bluuebunny/crossref_metadata_2025 huggingface dataset:
Combined dermatofibroma: co‐existence of two or more variant patterns in a single lesion
Authors: Zelger B G , Sidoroff A , Zelger B
Published: March 2003

Aims: Based on a series of 25 cases, we define and characterize combined dermatofibroma, a tumour comprising two or more variant patterns of dermatofibroma in a single lesion.

Method and results

Dermatofibroma may present with a wide variety of architectural, cellular or stromal peculiarities. Architectural peculiarities include deep penetration, atrophy, collarette formation, fascicular to plexiform architecture, massive haemorrhage, prominent haemangiopericytoma‐like vascularity and palisading; cellular peculiarities the presence of epithelioid cells, clear cells, granular cells, prominent myofibroblastic differentiation and atypical giant cells (‘monster cells’); or stromal peculiarities such as prominent sclerosis, mucin, haemosiderin and cholesterotic deposits. In combined dermatofibromas two or more of these features are seen in complex or inhomogenous combination such as the silhouette of a deep penetrating dermatofibroma with an ‘ordinary’ storiform pattern in the upper and granular cell differentiation in the lower part of the lesion; or a dermatofibroma with ordinary features in the upper, prominent sclerosis in the middle and clear cells in the lower portion of the lesion; or the characteristic epidermal collarette and cells of epithelial cell histiocytoma with a plexiform (‘neurothekeoma‐like’) architecture surrounded by a myxoid stroma with spindle‐shaped to stellate cells. Clinically, these lesions preferentially occur on the lower extremities of young to middle‐aged females, frequently with the diagnosis of a fibrohistiocytic lesion. Apart from one recurrence follow‐up was uneventful in all other cases. Immunohistochemically, lesions are consistently positive with KiM1p, variably positive for factor XIIIa, smooth muscle specific actin and with KP1 (CD68), NK1C3 and E9.

Conclusion

Recognition of combined dermatofibroma allows the histopathologist to apply a confident benign label to unusual lesions which might otherwise elude diagnosis, or tempt description of ‘new’ entities and to avoid a misdiagnosis of malignancy.
Poor agreement in recognition of abnormal mitoses: requirement for standardized and robust definitions
Authors: Barry M , Sinha S K , Leader M B , Kay E W
Published: March 2003

The finding of abnormal mitoses is a helpful feature in differentiating between benign and malignant neoplasia and has prognostic significance for some tumours. As the use of a histopathological variable is limited by the reproducibility of its recognition, we tested the interobserver agreement in the classification of abnormal mitoses among histopathologists.

Ten practising histopathologists were shown 30 potential mitotic figures and were asked to classify these as ‘normal mitoses’, ‘abnormal mitoses’ or ‘not mitoses’ according to the criteria each pathologist used in their routine practice. The results were analysed using kappa statistics. Overall agreement was only fair with a combined kappa of 0.31 and there was unanimous categorization of only four of 30 test items, none of which was called abnormal. The poorest result was obtained for the category ‘abnormal mitosis’ with only slight agreement (kappa 0.19). Agreement for the other categories varied from moderate (kappa = 0.45) for ‘not a mitosis’ to fair (kappa = 0.26) for ‘normal mitosis’. Comparison of the results for observer pairs showed that for 12 out of the 45 possible pairings, there was no more agreement than might be expected by chance alone.

Agreement is poor among practising histopathologists in the recognition of abnormal mitoses. A standardized and robust definition is needed if diagnostic and prognostic significance is accorded to the finding of an abnormal mitosis in the context of neoplasia.
Angiogenesis is redundant for tumour growth in lymph node metastases
Authors: Naresh K N , Nerurkar A Y , Borges A M
Published: March 2003

Angiogenesis is redundant for tumour growth in lymph node metastases

Aims: Angiogenesis is essential for the growth of solid tumours. As the role of angiogenesis is unclear in the pathogenesis of primary lymph node (LN) tumours, we wondered whether neoangiogenesis was important in supporting and promoting the growth of tumours in LNs.

Methods and results: We investigated 16 cases of squamous carcinoma involving oral cavity (n=9) and larynx/pyriform fossa (n=7), all of whom had lymph node metastases. Sections of the primary tumour, uninvolved mucosa, metastatic LN and nonmetastatic LN were double‐immunostained with factor VIII‐related antigen and MIB‐1. Proliferating blood vessels, i.e. neoangiogenesis, was identified by coexpression of factor VIII‐related antigen‐stained blood vessels and MIB‐1 staining of the endothelial cell nuclei. Counts were performed in an area of 4 mm2. Primary tumours (127–188.7), uninvolved mucosa (54–84.5) and metastatic LNs (123.5–167) had significantly lower vessel counts than nonmetastatic LNs (194–253.9) (P=0.003; P < 0.001; P < 0.001, respectively). With regard to neoangiogenesis, primary tumours (1.7–5) had significantly higher counts than uninvolved mucosa (0–0.4), nonmetastatic LNs (0–0.4) and metastatic LNs (0.9–2.4) (P < 0.001; P < 0.001; P=0.047, respectively).

Conclusions: Our data suggest that because of the rich native vascularity of lymph nodes, neoangiogenesis is redundant for the growth of metastatic tumour.
p27 immunostaining is related to prognosis in malignant mesothelioma
Authors: Beer T W , Shepherd P , Pullinger N C
Published: March 2003

p27 immunostaining is related to prognosis in malignant mesothelioma

Aims: To evaluate the prognostic value of the proliferation‐associated antigen p27 in malignant mesothelioma.

