bluuebunny/crossref_metadata_2025 huggingface dataset:
Matrix metalloproteinase‐2 expression correlates with morphological and immunohistochemical epithelial characteristics in synovial sarcoma
Authors: Saito T , Oda Y , Sakamoto A , Tamiya S , Iwamoto Y , Tsuneyoshi M
Published: March 2003

Matrix metalloproteinase‐2 expression correlates with morphological and immunohistochemical epithelial characteristics in synovial sarcoma

Aims: Synovial sarcoma is a unique mesenchymal tumour characterized by the presence of epithelial differentiation, although the mechanism involved in the epithelial morphology is still unclear. The aim of this study was to evaluate the function of matrix metalloproteinase‐2 (MMP‐2) in synovial sarcoma, in order to assess whether MMP‐2 expression plays an important role in epithelial differentiation, or whether it contributes to a poor clinical outcome.

Methods and results: Immunohistochemical stainings for MMP‐2, cytokeratins (CKs) 7, 8, 18 and 19, and E‐cadherin were performed for 58 (44 monophasic and 14 biphasic) cases of synovial sarcoma, and we compared the expression of these proteins with the histological and clinical findings. MMP‐2 and E‐cadherin expression was observed in 43 cases (74.1%) and in 18 cases (31.0%), respectively. Expression of these proteins was preferentially observed in the glandular components of biphasic tumours or the epithelioid areas of monophasic tumours. Statistically significant correlations were recognized between MMP‐2 expression and E‐cadherin expression of biphasic subtype. Moreover, there were statistically significant correlations between monophasic tumours with epithelioid areas and MMP‐2 expression or E‐cadherin expression. MMP‐2 expression was correlated with epithelial differentiation as assessed by CK immunoreactivity. The expression of MMP‐2 did not affect the overall survival rate in synovial sarcoma.

Conclusions: MMP‐2 expression seemed to have an important role to play in the epithelial differentiation of tumour cells in synovial sarcoma, through remodelling of the extracellular matrix and by changing the cytoskeletal interaction between the extracellular matrix and tumour cells.
PTEN is not altered in sporadic vestibular schwannomas
Authors: Mawrin C , Kirches E , Boltze C , Dietzmann K
Published: March 2003

PTEN is not altered in sporadic vestibular schwannomas

Aims: To investigate the role of the tumour suppressor gene PTEN in the tumorigenesis and growth of sporadic vestibular schwannomas, and to characterize the cellular distribution of the PTEN protein in relation to the MIB‐1 proliferation index in these tumour.

Methods and results: Immunoexpression of the PTEN protein was observed within the neoplastic Schwann cells in 21 out of 30 sporadic schwannomas examined (70%). PTEN expression was consistently stronger in Antoni A areas than in Antoni B areas. High levels of PTEN immmunoexpression in schwannomas were associated with an increased MIB‐1 labelling index. Occasionally, vascular endothelial cells also showed PTEN immunoreaction. By polymerase chain reaction‐single strand conformation polymorphism screening, no mutations were found in the complete protein coding region of the PTEN gene.

Conclusions: The PTEN tumour suppressor gene is expressed in the majority of sporadic schwannomas. The maintained expression of the PTEN protein, together with the lack of detectable mutations in this gene, suggests that the function of the PTEN tumour suppressor gene is not altered in sporadic vestibular schwannomas.
Unusual intraparenchymal growth patterns of malignant pleural mesothelioma
Authors: Nind N R , Attanoos R L , Gibbs A R
Published: March 2003

Aims:  Malignant pleural mesothelioma is known to mimic morphologically a number of diverse reactive and neoplastic conditions. We describe three unusual intraparenchymal growth patterns of malignant mesothelioma seen in a series of 200 malignant pleural mesotheliomas. The diagnostic pitfalls associated with these findings are described and their potential medico‐legal implications are highlighted.

Methods and results:  The study group comprised 200 malignant pleural mesotheliomas. In each case diagnosis was morphologically confirmed with ancillary immunohistochemistry using a broad panel of both mesothelial and epithelial markers. The patterns of intraparenchymal growth were documented and grouped as: direct subpleural; lymphangitic; and other. The 200 malignant pleural mesotheliomas comprised 118 epithelioid, 57 biphasic and 25 sarcomatoid, subtyped according to the WHO classification. Direct subpleural invasion was seen in 42 cases, lymphangitic spread in 27 cases. Other less well‐defined intraparenchymal patterns included three sarcomatoid subtype malignant mesotheliomas exhibiting an intra‐alveolar growth pattern mimicking epithelioid haemangioendothelioma. One epithelioid subtype malignant mesothelioma contained an intraparenchymal tumour nodule microscopically comprising lepidic spread of neoplastic cells over maintained alveolar structures mimicking bronchioloalveolar carcinoma. One epithelioid subtype malignant mesothelioma morphologically had areas in which alveoli were distended by discohesive epithelioid neoplastic cells with no interstitial invasion. The appearances mimicked desquamative interstitial pneumonia. Immunohistochemistry played an important role in the definitive diagnosis of each unusual parenchymal tumour deposit. In 126 malignant mesotheliomas no invasion of the subjacent lung parenchyma was identified.

