octavalent multiple antigen peptide (MAP) system either as trimers (VTEEI)3 (MAP1) or as an integral part of a naturally occurring Pf332 undecamer repeat sequence SVTEEIAEEDK (MAP2). Characteristics of the immunogenicity of these subunit constructs were evaluated in H‐2 congenic mice. MAP1 generated antibody responses in mice of the H‐2d, H‐2k and H‐2q haplotypes, but not in H‐2b or H‐2s mice, whereas MAP2 only induced antibodies in mice of H‐2k haplotype. When analysing T‐cell responses induced by the MAP, lymph node cells from responder strains primed in vivo with MAP1 proliferated in response to restimulation with both MAP1 and the peptide (VTEEI)3. MAP2, however, did not induce a detectable T‐cell proliferation. Additionally, the lack of antibody response to MAP1 in H‐2b mice could be circumvented by combining the MAP1 peptide and a H‐2b‐restricted T‐cell epitope in a diepitope MAP construct. Despite the fact that the motif VTEEI has not been identified in Pf332 sequences in the form of a trimer, MAP1 did induce Pf332 protein‐reactive antibodies. Assembly of multimers of short defined epitopes in MAP constitutes an interesting approach for the design of polyvalent subunit immunogens.
Induction of antibody‐secreting cells and T‐helper and memory cells in murine nasal lymphoid tissue
Authors: WU H.‐Y. , NIKOLOVA E. B. , BEAGLEY K. W. , RUSSELL M. W.
Published: November 2003

Intranasal (i.n.) immunization is an effective route for inducing mucosal immune responses especially in the upper respiratory tract and mouth. To characterize the cells involved in these responses, nasal lymphoid tissue (NALT; considered to be the equivalent of Waldeyer's ring in humans) of normal mice, and of mice immunized intranasally with a bacterial protein antigen conjugated to cholera toxin B subunit, was isolated and the lymphoid cells analysed according to surface phenotype, immunoglobulin and antibody secretion, and cytokine profile. Compared with cells obtained from Peyer's patches (PP), NALT cells contained a higher proportion of T cells, especially naive (CD45RB+hi) T‐helper cells, and fewer surface (s)IgA+ cells. Both tissues contained high proportions of sIgM+ IgD+ unswitched B cells. After i.n. immunization, IgA antibody‐secreting cells were increased, indicating that isotype switching and differentiation of B cells to IgA‐secreting cells occurred in NALT, whereas smaller numbers of antibody‐secreting cells were found in PP after intragastric (i.g.) immunization. Antigen‐specific memory cells persisted in NALT for at least 8 months after initial immunization. The cytokine expression profiles of antigen‐stimulated NALT and PP cells of immunized mice, revealed by reverse transcription polymerase chain reaction analysis of mRNA, were similar. Both NALT and PP cells tended to express type 2 earlier or for longer than type 1 cytokine mRNA, but NALT cells tended to express interleukin‐4 (IL‐4) earlier, and IL‐5 for a longer period, than PP cells. Thus NALT shares with PP cell populations typical of a mucosal inductive site, including unswitched B cells and naive T‐helper (Th) cells. After i.n. immunization, NALT has the capacity to provide help for B‐cell maturation and differentiation, as well as to maintain immune memory.
CD4+ cytolytic effectors are inefficient in the clearance of Listeria monocytogenes
Authors: SERODY J. S. , POSTON R. M. , WEINSTOCK D. , KURLANDER R. J. , FRELINGER J. A.
Published: November 2003