Methods and results: Sections from 36 patients with malignant mesothelioma were immunohistochemically stained for the p27 antigen. The results were quantified by recording the proportion of positively stained nuclei and these data were tested for association with patient survival. p27 expression was also compared with tumour type, MIB‐1 expression, apoptotic and mitotic indices. Patients with pleural malignant mesothelioma showing low p27 expression (< 53% of cells positive) survived for significantly shorter periods of time than those with high p27 expression (≥ 53% of cells positive) (P=0.04). The 50% survival time of subjects with low p27 was 4–6 months whereas for those with high p27 expression it was 10–11 months. There was no significant association between p27 expression and MIB‐1, apoptotic or mitotic indices.

Conclusions: Low p27 expression in pleural malignant mesothelioma is associated with a significantly worse prognosis. p27 appears to be an independent prognostic variable which may be of value in counselling patients with malignant mesothelioma.
Spindle cell squamous carcinoma of the oesophagus: an analysis of 17 cases, with new immunohistochemical evidence for a clonal origin
Authors: Handra‐Luca A , Terris B , Couvelard A , Molas G , Degott C , Flejou J‐F
Published: March 2003

Spindle cell squamous carcinoma of the oesophagus: an analysis of 17 cases, with new immunohistochemical evidence for a clonal origin

Aims: To study the morphological and immunohistochemical characteristics of spindle cell squamous carcinoma of the oesophagus, in order better to understand the histogenesis of this tumour.

Methods and results: In this study we analysed the morphological and immunohistochemical characteristics of 17 cases of spindle cell squamous carcinoma of the oesophagus. Most tumours were polypoid, but tumours with an ulcerated and infiltrative pattern were also observed. Histologically, most tumours were of superficial type, with a characteristic morphological aspect consisting of two types of tumour cells, i.e. differentiated squamous cells, and spindle cells with transition zones between the two components. On immunohistochemistry, the squamous cells were positive for cytokeratin and the spindle cells showed variable expression of cytokeratin, vimentin and smooth muscle actin. p53 protein was over‐expressed in 10 cases, both tumour cell types showing strong nuclear positivity. In most tumours, E‐cadherin was expressed in the squamous cells and absent in the spindle cells.

Conclusions: The similar pattern of p53 protein expression in the two tumour cell types of spindle cell squamous carcinoma of the oesophagus suggests their common origin. The change in adhesion molecule expression with loss of E‐cadherin expression may be associated with the acquisition of spindle cell morphology by the squamous tumour cells.
Malignant peripheral nerve sheath tumours: high Ki67 labelling index is the significant prognostic indicator
Authors: Watanabe T , Oda Y , Tamiya S , Kinukawa N , Masuda K , Tsuneyoshi M
Published: March 2003

Malignant peripheral nerve sheath tumours: high Ki67 labelling index is the significant prognostic indicator

Aims: We investigated p53, Ki67, MDM2, and p21WAF1/CIP1 in order to evaluate its relationship with prognosis in malignant peripheral nerve sheath tumours (MPNST).

Methods and results: In 49 cases of MPNSTs, the immunohistochemical studies of Ki67, p53, MDM2, p21WAF1/CIP1 and polymerase chain reaction single‐strand conformation polymorphism (PCR‐SSCP) with direct sequencing of p53 were performed with the formalin‐fixed paraffin‐embedded tissue. In 43 cases with survival data available, an evaluation of the prognostic significance of clinicopathological factors was also carried out. A high Ki67 labelling index (LI) (>25%) was correlated with a reduced survival rate in the 43 cases of MPNST (P=0.0106, log‐rank test). Furthermore, there was a significant correlation between the Ki67 LI and the immunohistochemical expression of p53 or MDM2. In 17 MPNST cases, PCR amplification of exons 5 through 8 of the p53 gene was successful. One case showed a base change of codon 240 (AGT→AGC), but translated amino acid (Ser) remained unchanged. Multivariate Cox analysis of our series showed that the association of von Recklinghausen’s disease, tumour depth, and the presence of rhabdomyoblasts (malignant triton tumour) each had an independent negative impact on overall survival.

Conclusion: High Ki67 LI (>25%) was of significant prognostic value in MPNST.
Evidence for local immunosuppression and demonstration of c‐myc amplification in pyothorax‐associated lymphoma
Authors: Yamato H , Ohshima K , Suzumiya J , Kikuchi M
Published: March 2003

Evidence for local immunosuppression and demonstration of c‐myc amplification in pyothorax‐associated lymphoma

Aims: Pyothorax‐associated lymphoma (PAL) develops in the pleural cavity of patients with a long history of pyothorax. Epstein–Barr virus (EBV) is also involved in PAL, similar to lymphomas in immunodeficient patients. Here we examined T‐lymphocyte subsets as well as c‐myc and REL gene amplification in PAL tissues.

Methods and results: We determined the number and distribution of CD4+ and CD8+ T‐lymphocytes, to evaluate T‐cells in the host immune reaction in seven cases of PAL. As controls, we also studied 10 cases of extranodal diffuse large B‐cell lymphoma (DLBL) and 10 cases of nodal DLBL. Chromosomal imbalances in PAL were determined by using comparative genomic hybridization (CGH) analysis. The mean numbers of CD4+ and CD8+ and their ratio were significantly lower in PAL than in nodal DLBL. CGH analysis of PAL showed amplification of the 8q24 chromosomal region. In addition, c‐myc amplification was found in four cases of PAL by Southern blot analysis.

Conclusions: Our results suggest that the development of PAL may involve a local immunosuppressive environment and that amplification of c‐myc might promote tumour progression, as has been described in the development of Burkitt’s lymphoma.
Immunohistochemical localization of CEA, CA19‐9 and DU‐PAN‐2 in hepatitis C virus‐infected liver tissues
Authors: Kitagawa Y , Iwai M , Muramatsu A , Tanaka S , Mori T , Harada Y , Okanoue T , Kashima K
Published: March 2003

Immunohistochemical localization of CEA, CA19‐9 and DU‐PAN‐2 in hepatitis C virus‐infected liver tissues

Aims: We investigated expression of CEA, CA19‐9 and DU‐PAN‐2 in liver tissues of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma, measuring their serum value to clarify their clinical significance, and proliferating cell nuclear antigen (PCNA) was assessed in serial sections to determine whether expression of these molecules in chronic liver disease was related to regeneration of biliary ducts.

