Library

Notes: Human mast cell tryptase was purified from lung tissue by high salt extraction, ammonium sulphate precipitation, octyl Sepharose and heparin-agarose chromatography. The tryptase isolated was a tetramer with a molecular weight of 132 kD on gel filtration, and on SDS-polyacrylamide gel electrophoresis was reduced to a single diffuse band with a mean molecular weight of 32·5 kD. Purified tryptase catalysed the cleavage of the tryptic substrates tosyl L-arginine methyl ester and benzoyl dl.-arginine p-nitroanilide; enzymatic activity was enhanced in the presence of heparin but markedly decreased in the presence of 2 M sodium chloride. Rabbit antisera and three new monoclonal antibodies (AA1, AA3 and AA5) were produced which were specific for tryptase in indirect ELISAs, immunoenzymatic overlay in crossed immunoelectrophoresis and by Western blotting. Additive and competitive ELISA experiments suggested that the three monoclonal antibodies all recognized epitopes within a single highly immunogenic area of the tryptase molecule, and enzyme assays indicated that this site was distant from the active site. Binding of monoclonal antibodies to tryptase was not affected by the presence of heparin, or by periodate treatment of the antigen suggesting that carbohydrate epitopes were not recognized. Western blotting indicated that some heterogeneity in molecular weight for monomeric tryptase was not reflected in antigenic differences. An immunofluorescence procedure with cytocentrifuge preparations of enzymatically dispersed lung, colon and skin revealed highly specific localization of tryptase to the granules of all mast cells, but there was no binding to other cells in these preparations, to cultured keratinocytes, to basophils or to any other blood leucocyte.

Notes: Aims:  In the female genital tract CD10 has been used to assist in the evaluation of mesenchymal tumours of the uterus and in determining whether endometrial stroma is present. CD10 positivity has also been shown in cervical mesonephric remnants and this antibody has been suggested as a useful immunohistochemical marker of mesonephric lesions in the female genital tract. Calretinin has also been shown to be positive in mesonephric lesions. In this study the specificity of these two antibodies in evaluating cervical and uterine glandular lesions and the value of CD10 in determining whether stroma is endometriotic or not were investigated.Methods and results:  Cases of cervical tubo-endometrial metaplasia (TEM) (n = 11), microglandular hyperplasia (MGH) (n = 10), endometriosis (n = 8), mesonephric remnants/hyperplasia (n = 12), endocervical adenocarcinoma, usual type (n = 15), mucinous variant of minimal deviation adenocarcinoma (MDA) (n = 7) and mesonephric adenocarcinoma (n = 3) were stained with antibodies against CD10 and calretinin. Nine cases of endometrial adenocarcinoma of endometrioid type were also stained. In all the cervical cases normal endocervical glands were negative with both antibodies except for one case with strong positive luminal staining with CD10. All cases of TEM, MGH and endometriosis were negative with CD10 and calretinin except for focal staining with CD10 in one case each of MGH (cytoplasmic staining) and endometriosis (luminal staining). Most usual endocervical adenocarcinomas were negative with both antibodies, although one exhibited focal cytoplasmic staining with calretinin and five exhibited limited luminal positivity with CD10. All MDAs were negative with both antibodies. Ten of 12 mesonephric remnants/hyperplasia showed luminal positivity with CD10 and one exhibited cytoplasmic and nuclear staining with calretinin. Two of three mesonephric adenocarcinomas showed luminal positivity with CD10 and nuclear and cytoplasmic positivity with calretinin. Seven of nine endometrial adenocarcinomas were positive with CD10 (four cytoplasmic, two membranous and cytoplasmic, one luminal and cytoplasmic) and three with calretinin (two cytoplasmic, one nuclear and cytoplasmic). Positive staining of endometriotic stroma with CD10 was present in all endometriosis cases but normal cervical stroma was also strongly positive, especially around glands. Endometriotic stroma and cervical stroma were negative with calretinin.Conclusions:  We conclude that most endocervical glandular lesions, including mesonephric remnants/ hyperplasia, are negative with calretinin. However, the focal nuclear and cytoplasmic positivity with calretinin in two of three mesonephric adenocarcinomas suggests that this may be a useful indicator of a mesonephric origin of a cervical adenocarcinoma. Most mesonephric remnants/hyperplasias exhibit luminal positivity with CD10, although this is not invariable and staining is usually focal. Positive luminal staining of a benign endocervical glandular lesion with CD10 may help confirm mesonephric remnants. Although positive staining with CD10 was found in two of three mesonephric adenocarcinomas, the observed immunoreactivity of several conventional cervical adenocarcinomas limits the diagnostic value of CD10 in confirming a mesonephric origin for an adenocarcinoma. Since all cervical MDAs were negative with CD10, positivity with this antibody may be of value in distinguishing mesonephric hyperplasia from MDA, although this distinction rarely necessitates immunohistochemistry. Most endometrial adenocarcinomas of endometrioid type stain with CD10 and thus positivity with this antibody is not specific for a mesonephric origin of an endometrial adenocarcinoma. Positivity of normal cervical stroma limits the value of CD10 staining in confirming a diagnosis of cervical endometriosis.

Notes: Aims:  Although diffuse large B-cell lymphoma is categorized as a distinct entity in the REAL classification of lymphomas, it represents a heterogeneous group of neoplasms. A subgroup is probably of follicle centre cell origin and may evolve from a pre-existing follicular lymphoma. The t(14;18) chromosomal translocation can be demonstrated in the majority of follicular lymphomas and the aim of this study was to investigate the prevalence of t(14;18) translocation in a series of de novo nodal diffuse large B-cell lymphomas. We correlated this with the immunohistochemical expression of CD10, bcl2 and bcl6, markers which are usually expressed by the neoplastic cells in follicular lymphomas. We also correlated these parameters with the presence or absence of p53 protein expression by the neoplastic cells.Methods and results:  Nodal diffuse large B-cell lymphomas (n=34) were stained immunohistochemically with monoclonal antibodies to CD10, bcl2, bcl6 and p53 (D07). Polymerase chain reaction (PCR) for the t(14;18) translocation was also performed. Fourteen, 24 and 29 (41%, 71%, 85%) cases exhibited positivity for CD10, bcl2 and bcl6, respectively. In 12 cases there was positivity with D07 (35%). By PCR, the t(14;18) translocation was identified in five cases (15%), four of which were positive for CD10 and bcl2 and all of which were positive for bcl6. One of five cases positive for the chromosomal translocation exhibited positivity with D07.Conclusions:  In this study the t(14;18) translocation was identified in 15% of diffuse large B-cell lymphomas, all but one of which exhibited positivity for CD10, bcl2 and bcl6. These may represent cases of follicle centre cell origin which may or may not have evolved from a pre-existing follicular lymphoma. It is possible that positivity for CD10 especially may identify cases which are of follicle centre cell origin and that the absence of t(14;18) translocation in some of these cases may reflect the fact that the translocation cannot normally be demonstrated in all follicular lymphomas. Whether the presence or absence of the translocation and the immunophenotype are prognostically important should be investigated further.