Conclusions:  An awareness of the unusual parenchymal growth pattern in malignant mesothelioma is important to prevent misdiagnosis of other entities. In the medico‐legal setting, the presence of epithelioid haemangioendothelioma or bronchioloalveolar carcinoma (in the absence of asbestosis) may be deemed to impact upon the patient's anticipated life expectancy and thereby would decrease the compensation settlement.
TCR‐α chain‐like molecule is involved in the mechanism of antigen‐non‐specific suppression of a ubiquitin‐like protein
Authors: Nakamura M. , Tsunematsu T. , Tanigawa Y.
Published: March 2003

Although existence of suppressor T cells is a controversial issue in cellular immunology, several lines of evidence indicate that T‐cell‐receptor α‐chain (TCR‐α) is a critical component of suppressor factors produced by these cells. Monoclonal non‐specific suppressor factor (MNSF), a lymphokine produced by murine T‐cell hybridoma, possesses pleiotrophic antigen‐non‐specific suppressive functions. Recently, we have shown that the 70 000‐MW MNSF comprises an 8000‐MW ubiquitin‐like polypeptide and other subunit(s). Here we report that the 8000‐MW ubiquitin homologue is associated with an intracellular TCR‐α (but not TCR‐β) ‐like molecule and released from the cells. The affinity eluates obtained from the culture supernatants of E17 cells and concanavalin A (Con A)‐activated splenocytes with anti‐TCR‐α monoclonal antibody (mAb) showed an antigen‐non‐specific, major histocompatibility complex (MHC)‐non‐restricted suppression. Immunoblot analysis demonstrated that anti‐TCR‐α, but not anti‐TCR‐β, mAb recognizes native 70 000‐MW MNSF. In addition, we found the dissociation of the 8000‐MW polypeptide from the 62 000‐MW TCR‐α cross‐reactive protein by hydrolase which cleaves isopeptide bonds. Thus the covalent attachment of ubiquitin‐like protein(s) may be involved in the underlying mechanism of suppressor T‐cells and TCR‐α‐like molecule(s) might be a main link between antigen‐specific and non‐specific suppression.

hNSF, human non‐specific suppressor factor MNSF, monoclonal non‐specific suppressor factor Ubi‐L, ubiquitin‐like moiety.
Inhibition of interleukin‐2 by a Gram‐positive bacterium, Streptococcus mutans
Authors: PLITNICK  , BANAS  , JELLEY‐GIBBS  , O’NEIL  , CHRISTIAN  , MUDZINSKI  , GOSSELIN
Published: March 2003

Generation of an effective cellular immune response is key to the successful development of both humoral and cellular immune defences against most pathogens. However, while the type of cellular immune response elicited by any given pathogen is dictated by the entire array of antigens and molecules which comprise that pathogen, most studies of human immune responses to bacterial pathogens tend to focus on selected antigens. This is a result, in part, of a desire to find those antigens that will generate a desired immune response, as well as limited technology for monitoring the complex array of responses generated by an intact organism. Utilizing Streptococcus mutans as a model Gram‐positive organism, a novel flow cytometric assay that permits the identification of individual cells within a responding population, and highly sensitive cytokine assays, we show for the first time that CD8 T cells and natural killer (NK) cells comprise a significant component of the response to this organism in humans. This is despite the fact that CD8 T cells are traditionally thought to respond to endogenously derived antigens only. In addition, we provide the first evidence that a Gram‐positive organism can actively inhibit interleukin‐2 (IL‐2), an important autocrine growth factor for T cells. The latter observation could represent an additional mechanism by which Gram‐positive organisms evade host defences.
Augmentation of natural killer cell activity in mice by oral administration of transforming growth factor‐β
Authors: ISHIZAKA  , KIMOTO  , KANDA  , SAITO
Published: March 2003

The latent form of transforming growth factor‐β (TGF‐β) in human milk and platelets was converted to the active form when conscious, pylorus‐ligated mice were given human milk and platelets by intragastric intubation. Oral administration of TGF‐β exerted enhancing effects on the natural killer (NK)‐cell activities in spleen and liver. Augmentation of NK‐cell activities in spleen was observed for 7 days after oral administration of TGF‐β. TGF‐β at concentrations of 5 and 20 ng produced the greatest augmentation of NK‐cell activities in spleen. However, NK‐cell activities in spleen were unaffected when TGF‐β was given intravenously. Interleukin (IL)‐12 production in spleen was enhanced by oral administration of TGF‐β, but not by intravenous administration of TGF‐β. These findings suggest that large amounts of TGF‐β in human milk are involved in early antiviral protection through the augmentation of NK‐cell activities.
Effect of the synthetic immunomodulator, Linomide, on experimental models of thyroiditis
Authors: HUTCHINGS  , HEDLUND  , DAWE  , HOWLETT  , COOKE
Published: March 2003

The drug Linomide is an immunomodulator showing marked down‐regulation of several experimental autoimmune diseases. In this study, its effect on three different experimental models of thyroid disease and on spontaneous infiltration of salivary glands (sialoadenitis), was investigated. Although very effective at preventing thyroid infiltrates in mice immunized with mouse thyroglobulin and complete Freund’s adjuvant and in spontaneous models of thyroiditis and sialoadenitis, it completely failed to modify experimental autoimmune thyroiditis (EAT) induced in mice immunized with mouse thyroglobulin and lipopolysaccharide. There was no significant shift in the observed isotypes of anti‐mouse thyroglobulin antibodies and only anti‐mouse thyroglobulin antibodies in the spontaneous model were completely down‐modulated by the drug. One surprising fact to emerge was that Linomide‐treated donor mice, although protected from thyroid lesions themselves, were still able to transfer EAT showing that they must have been effectively primed while being treated with Linomide. It is possible that the drug down modulated EAT by interfering with the trafficking of primed effector cells.
Dominant recognition of a cross‐reactive B‐cell epitope in Mycobacterium leprae 10 K antigen by immunoglobulin G1 antibodies across the disease spectrum in leprosy
Authors: HUSSAIN  , DOCKRELL  , CHIANG
Published: March 2003