Methods and results: Liver tissues were biopsied under peritoneoscopy or echo‐guidance and resected surgically among 63 patients with anti‐hepatitis C virus‐positive sera. There were 26 cases of chronic hepatitis, 21 cases of liver cirrhosis and 16 cases of hepatocellular carcinoma (four cases of mixed type). They were simultaneously used for immunocytochemistry for CEA, CA19‐9 and DU‐PAN‐2, and PCNA was demonstrated in serial liver tissues by immunohistochemistry. Serum CEA, CA19‐9 and DU‐PAN‐2 were measured by radioimmunoassay or enzyme immunoassay. In chronic hepatitis and liver cirrhosis CEA immunoreactivity was seen on membranes facing bile canaliculi and in bile ductules. Both CA19‐9 and DU‐PAN‐2 immunoreactivity were observed in bile ductules in chronic hepatitis liver cirrhosis and non‐neoplastic areas surrounding hepatocellular carcinoma, and CA19‐9 was also present in interlobular bile ducts. PCNA immunoreactivity was not detected in marker‐positive bile ductules or interlobular bile ducts. In hepatocellular carcinoma CEA immunoreactivity was seen on membrane facing dilated bile canaliculi in glandular structures, and CEA, CA19‐9 and DU‐PAN‐2 immunoreactivity was observed in cholangiolar areas in mixed type of hepatocellular carcinoma.

Conclusions: CEA in chronic hepatitis and liver cirrhosis is expressed not only in bile ductules but also on membrane facing bile canaliculi, and both CA19‐9 and DU‐PAN‐2 were seen in different levels of biliary ducts. These molecules were expressed in bile ductules in surrounding non‐neoplastic areas of hepatocellular carcinoma, and their expression was not associated with regeneration of biliary ducts. CEA expression was present in the trabecular type of hepatocellular carcinoma, and CA19‐9 and DU‐PAN‐2 were observed in cholangiolar areas in mixed type of hepatocellular carcinoma.
Abnormal α‐catenin expression in invasive breast cancer correlates with poor patient survival
Authors: Nakopoulou L , Gakiopoulou‐Givalou H , Karayiannakis A J , Giannopoulou I , Keramopoulos A , Davaris P , Pignatelli M
Published: March 2003

Abnormal α‐catenin expression in invasive breast cancer correlates with poor patient survival

Aims: α‐Catenin is a member of the E‐cadherin‐catenin family of adhesion molecules whose role is essential for the function of the E‐cadherin complex. In this study, we have evaluated the expression of α‐catenin but also of the other catenins (β‐, γ‐ and p120‐catenin) and E‐cadherin in invasive breast cancer and statistically analysed these expressions with known clinicopathological parameters, c‐erbB‐2 oncoprotein expression and patient survival.

Methods and results: Abnormal E‐cadherin and β‐catenin expression, especially loss of expression, was associated with lobular histological type of breast carcinomas (P=0.03 and P=0.01, respectively). Abnormal E‐cadherin and α‐catenin expression was associated with high histological grade ductal carcinomas (P=0.01 and P=0.03, respectively). Abnormal E‐cadherin and β‐catenin expression was correlated with lymph node metastases (P=0.02 and P=0.05, respectively), while abnormal α‐ and β‐catenin were correlated with the advanced stage of the disease (P=0.04 and P=0.05, respectively). Abnormal p120‐catenin expression was associated with loss of PR (P=0.008). Survival analysis demonstrated a statistically significant association between abnormal α‐catenin expression and poor patient survival (P=0.02). When survival analysis was performed according to the different patterns of abnormal expression, statistically significant associations were seen between cytoplasmic α‐ and β‐catenin expression and poor survival (P=0.006 and P=0.04, respectively).

Conclusions: α‐Catenin, especially its cytoplasmic expression, seems to be a more sensitive prognostic marker than the other members of the E‐cadherin complex in invasive breast cancer.
Calreticulin associates with non‐HLA‐A,‐B class I proteins in the human choriocarcinoma cell lines JEG‐3 and BeWo
Authors: WAINWRIGHT  , SIMPSON  , HOLMES
Published: March 2003

Human placental trophoblast expresses an unusual repertoire of major histocompatibility complex (MHC) class I products that appears to reflect the unique role of this epithelium in mediating feto–maternal relations during pregnancy. Trophoblast is devoid of human leucocyte antigen (HLA) ‐A,‐B antigens but can express one or more non‐HLA‐A,‐B class I proteins. The human choriocarcinoma cell lines JEG‐3, BeWo and JAR are widely used as models to study trophoblast. During attempts to isolate non‐HLA‐A,‐B class I from JEG‐3 and BeWo by immunoaffinity chromatography using a monoclonal antibody to β2‐microglobulin we observed a 55 000 MW protein co‐purifying with class I. N‐terminal amino acid sequencing and immunoblotting using a specific antiserum identified this product as calreticulin, a molecule recently shown to be involved in the assembly of classical class I in human B‐lymphoblastoid cells. In our hands JEG‐3 and BeWo were found to express 45 000 MW non‐HLA‐A,‐B class I proteins while the 40 000 MW HLA‐G product was identified only in JEG‐3. Our data suggest that calreticulin associates with non‐HLA‐A,‐B class I heterodimers and with free 45 000 MW non‐HLA‐A,‐B class I H chains in JEG‐3. JAR was found to be devoid of detectable class I H chains but contained β2‐microglobulin and calreticulin. However, calreticulin‐β2‐microglobulin complexes were not detected in JAR. Calreticulin and class I were apparently co‐localized within the endoplasmic reticulum of JEG‐3 cells whereas only class I was expressed at the cell surface. These studies demonstrate that calreticulin is associated with non‐HLA‐A,‐B class I products in human choriocarcinoma cells.
The CD8+ granzyme B+ T‐cell subset in peripheral blood from healthy individuals contains activated and apoptosis‐prone cells
Authors: WEVER  , VAN DER VLIET  , SPAENY  , WOLBINK  , VAN DIEPEN  , FROELICH  , HACK  , TEN BERGE
Published: March 2003