Cytotoxic T lymphocytes (CTL) recognize and lyse target cells through the interaction of the T‐cell receptor complex with the class I or class II major histocompatibility complex (MHC). The production of class I‐restricted CTL has been shown to be critical to the elimination of specific pathogens including Listeria monocytogenes. However, the function of class II‐restricted CTL in the clearance of intracellular pathogens is poorly understood. H‐2bβ2‐microglobulin‐deficient mice (β2M−/−) are not able to produce CD8+ CTL in response to infection with L. monocytogenes. We used this model to evaluate the efficacy of class II‐restricted CTL, in the absence of a class I‐restricted response, during a primary infection with L. monocytogenes. We demonstrate that, despite their effectiveness in adoptive transfer of protection, Listeria‐specific CD4+ class II‐restricted cytotoxic lymphocytes are ineffective in decreasing titres of L. monocytogenes in the spleen after an established infection. In β2M−/− mice, persistence of L. monocytogenes in the spleen was found preferentially in class II‐negative cells. Surprisingly, class I‐restricted CTL from C57BL/6 mice were capable of decreasing bacterial titres during an established infection even in the absence of detectable class I on the surface of cells from β2M−/− mice. These data strongly suggest that, in the absence of a class I‐restricted response, pathogens that elicit a class II‐restricted cytotoxic response may escape prompt eradication by the immune system.
Induction of cytotoxic T‐cell response by optimal‐length peptides does not require CD4+ T‐cell help
Authors: FAYOLLE C. , ABDEL‐MOTAL U. M. , BERG L. , DERIAUD E. , JONDAL M. , LECLERC C.
Published: November 2003

In several experimental models, synthetic peptides were shown to activate efficiently cytotoxic T‐lymphocyte (CTL) responses and therefore represent an attractive strategy to develop new vaccines. However, the mechanisms by which they induce CTL responses are not yet fully understood. Several studies using 15–16‐mer peptides previously demonstrated that CD4+ helper T cells are required to induce optimal CTL responses with synthetic peptides. However, recently, it was suggested that shorter 8–12‐mer peptides could have an increased in vivo immunogenicity. In the present study, we therefore investigated if such optimal‐length peptides still require CD4+ T‐cell help to activate CTL responses. To address this question, three synthetic peptides containing different viral CTL epitopes were injected into mice depleted of CD4+ or CD8+ T cells using specific monoclonal antibodies or into mice genetically deficient in those T‐cell populations. Our results clearly established that activation of CTL responses by those short optimal peptides does not require CD4+ T‐cell help and therefore suggested that high‐density binding of peptides to major histocompatibility complex class I molecules on the surface of antigen‐presenting cells is required for direct activation of CD8+ T cells, independently of CD4+ T‐cell help.
Role of Th1 and Th2 cells in anterior chamber‐associated immune deviation
Authors: LI X.‐Y. , D'ORAZIO T. , NIEDERKORN J. Y.
Published: November 2003

The immunological privilege of the anterior chamber (AC) of the eye is due, at least in part, to a selective antigen‐specific down‐regulation of delayed‐type hypersensitivity (DTH) and a normal induction of antibody responses: a phenomenon that has been termed anterior chamber‐associated immune deviation (ACAID). This dichotomy in the systemic immune responses is suggestive of a T‐helper type‐2 (Th2)‐dominated immune phenotype in which a Th2 cell population is preferentially activated and cross‐regulates T‐helper type‐1 (Th1) effector elements. This hypothesis was tested by comparing the cytokine pattern of antigen‐pulsed spleen cells from mice primed in the anterior chamber with antigens that induce ACAID with responses in hosts primed with antigens that do not induce ACAID. The results indicated that CD4+ spleen cells from hosts primed in the AC with antigens that induce ACAID produced significant quantities of interleukin‐10 (IL‐10) but insignificant levels of IL‐2, IL‐4 and interferon‐γ (IFN‐γ). In contrast, hosts primed in the AC with antigens that do not induce ACAID, but instead elicit normal DTH, displayed cytokine patterns indicative of a Th1 response: significant quantities of IL‐2 and IFN‐γ were produced while IL‐4 and IL‐10 secretion was insignificantly different from normal controls. The immunological phenotype of the AC‐primed hosts could be altered by systemic treatment with antibodies against either a Th1 cytokine (IFN‐γ) or a Th2 cytokine (IL‐10). Hosts treated with anti‐IL‐10 antibody and subsequently primed in the AC with ACAID‐inducing antigens developed normal DTH responses, while hosts treated with anti‐IFN‐γ antibody and primed in the AC with antigens that normally produce positive DTH responses failed to develop positive DTH. Collectively the results support the proposition that immune privilege in the AC of the eye is due to the selective activation of a Th2 population that cross‐regulates Th1 responses.
An early antigen‐presenting cell defect in HIV‐1‐infected patients correlates with CD4 dependency in human T‐cell clones
Authors: FIDLER S. J. , DORRELL L. , BALL S. , LOMBARDI G. , WEBER J. , HAWRYLOWICZ C. , REES A. D. M.
Published: November 2003