Mycobacterium leprae‐specific immunoglobulin G1 (IgG1) antibodies in patients with leprosy show a direct correlation with bacterial load (ρ=0·748; P<0002) suggesting that IgG1 B‐cell responses may be surrogate markers of disease progression. To investigate if this upregulation was a general feature of IgG1 responses to all M. leprae (ML) antigens, we analysed responses to several recombinant purified ML heat‐shock proteins (HSP). Three recombinant HSPs (ML10 K, ML 18 K and ML 65 K) were tested for their ability to induce various IgG subclasses in patients with either the lepromatous (LL/BL, n=26) or tuberculoid form (BT/TT, n=39) of the disease as well as in healthy households (HC, n=14) and endemic controls (EC=19). Our major findings were: (1) selective augmentation of IgG1 antibody responses to ML10 K; (2) recognition of a restricted number of epitopes across the disease spectrum and healthy controls by IgG1 antibodies; (3) dominant recognition of cross‐reactive epitopes which were common to both ML and MT 10 K. This response was not related to contamination with endotoxin. Epitope mapping using 15‐mer overlapping peptides spanning the ML 10 000 MW revealed an immunodominant IgG1 binding peptide (aa41–55) in patients as well as healthy controls. This peptide is a shared epitope with M. tuberculosis 10 K suggesting that postswitched IgG1 B cells recognizing this epitope rather than naive B cells are being expanded.
Decomplementation with cobra venom factor prolongs survival of xenografted islets in a rat to mouse model
Authors: OBERHOLZER  , YU  , TRIPONEZ  , CRETIN  , ANDEREGGEN  , MENTHA  , WHITE  , BUEHLER  , MOREL  , LOU
Published: March 2003

Although the involvement of complement in hyperacute rejection of xenotransplants is well recognized, its role in rejection of devascularized xenografts, such as pancreatic islets, is not completely understood. In this study, we investigated whether complement participates in the immunopathology of xeno‐islet transplantation in a concordant rat to mouse model. Rat pancreatic islets were implanted under the kidney capsule of normal and cobra venom factor (CVF)‐decomplementized diabetic C57BL/6 mice. Graft survival was monitored by blood glucose levels. Deposition of IgM and C3 on grafted islets in vivo or on isolated islets in vitro (after incubation with normal and decomplementized mouse serum), as well as CD4‐ and CD8‐positive leucocyte infiltration of grafts, was checked by immunohistochemistry. In addition, complement‐mediated cytotoxicity on rat islet cells was evaluated by a 3‐(4,5‐dimethythiazolyl)‐2.5‐diphenyl‐2H‐tetrazolium‐bromide (MTT) assay. A significant C3 deposition was found on grafted islets from the first day after transplantation in vivo, as well as on isolated islets after incubation with mouse serum in vitro. By MTT assay, complement‐mediated cytotoxicity for islet cells was found. Decomplementation by CVF decreased C3 deposition on either isolated or grafted islets, delayed CD4‐ and CD8‐positive leucocyte infiltration, led to significant inhibition of complement‐mediated cytotoxicity for islet cells, and prolonged graft survival (mean survival time 21·3 versus 8·5 days; P<0·01). Our results indicate that decomplementation can prolong the survival time of devascularized xenografts across concordant species. The deposition of complement on transplanted islets may contribute to xenograft rejection by direct cytotoxicity and by promoting leucocyte infiltration.
Fyn and Lck tyrosine kinases regulate tyrosine phosphorylation of p105CasL, a member of the p130Cas docking protein family, in T‐cell receptor‐mediated signalling
Authors: KANDA  , MIMURA  , HAMASAKI  , YAMAMOTO  , YAZAKI  , HIRAI  , NOJIMA
Published: March 2003

We have previously shown that engagement of the T‐cell receptor (TCR)/CD3 complex with anti‐CD3 antibody induces tyrosine phosphorylation of p105CasL (CasL), a member of the p130Cas docking protein family. In the present work, we attempted to determine which protein tyrosine kinases (PTKs) regulate TCR‐mediated phosphorylation of CasL. We show here that an association between CasL and two types of Src family PTKs, Fyn and Lck, is induced by anti‐CD3 cross‐linking of human H9 T cells. In contrast, ZAP‐70, another PTK that also plays a critical role in the TCR signalling, failed to bind CasL, even after anti‐CD3 stimulation. In vitro kinase assays revealed that Fyn and Lck, but not ZAP‐70, were capable of phosphorylating CasL. Moreover, we found that CasL was constitutively hyperphosphorylated in vivo in splenocytes of MRL‐MP‐lpr/lpr mice, in which overproduction and excessive activation of Fyn and Lck have previously been shown to occur. Constitutive in vivo binding of CasL to both kinases was also demonstrated in lpr splenocytes. These results strongly suggest that CasL is a substrate for Fyn and Lck PTKs in TCR signal transduction.
Ileal and jejunal Peyer’s patches play distinct roles in mucosal immunity of sheep
Authors: Mutwiri  , Watts  , Lew  , Beskorwayne  , Papp  , Baca‐Estrada  , Griebel
Published: March 2003

The majority of pathogens enter the body through mucosal surfaces and it is now evident that mucosal immunity can provide effective disease protection. However, the induction of mucosal immunity will require efficient targeting of mucosal vaccines to appropriate mucosa‐associated lymphoid tissue. An animal model, based upon the surgical preparation of sterile intestinal ‘loops’ (blind‐ended segments of intestine), was developed to evaluate mucosal and systemic immune responses to enteric vaccines in ruminants. The effectiveness of end‐to‐end intestinal anastomoses was evaluated and fetal surgery did not disrupt normal intestinal function in lambs up to 6–7 months after birth. The immunological competence of Peyer’s patches (PP) within the intestinal ‘loops’ was evaluated with a human adenovirus 5 vector expressing the gD gene of bovine herpesvirus‐1. This vaccine vector induced both mucosal and systemic immune responses when injected into intestinal ‘loops’ of 5–6‐week‐old lambs. Antibodies to the gD protein were detected in the lumen of intestinal ‘loops’ and serum and PP lymphocytes proliferated in response to gD protein. The immune competence of ileal and jejunal PP was compared and these analyses confirmed that jejunal PP are an efficient site for the induction of mucosal immune responses. This was confirmed by the presence of gD‐specific antibody‐secreting cells in jejunal but not ileal PP. Systemic but not mucosal immune responses were detected when the vaccine vector was delivered to the ileal PP. In conclusion, this model provided an effective means to evaluate the immunogenicity of potential oral vaccines and to assess the immunological competence of ileal and jejunal Peyer’s patches.
Regulation of macrophage inflammatory protein‐2 gene expression by oxidative stress in rat alveolar macrophages
Authors: Shi  , Chong  , Godleski  , Paulauskis
Published: March 2003