Granzyme B (GrB) has been implicated in induction of apoptosis in target cells. The presence of GrB in peripheral blood CD8+ T cells from healthy individuals was analysed in immunocytochemical and flow cytometric studies. Furthermore, CD8+ GrB− T cells and CD8+ GrB+ T cells were compared regarding phenotypical characteristics and susceptibility to both spontaneous and Fas‐mediated apoptosis. GrB was expressed by approximately one‐fifth of CD8+ T cells. Compared with the CD8+ GrB− T‐cell subset, the CD8+ GrB+ T‐cell subset contained cells that were relatively more activated and more prone to spontaneous apoptosis. Culturing of cells with immunoglobulin M (IgM) anti‐Fas monoclonal antibody had no additional effect on the number of CD8+ GrB+ T cells undergoing apoptosis. We suggest that the presence of CD8+ GrB+ T cells in peripheral blood from healthy individuals results from immune surveillance or contact with infectious agents, and that spontaneous apoptosis of these cells might serve as a mechanism for their eventual clearance.
Differential distribution of FcγRIIIa in normal human tissues and co‐localization with DAF and fibrillin‐1: implications for imm_unological microenvironments
Authors: BHATIA  , BLADES  , CAMBRIDGE  , EDWARDS
Published: March 2003

FcγRIIIa is a cytokine‐inducible IgG Fc receptor implicated in the activation of macrophages by imm_une complexes. Differential expression of FcγRIIIa by macrophages in different tissues may therefore modulate local imm_une responsiveness. FcγRIIIa expression in normal human tissues was assessed semiquantitatively using microdensitometry. Synovial intimal, serosal, alveolar, salivary gland and placental macrophages, Kupffer cells, and macrophages in mechanically stressed dermis expressed high levels of FcγRIIIa. Less consistent expression was seen in skeletal muscle and lymphoid organs. No significant expression was observed in brain, thyroid, spine, intestine, myocardium, prostate, uterus, flexor forearm dermis, uterus, or kidney. Staining for FcγRIII was also observed on extracellular matrix, and co‐localized with both complement decay‐accelerating factor and fibrillin‐1. It is proposed that differential levels of both cellular and extracellular FcγRIIIa, by modulating the response to imm_une complexes, may contribute to relative tissue susceptibility to infection and autoimm_une disease.
Uveitogenic epitopes of retinal S‐antigen are generated in vivo via an alternative antigen‐presentation pathway
Authors: Liversidge J. , Dawson R. , Dick A. D. , Forrester J. V.
Published: March 2003

We have found that different antigen‐processing pathways are involved in the induction of experimental autoimmune uveoretinitis (EAU) by the retinal autoantigens S‐antigen and interphotoreceptor retinoid‐binding protein (IRBP). Although in vitro T‐cell proliferative responses to IRBP were completely inhibited in the presence of irreversible cysteine protease inhibitors, no significant reduction of S‐antigen proliferative responses was found. Furthermore, acidic proteolysis of S‐antigen by the cysteine protease cathepsin B prior to immunization radically reduced pathogenicity (disease severity). In addition, in vitro processing of S‐antigen, but not IRBP, was also found to be resistant to the action of cycloheximide and lysosomotropic agents, inhibition of proliferation only occurring after extended exposure of antigen‐presenting cells to methyl amine or high concentrations of chloroquine. These data indicate that an alternative pathway of antigen processing exists for S‐antigen, which is independent of processing within the normal endo‐lysosomal pathway and that uveitogenic peptides of naturally processed S‐antigen bind to major histocompatibility complex class II antigens either at the cell surface or within very early endosomes where cathepsin B is inactive.
Treatment with recombinant granulocyte colony‐stimulating factor (FilgrastinTM) stimulates neutrophils and tissue macrophages and induces an effective non‐specific response against Mycobacterium avium in mice
Authors: BERMUDEZ L. E. , PETROFSKY M. , STEVENS P.
Published: March 2003

A role of neutrophils in the host response against Mycobacterium avium (MAC) has recently been suggested. To investigate this matter further, we determined the effect of granulocyte colony‐stimulating factor (G‐CSF) on the outcome of MAC infection in mice. C57BL/6 bg+/bg− black mice were intravenously infected with 1×107 MAC and then divided into four experimental groups to receive G‐CSF as follows: (i) 10 μg/kg/day; (ii) 50 μg/kg/day; (iii) 100 μg/kg/day; (iv) placebo control. Mice were killed at 2 and 4 weeks of treatment to determine the bacterial load of liver and spleen. Treatment with G‐CSF at both 10 and 50 μg/kg/day doses significantly decreased the number of viable bacteria in liver and spleen after 2 weeks (≈70·5% and 69·0%, respectively), and after 4 weeks (≈53% and 52%, respectively, P<0·05 compared with placebo control). Treatment with 100 μg/kg/day did not result in decrease of bacterial colony‐forming units in the liver and spleen after 4 weeks. Administration of G‐CSF induced interleukin‐10 (IL‐10) and IL‐12 production by splenocytes. To examine if the protective effect of G‐CSF was accompanied by the activation of phagocytic cells, blood neutrophils and splenic macrophages were purified from mice receiving G‐CSF and their ability to kill MAC was examined ex vivo. Neutrophils and macrophages from G‐CSF‐treated mice were able to inhibit the growth of or to kill MAC ex vivo, while phagocytic cells from untreated control mice had no anti‐MAC effect. These results suggest that activation of neutrophils appears to induce an effective non‐specific host defence against MAC, and further studies should aim for better understanding of the mechanisms of protection.
Role of GM1 binding in the mucosal immunogenicity and adjuvant activity of the Escherichia coli heat‐labile enterotoxin and its B subunit
Authors: DE HAAN L. , VERWEIJ W. R. , FEIL I. K. , HOLTROP M. , HOL W. G. J. , AGSTERIBBE E. , WILSCHUT J.
Published: March 2003