We have used a defined panel of nine HIV peptide‐specific T‐cell clones (TLC) generated from a healthy volunteer to evaluate the antigen‐presenting cell (APC) function of human immunodeficiency virus‐1 (HIV‐1)‐infected patients. Peripheral blood mononuclear cells (PBMC) from HLA‐matched seropositive and uninfected volunteers were compared for their capacity to present peptide to TLC specific for the V3 loop of HIV‐1 envelope glycoprotein gp120, influenza haemagglutinin or the mycobacterial 19 000 MW antigen. APC from uninfected volunteers (HIV−APC) invariably presented peptides to all TLC with comparable efficiency. In contrast, using APC from HIV‐1‐infected subjects (HIV+APC), three patterns of responsiveness were observed. The first group of TLC was not stimulated by HIV+APC, even early in infection. The second responded to all APC comparably. The third, and intermediate group, responded to APC from some clinically asymptomatic, but not acquired immune deficiency syndrome (AIDS), patients. The two additional TLC, derived from other donors and with specificity for non‐HIV peptides, showed similar variation in response to HIV+APC. The different patterns of response to HIV+APC did not correlate with the fine specificity or cytokine phenotypes of the TLC. Neither was the defect due to decreased levels of expression of APC molecules involved in delivering the first or second signal required for T‐cell activation. APC mixing experiments showed no evidence of APC‐derived inhibitory factors. Furthermore, the defect was independent of T cells or their products and was equally expressed in monocytes and dendritic cells. Instead, responsiveness was inversely related to the degree of CD4 dependency, suggesting that the underlying mechanism was a CD4 APC‐associated–gp120 interaction. The early appearance of this defect in HIV‐1 infection co‐incident with the loss of recall responses is consistent with a role for APC dysfunction in pathogenesis.
Cytotoxic activity against tumour cells mediated by intermediate TCR cells in the liver and spleen
Authors: KAWAMURA T. , KAWACHI Y. , MORODA T. , WEERASINGHE A. , IIAI T. , SEKI S. , TAZAWA Y. , TAKADA G. , ABO T.
Published: November 2003

Morphological and phenotypic characterization in previous studies has indicated that intermediate (int) T‐cell receptor (TCR) cells or T natural killer (TNK) cells may stand at an intermediate position between NK cells and high TCR cells of thymic origin in phylogenetic development. In this study, a functional study on cytotoxic activity against various tumour targets was performed in each purified subset. When a negative selection method entailing in vivo injection of anti‐asialo GM1 antibody or anti‐interleukin (IL)‐2Rβ monoclonal antibody (mAb) was applied, IL‐2Rβ+ CD3− NK cells were found to have the highest NK activity while IL‐2Rβ+ int CD3 (or TCR) cells had a lower level of the NK activity. High CD3 cells (freshly isolated) did not have any such activity. Sorting experiments further revealed that the NK function mediated by int CD3 cells was augmented when they were exposed to anti‐CD3 mAb, anti‐TCRαβ, or anti‐TCRγδ mAb. This phenomenon was not observed in NK cells and high CD3 cells. More importantly, when anti‐CD3 mAb (or anti‐TCR mAb) was added to the assay culture, int CD3 cells became cytotoxic against even NK‐resistant tumour (FcγR−, Fas+) targets. Liver mononuclear cells or int CD3 cells exposed to anti‐CD3 mAb for 6 hr showed an elevated level of perforin in their cytoplasms. The present results suggest that int CD3 cells are usually non‐cytotoxic against various tumours but become functional after being stimulated via the TCR–CD3 complex.