Chemokines are important mediators in the development of inflammation. Our previous work demonstrated that an oxidative stress can up‐regulate mRNA expression of a CC chemokine macrophage inflammatory protein (MIP)‐1α in rat alveolar macrophages. In the present study, we further investigate whether an oxidative stress can regulate the gene expression of a related CXC chemokine MIP‐2, involved in both neutrophil chemotaxis and activation. A rat alveolar macrophage cell line (NR8383) was exposed to 10 μg/ml bacterial lipopolysaccharide (LPS) and MIP‐2 mRNA levels dramatically increased after 4 hr of stimulation. This increase by LPS was attenuated by co‐treatment with the antioxidants N‐acetylcysteine and dimethylsulphoxide, suggesting that the induction of MIP‐2 mRNA is mediated via the generation of reactive oxygen species. To assess directly the role of oxidative stress on regulation of MIP‐2 mRNA expression, macrophages were exposed to H2O2. MIP‐2 mRNA levels had significantly increased after 1 hr exposure to 0·5 mm H2O2, were maximally increased after 4 hr and decreased after 6 hr. Co‐treatment of macrophages with the transcriptional inhibitor actinomycin D eliminated the H2O2‐induction of MIP‐2 mRNA, implicating a role for transcriptional activation in increased expression of MIP‐2. Genomic cloning of the rat MIP‐2 gene 5′‐flanking region has identified a consensus nuclear factor‐κB (NF‐κB) binding site. Gel‐mobility shift assays revealed NF‐κB binding to the MIP‐2 promoter/enhancer sequence was induced by H2O2. LPS treatment for 4 hr also significantly activated NF‐κB binding, which could also be attenuated by pretreatment with N‐acetylcysteine at the doses that reduced MIP‐2 mRNA expression. The half‐life of MIP‐2 mRNA transcripts was also increased by H2O2 treatment. These observations indicate that MIP‐2 gene expression is subject to both transcriptional and post‐transcriptional control in response to an H2O2 oxidative stress.
Ly49A inhibitory receptors redistribute on natural killer cells during target cell interaction
Authors: Eriksson  , Ryan  , Nakamura  , Sentman
Published: March 2003

When T effector cells meet antigen‐bearing target cells, there is a specific accumulation of T‐cell receptors, co‐receptors and structural proteins at the point of cell–cell contact. Ly49 inhibitory receptors bind to murine major histocompatibility complex (MHC) class I molecules and prevent natural killer‐(NK) cell cytotoxicity. In this study we have tested whether inhibitory receptors accumulate at the point of cell–cell contact when NK cells encounter target cells bearing MHC class I ligands for those inhibitory receptors. We have used RNK‐16 effector cells that express Ly49A receptors and have found that there was a specific accumulation of Ly49A receptors at the point of NK cell–target cell contact when the target cells expressed H‐2Dd. We also observed that engagement of Ly49A on NK cells resulted in an altered redistribution of potential triggering receptors CD2 and NKR‐P1. These data indicate that inhibitory receptors, like activating receptors, may specifically aggregate at the point of cell–cell contact which may be necessary for them to mediate their full inhibitory effect.
Phenotypic identification of the subgroups of murine T‐cell receptor αβ+ CD4+ CD8− thymocytes and its implication in the late stage of thymocyte development
Authors: Ge  , Chen
Published: March 2003

Phenotypic analysis of the medullary‐type CD4+ CD8− [CD4 single‐positive (SP)] thymocytes has revealed phenotypic heterogeneity within this cell population. The characteristic phenotype of mature peripheral T cells can be uniquely marked as Qa‐2+ HSA− CD69−, whereas in the medullary‐type CD4 SP thymocytes, the expression patterns of many markers were quite different. This suggests that there are many subgroups in the population, which reflects that medullary‐type CD4 SP thymocytes may undergo phenotypic maturation. According to the results of two‐colour flow cytometry, seven discrete phenotypes were identified by the expression capacity of Qa‐2, HSA, CD69, 3G11 and 6C10 molecules. Consequently, the phenotypic precursor–progeny relationship can be envisaged as: 3G11− 6C10+ CD69+ HSAhi→3G11+ 6C10+ CD69+ HSAhi→ 3G11+ 6C10− CD69+ HSAint→3G11+ 6C10− CD69− HSAint Qa‐2−→3G11+ HSA−/lo Qa‐2lo. At the stage of 3G11+ 6C10− CD69− HSAint Qa‐2−, a branch pathway could be initiated, which gave rise to 3G11− HSAl°Qa‐2− cells, which then, in turn, developed into 3G11− HSA−/loQa‐2hi cells, a minor subgroup of the most mature CD4 SP cells. Consistent with this predicted pathway, experiments indicated that the first two subgroups were still cortisone sensitive, whereas the others were cortisone resistant. The cells in the last two Qa‐2‐positive subgroups are probably ready for emigration into the periphery.
Therapy of established tumour with a hybrid cellular vaccine generated by using granulocyte–macrophage colony‐stimulating factor genetically modified dendritic cells
Authors: Cao  , Zhang  , Wang  , Zhang  , Huang  , Hamada  , Chen
Published: March 2003