Escherichia coli (E. coli ) heat‐labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant towards co‐administered antigens. LT is composed of one copy of the A subunit, which has ADP‐ribosylation activity, and a homopentamer of B subunits, which has affinity for the toxin receptor, the ganglioside GM1. Both the ADP‐ribosylation activity of LTA and GM1 binding of LTB have been proposed to be involved in immune stimulation. We investigated the roles of these activities in the immunogenicity of recombinant LT or LTB upon intranasal immunization of mice using LT/LTB mutants, lacking either ADP‐ribosylation activity, GM1‐binding affinity, or both. Likewise, the adjuvant properties of these LT/LTB variants towards influenza virus subunit antigen were investigated. With respect to the immunogenicity of LT and LTB, we found that GM1‐binding activity is essential for effective induction of anti‐LTB antibodies. On the other hand, an LT mutant lacking ADP‐ribosylation activity retained the immunogenic properties of the native toxin, indicating that ADP ribosylation is not critically involved. Whereas adjuvanticity of LTB was found to be directly related to GM1‐binding activity, adjuvanticity of LT was found to be independent of GM1‐binding affinity. Moreover, a mutant lacking both GM1‐binding and ADP‐ribosylation activity, also retained adjuvanticity. These results demonstrate that neither ADP‐ribosylation activity nor GM1 binding are essential for adjuvanticity of LT, and suggest an ADP‐ribosylation‐independent adjuvant effect of the A subunit.
Modulation of Ly49A receptors on mature cells to changes in major histocompatibility complex class I molecules
Authors: KHOO*  , FAHLÉN*  , SENTMAN
Published: March 2003

The expression of murine Ly49 receptors on natural killer (NK) cells and NK1.1+ T cells is believed to prevent these cells from responding against normal self‐tissues. In this report we investigated whether the expression level of Ly49A was fixed on mature cells or if it could be adapted as the major histocompatibility complex (MHC) class I environment changed in vivo. By transferring peripheral T cells from Ly49A transgenic mice into BALB/c nude/nude and B6 nude/nude mice, we demonstrated that mature cells modulate their Ly49A receptor expression relative to the in vivo MHC class I environment. These results indicated that the expression of the inhibitory Ly49A receptor is not permanently fixed during a maturation and/or education process but rather is adapted to MHC class I changes on the surrounding cells.
The Ii41 isoform of invariant chain mediates both positive and negative selection events in T‐cell receptor transgenic mice
Authors: WRIGHT  , BIKOFF  , STOCKINGER
Published: March 2003

The functional role of invariant chain in T‐cell selection events and antigen presentation is well established. The invariant chain gene encodes differentially spliced isoforms, Ii31 and Ii41. The Ii41 isoform has been described to increase the efficiency of antigen presentation. We have analysed the effect of the Ii41 isoform on positive and negative selection of transgenic CD4 T cells with specificity for a natural self antigen (C5) which are crucially dependent on invariant chain for their development and functional antigen recognition. The data show that Ii41 fully substitutes for wild‐type invariant chain in both positive and negative selection events during functional maturation of T cells with specificity for a natural, blood‐borne self antigen.
Cross‐linking of the CAMPATH‐1 antigen (CD52) mediates growth inhibition in human B‐ and T‐lymphoma cell lines, and subsequent emergence of CD52‐deficient cells
Authors: ROWAN  , TITE  , TOPLEY  , BRETT
Published: March 2003

The CAMPATH‐1H (CD52) antigen is a 21 000–28 000 MW glycopeptide antigen that is highly expressed on T and B lymphocytes and is coupled to the membrane by a glycosylphosphatidylinositol (GPI) anchoring structure. The humanized CAMPATH‐1H anti‐CD52 antibody is extremely effective at mediating depletion of both normal and tumorigenic lymphocytes in vivo and has been used in clinical trials for lymphoid malignancy and rheumatoid arthritis. Cross‐linking GPI‐anchored molecules, including CD52, on the surface of T lymphocytes in the presence of phorbol 12‐myristate 13‐acetate or anti‐CD3, results in cellular activation. In the present study we have investigated the functional effects of cross‐linking CD52 on T and B tumour cell lines. Cross‐linking CD52 on either a B‐cell line, Wien 133, which expresses high levels of endogenous CD52 or Jurkat T cells transfected and selected to express high levels of CD52 resulted in growth inhibition. This effect showed slower kinetics and occurred in a lower percentage of cells than growth inhibition stimulated via T‐ or B‐cell receptors. Growth inhibition of the Wien 133 line was followed by the induction of apoptosis, which appeared independent of the Fas/Fas L pathway. Wien 133 cells surviving anti‐CD52 treatment were selected and cloned and found to have down‐regulated CD52 expression, with a characteristic biphasic pattern of 10% CD52‐positive, 90% negative by fluorescence‐activated cell sorter analysis. Interestingly, surface expression of other GPI‐linked molecules, such as CD59 and CD55, was also down‐regulated, but other transmembrane molecules such as surface IgM, CD19, CD20, HLA‐DR were unaffected. The present study and previous work show that this is due to a defect in the synthesis of mature GPI precursors. Separation of CD52‐positive and negative populations in vitro resulted in a rapid redistribution to the mixed population. Injection of CD52‐negative cells into nude mice to form a subcutaneous tumour resulted in a substantial increase in expression of CD52. These results suggest that the defect in the Wien 133 cells is reversible, although the molecular mechanism is not clear. These observations have relevance to the clinical situation as a similar GPI‐negative phenotype has been reported to occur in lymphocytes following CAMPATH‐1H treatment in vivo.
The working mechanism of an immune complex vaccine that protects chickens against infectious bursal disease
Authors: JEURISSEN  , JANSE  , LEHRBACH  , HADDAD  , AVAKIAN  , WHITFILL
Published: March 2003