Dendritic cells (DCs) are the most powerful of all antigen‐presenting cells and play a critical role in the induction of primary immune responses. DC‐based vaccination represents a potentially powerful strategy for cancer immunotherapy. In this study, a new approach for a DC‐based melanoma vaccine was described. Splenic DCs from C57BL/6 mice were fused with B16 melanoma cells, and the resultant B16/DC hybrid cells expressed major histocompatibility complex (MHC) molecules – B7 as well as the B16 tumour marker M562 – which were enriched by Ia‐mediated positive selection with a MiniMACS column. The fusion rates were 12·7–26·8%. To generate hybrid tumour vaccines with potentially greater potent therapeutic efficacy, we genetically engineered DCs with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) prior to cell fusion. Recombinant adenovirus vector was used to mediate gene transfer into DCs with high efficiency and DCs expressed GM‐CSF at 96–138 ng/105 cells/ml 24 hr after GM‐CSF gene transfer. GM‐CSF gene‐modified DCs (DC.GM) exhibited higher expression of B7 and co‐stimulatory capacity in mixed lymphocyte reaction (MLR). Fusion of DC.GM with B16 cells generated B16/DC.GM hybrid cells secreting GM‐CSF at 59–63 ng/105 cells/ml. Immunization of C57BL/6 mice with the B16/DC hybrid vaccine elicited a specific cytotoxic T‐lymphocyte (CTL) response and protected the immunized mice from B16 tumour challenge, reduced pulmonary metastases and extended the survival of B16 tumour‐bearing mice. The B16/DC.GM hybrid vaccine was able to induce a CTL response and protective immunity more potently and tended to be therapeutically more efficacious than the B16/DC vaccine. In vivo depletion of T‐cell subsets demonstrated that both CD8+ and CD4+ T cells were essential for the therapeutic effects of B16/DC and B16/DC.GM hybrid vaccines. Additionally, other non‐specific effector cells may also contribute to tumour rejection induced by the B16/DC.GM hybrid vaccine. These data indicate that a DC‐based hybrid tumour vaccine may be an attractive strategy for cancer immunotherapy, and that GM‐CSF gene‐modified DCs may lead to the generation of hybrid vaccines with potentially increased therapeutic efficacy.
Activation sensitizes human memory B cells to B‐cell receptor‐induced apoptosis
Authors: Berard  , Casamayor‐Pallejà  , Billian  , Bella  , Mondière  , Defrance
Published: March 2003

The outcome of antigen receptor (B‐cell receptor; BCR) ligation on B‐cell survival can be influenced by multiple parameters. They are linked to the physical properties of the antigen itself, the maturational stage of the cells and the costimuli provided by different components of the innate and acquired immunity. Here we report that apoptosis prevails over stimulation when a BCR agonist is applied to human memory B cells which have been preactivated by CD40 ligand or anti‐immunoglobulin antibodies. The susceptibility of activated memory B cells to BCR‐induced killing is correlated with their enhanced expression of the transcripts encoding the pro‐apoptotic molecules Bax, c‐Myc and p53. The BCR‐mediated apoptosis of activated memory B cells does not require extensive cross‐linking of the antigen receptors and relies neither on engagement of the FcγRII nor on the Fas/Fas ligand (Fas‐L) system. Our findings suggest that activation stimuli open the BCR‐induced apoptotic pathway in memory B cells. Therefore we propose that the concept of activation‐induced cell death (AICD), originally described for T cells, also applies to mature B lymphocytes. The functions fulfilled by the AICD of mature B cells in the regulation of B‐cell responses are discussed.
Characteristics of IgVH genes used by human intestinal plasma cells from childhood
Authors: Boursier  , Dunn‐Walters  , Spencer
Published: March 2003

Plasma cells secreting immunoglobulin M (IgM) and IgA in human intestinal mucosa are the largest antibody‐producing population in the human body. Despite this there have been relatively few studies of the characteristics and maturation of the genes which encode the mucosal immunoglobulins. We have previously demonstrated that intestinal plasma cells use highly mutated IgVH genes, likely to reflect germinal centre origin. Here we show that IgVH genes used by intestinal lamina propria plasma cells secreting IgM and IgA are highly mutated from childhood, with no change in the frequency of mutation through to adulthood, though IgVH genes used by IgM are significantly less mutated than those used by IgA. There was no difference between the IgA subclasses in either the frequency or distribution of mutations. The frequency of mutation in IgVH4–34 genes used by IgG was also studied in the adult biopsies, and was found to be of the same order as that observed in IgA and was significantly higher than that observed in IgM. We have identified IgM and IgA sequences which share identical CDR3 and distribution of mutations. Isotype switching may therefore occur after extensive mutation of IgM sequences, and IgM‐ and IgA‐secreting plasma cells with the same specificity may occur within the same microenvironment. IgM should therefore be considered to be a component of secondary immune responses in the gut.
ATP‐induced Ca2+ response mediated by P2U and P2Y purinoceptors in human macrophages: signalling from dying cells to macrophages
Authors: Oshimi  , Miyazaki  , Oda
Published: March 2003

The activation of macrophages by various stimuli leading to chemotactic migration and phagocytosis is known to be mediated by an increase in intracellular Ca2+ concentration ([Ca2+]i). We measured changes in [Ca2+]i using a Ca2+ imaging method in individual human macrophages differentiated from freshly prepared peripheral blood monocytes during culture of 1–2 days. A transient rise in [Ca2+]i (duration 3–4 min) occurred in 10–15 macrophages in the vicinity of a single tumour cell that was attacked and permeabilized by a natural killer cell in a dish. Similar Ca2+ transients were produced in 90% of macrophages by application of supernatant obtained after inducing the lysis of tumour cells with hypo‐osmotic treatment. Ca2+ transients were also evoked by ATP in a dose‐dependent manner between 0·1 and 100 μm. The ATP‐induced [Ca2+]i rise was reduced to less than one‐quarter in Ca2+‐free medium, indicating that it is mainly due to Ca2+ entry and partly due to intracellular Ca2+ release. UTP (P2U purinoceptor agonist) was more potent than ATP or 2‐chloro‐ATP (P2Y agonist). Oxidized ATP (P2Z antagonist) had no inhibitory effect. Both cell lysate‐ and ATP‐induced Ca2+ responses were inhibited by Reactive Blue 2 (P2Y and P2U antagonist) to the same extent, but were not affected by PPADS (P2X antagonist). Sequential stimuli by cell lysate and ATP underwent long‐lasting desensitization in the Ca2+ response to the second stimulation. The present study supports the view that macrophages respond to signal messengers discharged from damaged or dying cells to be ingested, and ATP is at least one of the messengers and causes a [Ca2+]i rise via P2U and P2Y receptors.
Differential modes of regulation of interleukin‐1β expression by extracellular matrices
Authors: Roman  , Ritzenthaler  , Perez  , Roser
Published: March 2003

Extracellular matrices (ECMs) can stimulate human monocytic cells to express interleukin‐1β (IL‐1β), a proinflammatory cytokine implicated in the regulation of tissue inflammation. In this study, we explored the intracellular mechanisms responsible for ECM induction of IL‐1β using human promonocytic U937 cells transfected with the full‐length human IL‐1β gene promoter connected to a reporter gene. Using this system, we demonstrated that the ECM molecules fibronectin (FN), type I collagen (Coll), fibrin (Fb) and laminin (Lm) induced transcription of the IL‐1β gene (which was associated with a modest increase in IL‐1β protein secretion) in suspended cells, when used in their soluble monomeric form. This effect was mimicked or blocked by anti‐integrin monoclonal antibodies (mAbs) and was dependent on the activation of protein kinase C (PKC). Three of the ECMs tested (FN, Coll and Fb) induced the activation of mitogen‐activated protein kinases (MAPKs), whereas Lm had no effect. FN, Coll and Fb (but not Lm) also induced DNA binding of the transcription factor activator protein‐1 (AP‐1), but not that of nuclear factor‐κB. Co‐transfection of U937 cells with a competing AP‐1 oligomer blocked the IL‐1β response induced by FN, but not that induced by the other ECMs. By inhibiting AP‐1 translocation, glucocorticoids also blocked the FN‐induced response, but not that of the other ECMs. These studies suggest that the signalling pathways which mediate ECM induction of IL‐1β expression in human monocytic cells converge at the level of PKC activation. However, they diverge in other aspects, as demonstrated by the differential activation of MAPKs and the need for diverse transcription factors.
Intermediate stages in monocyte–macrophage differentiation modulate phenotype and susceptibility to virus infection
Authors: Mccullough  , Basta  , Knötig  , Gerber  , Schaffner  , Kim  , Saalmüller  , Summerfield
Published: March 2003

The kinetics of monocyte–macrophage differentiation was analysed using two Swine Workshop Cluster (SWC) CD molecules: SWC1 and SWC9. Myeloid cells were selected by labelling for the common myeloid antigen, SWC3. Confirmation of macrophage identification used acid phosphatase and phagocytosis activities. During differentiation, SWC1 was gradually lost. SWC9 was absent on monocytes but up‐regulated early. Consequently, monocytes were SWC1+ SWC9− and macrophages were SWC1− SWC9+. An additional, intermediate, cell population was identified as SWC1+ SWC9+. Size and granularity characteristics mirrored the monocyte, macrophage and intermediate‐cell phenotypes. Overall, SWC9 up‐regulation was central in macrophage differentiation and dependent on plasma factors. The concomitant loss of SWC1 was independent of these factors, but always associated with mature macrophages. Upon up‐regulation of SWC9, the SWC1+ SWC9+ intermediate monocytic cells became susceptible to African swine fever virus infection. These results demonstrate the heterogeneity of monocytic cell differentiation and the importance of these characteristics for interaction with monocytotropic viruses.
Analysis of immunoglobulin E VH transcripts in a bronchial biopsy of an asthmatic patient confirms bias towards VH5, and indicates local clonal expansion, somatic mutation and isotype switch events
Authors: Snow R. E. , Djukanovic R. , Stevenson F. K.
Published: March 2003

Immunoglobulin E (IgE)‐dependent mechanisms play a pivotal role in mediating allergic disease. Previously, VH‐Cε transcripts from blood or spleen of atopic asthmatics have been analysed for VH gene usage and patterns of somatic mutation. An over‐representation of the minor VH5 family has been observed, consistent with a superantigen drive. As local mucosal events in IgE production may be more significant in the disease process, we have analysed VH‐Cε transcripts from a bronchial biopsy of a patient with severe asthma. VH5 predominance was confirmed with 10 of 30 unique clones derived from this family. Repeated sequences, some with intraclonal variation, revealed clonal expansion and continuing mutational activity at the site. Unexpectedly, three unmutated VH‐Cε sequences were found, indicating that isotype switching to IgE can occur without mutation. Detection of a sister clone with extensive mutations was again consistent with local mutational activity. Evidence for local isotype switching was obtained by identification of clonally related immunoglobulin M (IgM), immunoglobulin G (IgG) and immunoglobulin E (IgE) sequences. However, in contrast to findings in blood, no IgG4 transcripts clonally related to IgE were detected, suggesting that the balance between synthesis of IgG4 and IgE may differ between systemic and local sites. These data confirm a VH5 bias in IgE, and support the concept that IgE‐synthesizing B cells arise via local differentiation.
Dimerization of major histocompatibility complex class I on the surface of THP‐1 cells stimulates the expression of inducible nitric oxide synthase and subsequent nitric oxide release
Authors: Bottley G. , Fernández N.
Published: March 2003