The role of immune complexes (Icx) in B‐cell memory formation and affinity maturation allow for their potential use as vaccines. Recently, a new immune complex vaccine has been developed that is currently under field trials conducted in commercial poultry. This immune complex vaccine is developed by mixing live intermediate plus infectious bursal disease virus (IBDV) with hyperimmune IBDV chicken serum (IBDV–Icx vaccine). Here we have investigated the infectivity of this vaccine as well as the native IBDV (uncomplexed) vaccine in terms of differences in target organs, in target cells and speed of virus replication. At various days after inoculation on day 18 of incubation (in ovo) with either one dose of virus alone or the IBDV–Icx vaccine, the replication of IBDV and the frequency of B cells and other leucocyte populations were examined in the bursa of Fabricius, spleen, and thymus using immunocytochemistry. With both vaccines, IBDV was detected associated with B cells, macrophages and follicular dendritic cells (FDC) in bursa and spleen, although complexing IBDV with specific antibodies caused a delay in virus detection of about 5 days. Most remarkable was the low level of depletion of bursal and splenic B cells in IBDV–Icx vaccinated chickens. Furthermore, in ovo inoculation with the IBDV–Icx vaccine induced more germinal centres in the spleen and larger amounts of IBDV were localized on both splenic and bursal FDC. From these results we hypothesize that the working mechanism of the IBDV–Icx vaccine is related to its specific cellular interaction with FDC in spleen and bursa.
Specific NF‐κB blockade selectively inhibits tumour necrosis factor‐α‐induced COX‐2 but not constitutive COX‐1 gene expression in HT‐29 cells
Authors: JOBIN  , MORTEAU  , HAN  , BALFOUR SARTOR
Published: March 2003

Cyclo‐oxygenase (COX) is the key regulatory enzyme of the prostaglandin/eicosanoid pathway. While COX‐1 is mostly constitutively expressed, the COX‐2 isoform is inducible by proinflammatory cytokines. We used an adenoviral vector containing an NF‐κB super‐repressor (Ad5IκB) to investigate the role of NF‐κB in tumour necrosis factor‐α (TNF‐α)‐mediated COX‐2 gene expression in a colonic epithelial cell line. COX‐1 mRNA and protein were constitutively expressed in uninfected, control Ad5LacZ‐ or Ad5IκB‐infected HT‐29 cells with no apparent change following TNF‐α exposure. COX‐2 mRNA and protein expression was undetectable in unstimulated cells but was strongly up‐regulated after TNF‐α stimulation in uninfected and Ad5LacZ‐infected HT‐29 cells. This induction was prevented in Ad5IκB cells. TNF‐α increased prostaglandin E2 production by 20‐fold in Ad5LacZ‐infected HT‐29 cells compared with uninfected cells and was significantly inhibited in Ad5IκB‐infected cells in agreement with the COX‐2 mRNA findings. We conclude that NF‐κB activation is critical in mediating COX‐2, but not COX‐1 gene expression in HT‐29 cells. Selective inhibition of COX‐2 expression with the NF‐κB super‐repressor may be useful in distinguishing the role of inducible versus constitutive prostaglandins in intestinal function and provides greater specificity than pharmacological inhibitors.
Phenotyping of peripheral blood lymphocytes in adult coeliac disease
Authors: DI SABATINO  , BERTRANDI  , CASADEI MALDINI  , PENNESE  , PROIETTI  , CORAZZA
Published: March 2003

In coeliac disease immunological abnormalities are not confined to the small bowel and it has been suggested that changes in peripheral blood lymphocytes may predispose to autoimmune or malignant complications. Using dual‐colour immunofluorescence with labelled monoclonal antibodies, multiparameter flow cytometry was used to analyse peripheral blood lymphocytes in 32 untreated coeliacs, 29 treated coeliacs and 20 healthy volunteers. When the absolute numbers were considered, a decrease of CD3+, CD4+, CD8+ and CD19+ lymphocytes was found in untreated coeliacs compared with treated coeliacs and healthy volunteers. The proportion of CD3+ was significantly higher in untreated coeliacs (P<0·05) than in healthy volunteers. No differences were observed in CD4+, CD8+ and CD19+ subsets between the three groups studied. The proportion of CD3+ CD25+ and CD3+ HLA‐DR+ cells were higher in untreated coeliacs (P<0·001 and P>0·005) and in treated coeliacs (P<0·005 and P<0·05) than in healthy volunteers. On the contrary, natural killer cells and cytotoxic cells were lower in untreated and treated coeliacs than in healthy volunteers. As regards B‐cell subsets, the only difference was the increase in FcεR+ B cells in untreated coeliacs. The absolute reduction of peripheral lymphocytes in coeliac disease probably reflects their compartimentalization in intestinal mucosa. The decrease of natural killer cells and cytotoxic cells may be in keeping with the increased prevalence of malignancy in this condition. Finally, the phenotypic changes found in untreated coeliacs indicate T‐cell activation.
Suppression of chronic experimental autoimmune neuritis by nasally administered recombinant rat interleukin‐6
Authors: DERETZI  , PELIDOU  , ZOU  , QUIDING  , MIX  , LEVI  , WAHREN  , ZHU
Published: March 2003