We show that dimerization of major histocompatibility complex (MHC) class I on a human monocytic cell line, THP‐1, induces nitric oxide (NO) synthesis. Cells cultured in the presence of a human MHC class I‐specific monoclonal antibody produced significant amounts of NO after 72 hr. Reverse transcription–polymerase chain reaction and flow cytometry analysis revealed that the cells synthesized detectable levels of inducible NO synthase mRNA and protein. These effects were not seen after treatment with monovalent Fab fragments or Fc fragments of the same antibody, or after treatment with a control antibody. These data show a link between innate and acquired immune mechanisms mediated by NO and MHC class I.
Cyclooxygenase‐independent inhibition of dendritic cell maturation by aspirin
Authors: Matasić R. , Dietz A. B. , Vuk‐Pavlović S.
Published: March 2003

When immature human myeloid dendritic cells were differentiated in vitro in the presence of aspirin, they were unable to stimulate T‐cell proliferation. Aspirin and its major metabolite salicylate changed the surface marker phenotype of dendritic cells. The drugs particularly suppressed the levels of CD83 and the secreted p40 unit of interleukin‐12 (IL‐12), both markers of mature dendritic cells; 50% inhibitory concentration (IC50) values were 2·5 m
m, a concentration more than 100 times greater than the concentration at mid‐point inhibition (ID50) value for inhibition of prostaglandin synthesis. Concomitantly, the levels of CD14, a marker of monocytes/macrophages, increased above the levels found in immature dendritic cells. Cyclooxygenase inhibitors ketoprofen, indomethacin and NS‐398 had no effect at concentrations more than a thousand‐fold higher than their IC50 values. The effects were independent of the presence of prostaglandin E2 in the medium. Salicylates suppressed activation of the nuclear transcription factor κB, which regulates dendritic cell differentiation, but their effects on mature dendritic cells were negligible. Hence, aspirin inhibits dendritic cell function by inhibiting their terminal differentiation at concentrations achieved in the blood of patients chronically treated with high‐dose aspirin.
The contribution of both oxygen and nitrogen intermediates to the intracellular killing mechanisms of C1q‐opsonized Listeria monocytogenes by the macrophage‐like IC‐21 cell line
Authors: Álvarez‐Domínguez C. , Carrasco‐Marín E. , López‐Mato P. , Leyva‐Cobián F.
Published: March 2003

Listeria monocytogenes is a facultative intracellular pathogen which is internalized by host mammalian cells upon binding to their surface. Further listerial growth occurs in the cytosol after escape from the phagosomal–endosomal compartment. We have previously reported that C1q is able to potentiate L. monocytogenes phagocytosis upon bacterial opsonization by ingestion through C1q‐binding structures. In this report, we analysed the post‐phagocytic events upon internalization of C1q‐opsonized L. monocytogenes and found an induction of macrophage (Mφ)‐like IC‐21 cell bactericidal mechanisms displayed by the production of oxygen and nitrogen metabolites. Both types of molecules are effective in L. monocytogenes killing. Further analysis of the cellular responses promoted by interaction of C1q with its surface binding structures, leads us to consider C1q as a collaborative molecule involved in Mφ activation. Upon interaction with surface binding structures, C1q was able to trigger and/or amplify the production of reactive oxygen and nitrogen intermediates induced by stimuli such as interferon‐γ and L. monocytogenes phagocytosis.
M‐ficolin is expressed on monocytes and is a lectin binding to N‐acetyl‐	d‐glucosamine and mediates monocyte adhesion and phagocytosis of Escherichia coli
Authors: Teh C. , Le Y. , Lee S. H. , Lu J.
Published: March 2003

Ficolins are a group of multimeric proteins that contain collagen‐like and fibrinogen‐like (FBG) sequences. Three types of ficolins have been characterized: H‐, L‐ and M‐ficolins. Both H‐ and L‐ficolins have demonstrated lectin activities. In the present study, the FBG domain of M‐ficolin was expressed and shown to bind to N‐acetyl‐
d‐glucosamine. M‐ficolin mRNA was expressed in monocytes but not in the more differentiated macrophages and dendritic cells. By flow cytometry, surface biotinylation and immunoprecipitation, we showed that M‐ficolin was associated with the surface of promonocytic U937 cells. M‐ficolin transiently expressed in COS‐7 cells was also clearly detected on the cell surface by immunoprecipitation. By flow cytometry, M‐ficolin was detected on peripheral blood monocytes but not on lymphocytes or granulocytes. Immobilized rabbit anti‐M‐ficolin F(ab′)2 mediated U937 cell adhesion, and the antibody also inhibited phagocytosis of Escherichia coli K‐12 by U937 cells. Therefore, M‐ficolin might act as a phagocytic receptor or adaptor on circulating monocytes for micro‐organism recognition and may potentially mediate monocyte adhesion.
Modulation of CD11C+ splenic dendritic cell functions in murine visceral leishmaniasis: correlation with parasite replication in the spleen
Authors: Basu A. , Chakrabarti G. , Saha A. , Bandyopadhyay S.
Published: March 2003

BALB/c mice resolve Leishmania donovani infection in the liver over an 8–12‐week period. However, after an initial phase of 2–4 weeks where increases in parasite load are not readily detectable, parasite numbers in the spleen begin to increase reaching maximum levels at 16 weeks post‐infection. Thereafter, parasite replication in the spleen is controlled and BALB/c mice maintain this residual parasite load in the spleen for many months, without further increase. We evaluated functions of CD11C+ splenic dendritic cells throughout the course of L. donovani infection in the spleen of BALB/c mice. Unlike the dendritic cell (DC)‐specific antigen DEC‐205, CD11C was not up‐regulated on macrophages during visceral leishmaniasis. No appreciable impairment of splenic DC functions was observed when this antigen‐presenting cell subset was purified from 30‐day post‐infected mice. Significant impairment in inducing allogeneic mixed lymphocyte reaction (MLR) and presenting L. donovani antigens or keyhole limpet haemocyanin (KLH) to specific T cells was observed with CD11C+ splenic DC purified from 60‐day post‐infected mice. Functional impairment of splenic DC at 60 days post‐infection correlated with their reduced surface expression of major histocompatibility complex (MHC) class II molecules, impairment of interleukin‐12 (IL‐12) production and to their ability to suppress interferon‐γ (IFN‐γ) production by Leishmania antigen‐primed T cells. Of interest, the impairment of splenic DC in presenting Leishmania antigens or KLH to specific T cells was corrected at 120 days post‐infection, and correlated with their up‐regulation of MHC class II expression, IL‐12 production, induction of IFN‐γ by Leishmania antigen‐primed T cells and the onset of control over splenic parasite replication in vivo. These results indicate that functional integrity of DC may be important in controlling L. donovani infection.
Production and functional analysis of rat CD59 and chimeric CD59‐Crry as active soluble proteins in Pichia pastoris
Authors: Quigg R. J. , He C. , Hack B. K. , Alexander J. J. , Morgan B. P.
Published: March 2003

Crry (CR1‐related gene/protein) is a rodent complement regulator that inhibits C3 convertases. CD59 is a conserved protein inhibitor active towards C8 and C9. We have previously produced rat Crry as a recombinant soluble (rs) protein in Pichia pastoris. In this study we produced functionally active rat rsCD59 and a chimeric rsCD59‐Crry protein in P. pastoris. The GPI anchor addition site of rat CD59 (Asn‐79) was replaced either by a stop codon to produce rsCD59, or with the sequence of the first five short consensus repeats of Crry to produce rsCD59‐Crry. Proteins were generated by fermentation and purified by affinity chromatography on an anti‐CD59 column. In a standard classical pathway haemolysis assay, all three rs proteins had inhibitory activity, with 50% inhibition at 0·5 µm (rsCrry and rsCD59‐Crry) and 4·4 µm (rsCD59). In an assay examining inhibition of C5b‐9, in which C5b‐7 was first formed, followed by purified C8 and C9, rsCD59 and rsCD59‐Crry were active with 50% inhibition at 0·8 µm (rsCD59‐Crry) and 1·3 µm (rsCD59). The degree of inhibition was independent of whether the C8 and C9 were of rat or human origin. Therefore, we have produced rsCD59 and rsCD59‐Crry in P. pastoris. The rsCD59 retains its inhibitory activity towards C5b‐9, while rsCD59‐Crry appears to have the combined activities of Crry and CD59. In a haemolytic assay, the inclusion of CD59 to Crry is of no additional benefit to Crry, which may illustrate the overall importance of the C3 convertase step. Yet, inclusion of Crry to CD59 increases the potency of CD59 towards C5b‐9.
CXC chemokine receptor 4 expression and stromal cell‐derived factor‐1α‐induced chemotaxis in CD4+ T lymphocytes are regulated by interleukin‐4 and interleukin‐10
Authors: Jinquan T. , Quan S. , Jacobi H. H. , Madsen H. O. , Glue C. , Skov P. S. , Malling H.‐J. , Poulsen L. K.
Published: March 2003

We report that interleukin (IL)‐4 and IL‐10 can significantly up‐ or down‐regulate CXC chemokine receptor 4 (CXCR4) expression on CD4+ T lymphocytes, respectively. Stromal cell‐derived factor‐1α (SDF‐1α)‐induced CD4+ T‐lymphocyte chemotaxis was also correspondingly regulated by IL‐4 and IL‐10. IL‐4 and IL‐10 up‐ or down‐regulated CXCR4 mRNA expression in CD4+ T lymphocytes, respectively, as detected by real‐time quantitative reverse transcription–polymerase chain reaction (RT–PCR). Scatchard analysis revealed a type of CXCR4 with affinity (Kd≈ 6·3 n
m), and ≈ 70 000 SDF‐1α‐binding sites per cell, among freshly isolated CD4+ T lymphocytes, and two types of CXCR4 with different affinities (Kd1≈ 4·4 n
m and Kd2≈ 14·6 n
m), and a total of ≈ 130 000 SDF‐1α‐binding sites per cell, among IL‐4‐stimulated CD4+ T lymphocytes. The regulation of CXCR4 expression in CD4+ T lymphocytes by IL‐4 and IL‐10 could be blocked by a selective inhibitor of protein kinase (staurosporine) or by a selective inhibitor of cAMP‐ and cGMP‐dependent protein kinase (H‐8), indicating that these cytokines regulate CXCR4 on CD4+ T lymphocytes via both cAMP and cGMP signalling pathways. The fact that cyclosporin A or ionomycin were able to independently change the CXCR4 expression and block the effects of IL‐4 and IL‐10 on CXCR4 expression implied that the capacity of IL‐4 and IL‐10 to regulate CXCR4 on CD4+ T lymphocytes is not linked to calcium‐mobilization stimulation. These results indicate that the effects of IL‐4 and IL‐10 on the CXCR4–SDF‐1 receptor–ligand pair may be of particular importance in the cytokine/chemokine environment concerning the inflammatory processes and in the progression of human immunodeficiency virus (HIV) infection.
Mycobacterium tuberculosis‐activated dendritic cells induce protective immunityin mice
Authors: Tascon R. E. , Soares C. S. , Ragno S. , Stavropoulos E. , Hirst E. M. A. , Colston M. J.
Published: March 2003

Activated dendritic cells are critically important in the priming of T‐cell responses. In this report we show that the infection of a conditionally immortalized dendritic cell line (tsDC) with Mycobacterium tuberculosis resulted in the up‐regulation of B7‐1 and B7‐2 co‐stimulatory molecules and the induction of several inflammatory cytokines, including tumour necrosis factor‐α and interleukin‐6, ‐1β and ‐12. In addition, we show that these activated dendritic cells were capable of eliciting antigen‐specific T‐cell responses and potent anti‐mycobacterial protective immunity in a murine model of experimental tuberculosis infection.