Experimental autoimmune neuritis (EAN) is a CD4+ T‐cell‐mediated demyelinating disease of the peripheral nervous system (PNS) and serves as experimental model for human immune‐demyelinating neurophathies, especially the Guillain–Barré syndrome. In this study, we examined the effect of recombinant rat interleukin‐6 (rrIL‐6) on chronic EAN in Lewis rats induced by immunization with P2 peptide 57‐81 and Freund’s complete adjuvant (FCA). Nasal administration of rat rIL‐6 (1 μg/rat/day) beginning in the initial phase of EAN as a therapeutic agent, decreased the severity and the duration of clinical EAN. Low‐grade inflammation and suppression of regional demyelination within the sciatic nerves were seen in rrIL‐6‐treated rats. Hyporesponsiveness of lymph node T cells, down‐regulation of serum tumour necrosis factor‐α (TNF‐α) and increased levels of P2‐specific immunoglobulin G1 (IgG1) antibodies document that nasal administration of rrIL‐6 was effective systemically. However, because of the non‐specific nature of the treatment and multiple effects of IL‐6, more experience and great caution are needed, before nasal administration of IL‐6 can be considered as a treatment of human autoimmune demyelinating neurophathies.
Peptide‐receptive class I major histocompatibility complex molecules on TAP‐deficient and wild‐type cells and their roles in the processing of exogenous antigens
Authors: Song  , Porgador  , Harding
Published: March 2003

These studies addressed the nature and origin of peptide‐receptive class I major histocompatibility complex (MHC‐I) molecules used to present exogenous antigens. Peptide‐receptive Kb molecules in transporter for antigen presentation (TAP)1−/− and TAP1+/+ macrophages were quantitated by exposing cells to exogenous ovalbumin (OVA)(257–264) peptide and then measuring OVA(257–264):Kb complexes with a T hybridoma assay or flow cytometry (using a complex‐specific antibody). Relative to TAP1+/+ cells, TAP1−/− cells had decreased levels of pre‐existing cell‐surface peptide‐receptive MHC‐I molecules at 37°. With continued exposure of viable cells to peptide, however, TAP1−/− and TAP1+/+ cells formed similar levels of OVA(257–264):Kb complexes, suggesting that nascent labile MHC‐I molecules were captured and stabilized by exogenous peptide. Brefeldin A inhibited generation of OVA(257–264):Kb complexes on TAP1−/− (but not TAP1+/+) cells at 37°, confirming the importance of a flux of unstable nascent MHC‐I molecules in TAP1−/− cells at 37°. In contrast, at 26° both TAP1−/− and TAP1+/+ cells expressed brefeldin A‐resistant, peptide‐receptive MHC‐I molecules at similar levels. Alternate MHC‐I processing of exogenous particulate antigen correlated with ability to present exogenous peptide. Thus, processing was brefeldin A‐sensitive with TAP1−/− macrophages at 37°, but brefeldin A‐resistant with TAP1+/+ cells at 37°, as well as with TAP1+/+ or TAP1−/− cells at 26°. We conclude that alternate MHC‐I antigen processing normally utilizes pre‐existing MHC‐I molecules, but TAP1−/− cells at 37° mainly use nascent MHC‐I molecules, because of a lack of pre‐existing, stable, peptide‐receptive MHC‐I molecules. The results support a vacuolar processing mechanism with binding of peptides to MHC‐I molecules in post‐Golgi compartments or on the cell surface.
Kinetics and extent of protein tyrosine kinase activation in individual T cells upon antigenic stimulation
Authors: Müller  , Demotz  , Bulliard  , Valitutti
Published: March 2003

Using human CD4+ T‐cell clones and peptide‐pulsed antigen‐presenting cells (APC) we measured, at the single cell level, different steps in the T‐cell activation cascade. Simultaneous analysis of T‐cell antigen receptor (TCR) down‐regulation and interferon‐γ (IFN‐γ) production shows that both the level of TCR occupancy and the amount of IFN‐γ produced by single T cells increase in an antigen dose‐dependent fashion. Conversely, commitment of T cells to IFN‐γ production does not occur as soon as a defined number of TCR have been engaged, but requires the same duration of sustained signalling at low as well as at high antigen concentrations. Measurement of phosphotyrosine levels by flow cytometry reveals that, upon conjugation with APC, individual T cells undergo an antigen dose‐dependent activation of protein tyrosine kinases (PTK), which parallels the level of TCR occupancy. In antigen‐stimulated T cells the increased phosphotyrosine staining is localized in the area of contact with APC, as shown by confocal microscopy. PTK activation is sustained for at least 2 hr after conjugation, and is required to maintain a sustained increase in intracellular Ca2+ concentration. Our results show, for the first time, a direct correlation between the level of TCR occupancy and the activation of PTK in individual T cells and offer an explanation for how the number of triggered TCR can be ‘counted’ and integrated in a corresponding biological response.
Immunopathogenesis of classical swine fever: role of monocytic cells
Authors: Knoetig  , Summerfield  , Spagnuolo‐Weaver  , McCullough
Published: March 2003

Virulent classical swine fever (CSF) represents an immunomodulatory viral infection that perturbs immune functions. Circulatory and immunopathological disorders include leukopenia, immunosuppression and haemorrhage. Monocytic cells – targets for CSF virus (CSFV) infection – could play critical roles in the immunopathology, owing to their production of immunomodulatory and vasoactive factors. Monocytes and macrophages (Mφ) are susceptible to virus infection, as a consequence of which prostaglandin E2 (PGE2) production is enhanced. The presence of PGE2 in serum from CSFV‐infected pigs correlated with elevated PGE2 productivity by the peripheral blood mononuclear cells from these same animals. It was noted that these PGE2‐containing preparations did not inhibit, but actually enhanced, lymphocyte proliferation. The proinflammatory cytokines tumour necrosis factor‐α (TNF‐α) and interleukin (IL)‐6 were not involved, although elevated IL‐1 production could relate to lymphocyte activation. Nevertheless, IL‐1 was not the sole element: infected Mφ produced lympho‐stimulatory activity but little IL‐1. This release of immunomodulatory factors, following CSFV infection of monocytic cells, was compared with other characteristics of the disease. Therein, PGE2 and IL‐1 production was noted to coincide with the onset of fever and the coagulation disorders typical of CSF. Consequently, these factors are of greater relevance to the haemorrhagic disturbances, such as petechia and infarction, rather than the leukopenia found in CSF.
Expression and translocation of Rac2 in eosinophils during superoxide generation
Authors: Lacy  , Mahmudi‐Azer  , Bablitz  , Gilchrist  , Fitzharris  , Cheng  , Man  , Bokoch  , Moqbel
Published: March 2003

Eosinophils induce tissue injury by releasing granule‐associated cytotoxic proteins, lipid mediators and superoxide anions in response to appropriate stimuli. Superoxide generation associated with respiratory burst is largely dependent on the assembly of the NADPH oxidase complex in the membrane, consisting of membrane‐bound cytochrome b558 and translocated p47phox and p67phox. The activation of this complex is critically dependent on the translocation of GTP‐bound Rac1, or its homologue Rac2, from the cytosol to the membrane in neutrophils. Rac expression has not yet been fully characterized in eosinophils. We proposed that eosinophils translate and express Rac2 and its GDP‐dissociation inhibitor, RhoGDI. Furthermore, we hypothesized that Rac2 translocates along with p47phox and p67phox proteins from the cytosol to the plasma membrane during respiratory burst. By reverse transcription–polymerase chain reaction analysis and sequencing of the amplified product, guinea‐pig eosinophils were found to express Rac2 mRNA, exhibiting 93% homology with the human Rac2 sequence. Rac1 mRNA was also detected in eosinophils but not its translated product. In contrast, Rac2 protein expression was detected using a specific antibody. In subcellular fractions, Rac2 was found to translocate, along with p47phox and p67phox, from cytosol to plasma membrane‐associated fractions following phorbol myristate acetate stimulation, while RhoGDI remained within cytosolic fractions. These findings suggest that Rac2 is preferentially expressed and activated in eosinophils, and is likely to be a crucial regulator of the release of reactive oxygen species from these cells during inflammatory reactions.
Putative role for interleukin‐7 in the maintenance of the recirculating naive CD4+ T‐cell pool
Authors: Webb L. M. C. , Foxwell B. M. J. , Feldmann M.
Published: March 2003

The capacity of the immune system to respond efficiently to new antigens depends upon a continuous source of naive CD4+ T cells. Such cells exit from the thymus and join the recirculated T‐cell pool. Factors present at the sites of naive CD4+ T‐cell circulation must be responsible for their survival, since upon removal from their host, naive CD4+ T cells die. However, such factors remain unknown. The presence of the cytokine interleukin‐7 (IL‐7) in secondary lymphoid organs and the continuous expression of its receptor on naive CD4+ T cells prompted us to examine the possibility that IL‐7 might be a survival factor for naive CD4+ T cells. Using naive CD4+ T cells isolated from cord blood we show that IL‐7, but not IL‐2, can maintain naive CD4+ T‐cell viability in vitro for at least 15 days. In addition, we find that IL‐7 can induce modest proliferation of naive CD4+ T cells without affecting either their cell surface phenotype or their ability to respond to antigenic stimulation. We also find that after anti‐CD3 stimulation, naive CD4+ T cells lose that ability to respond to IL‐7. However, if cells are primed with IL‐7 prior to antigenic stimulation, their proliferative responses are enhanced. Together, these data suggest a novel and important role for IL‐7 in the maintenance and maturation of naive CD4+ T cells, ensuring that they can respond maximally when they first meet antigen in secondary lymphoid tissue.
Decay accelerating factor in guinea‐pig reproductive organs
Authors: He C. , Nonaka M. , Tada T. , Koji T. , Li W. , Okada N. , Okada H.
Published: March 2003

Decay accelerating factor (DAF, CD55) expressed in human reproductive organs and gametes is thought to play a pivotal role in protection against autologous complement activation in the genital tract. To further investigate the role of DAF in reproduction, we analysed DAF distribution in reproductive organs using guinea‐pigs that express multiple DAF isoforms. In males, significant staining was observed in testis on the elongated spermatids and spermatozoa. Levels of DAF mRNA with a shorter 3′ untranslated region were significantly enhanced in testis from 9 weeks of age, indicating the presence of DAF mRNA and protein synthesis of spermatozoa DAF in late haploid germ cells. Epididymal spermatozoa appeared to express DAF on the inner acrosomal membrane as well as over their entire surface. Significant DAF expression was also observed on the epithelium of seminal vesicles from 4 weeks of age, with no increase thereafter in the mRNA. C3 mRNA was not detected in this tissue. In females, DAF was detected on the plasma membranes of oocytes through follicle development and on the apical region of uterine epithelium, although the levels of DAF mRNA in these tissues were low. In addition, DAF was selectively expressed on the apical region of ciliated oviductal epithelial cells. The apical region of the ciliated cells comprising the efferent ductule epithelium was also stained significantly, even at 12 days of age, while other epididymal epithelial cells were hardly stained at any age, suggesting that DAF is constitutively expressed on cilia.