Margaret Jones

Locus Control Region Function and Heterochromatin-Induced Position Effect Variegation

Human CD2 locus control region (LCR) sequences are shown here to be essential for establishing an open chromatin configuration. Transgenic mice carrying an hCD2 minigene attached only to the 3′ CD2 transcriptional enhancer exhibited variegated expression when the transgene integrated in the centromere. In contrast, mice carrying a transgene with additional 3′ sequences showed no variegation even when the latter integrated in centromeric positions. This result suggests that LCRs operate by ensuring an open chromatin configuration and that a short region, with no enhancer activity, functions in the establishment, maintenance, or both of an open chromatin domain.

Heterogeneity of vascular endothelial cells with relevance to diagnosis of vascular tumours.

AIMS: To determine the distribution of factor VIII related antigen, CD31, CD34 and CD36 in normal and malignant human vascular tissues using a panel of well characterised monoclonal antibodies. METHODS: Frozen and fixed material from a wide range of normal tissues and routinely processed material from 43 benign and malignant vascular tumours were examined. Single immunocytochemical labelling was performed using the APAAP technique. Double staining involved the sequential use of APAAP with the peroxidase method. RESULTS: Human vascular endothelium was antigenically heterogeneous. One of the most restricted markers was factor VIII related antigen, despite its having been widely used in diagnostic pathology as a marker of vascular endothelium and of the tumours which arise from it. Three antibodies against factor VIII related antigen, CD31 (JC70) and CD34 (QBend 10) were identified as immunostaining routinely processed, formalin fixed, paraffin wax sections. Each antibody gave different staining when tested on a range of vascular tumours, both benign and malignant. CONCLUSIONS: A small panel of three reagents (factor VIII related antigen, CD31 (JC70) and CD34 (QBend 10)) should be used by diagnostic pathologists who want to show the presence of cells of endothelial origin in routine material.

Invariant NKT cells modulate the suppressive activity of IL-10-secreting neutrophils differentiated with serum amyloid A

Neutrophils are the main effector cells during inflammation, but they can also control excessive inflammatory responses by secreting anti-inflammatory cytokines. However, the mechanisms that modulate their plasticity remain unclear. We now show that systemic serum amyloid A 1 (SAA-1) controls the plasticity of neutrophil differentiation. SAA-1 not only induced anti-inflammatory interleukin 10 (IL-10)-secreting neutrophils but also promoted the interaction of invariant natural killer T cells (iNKT cells) with those neutrophils, a process that limited their suppressive activity by diminishing the production of IL-10 and enhancing the production of IL-12. Because SAA-1-producing melanomas promoted differentiation of IL-10-secreting neutrophils, harnessing iNKT cells could be useful therapeutically by decreasing the frequency of immunosuppressive neutrophils and restoring tumor-specific immune responses.

Fewer thymic changes in MuSK antibody-positive than in MuSK antibody-negative MG

In generalized myasthenia gravis (MG) patients without detectable acetylcholine receptor (AChR) antibodies (SNMG), the thymus is often reported as “normally involuted.” We analyzed thymic compartments in 67 patients with generalized MG, with AChR antibodies (AChR+, n = 23), with muscle‐specific kinase (MuSK) antibodies (MuSK+, n = 14) or with neither (MuSK−, n = 30), and in 11 non‐MG controls. Four of 14 MuSK+ thymi had rare small germinal centers, but overall they were not different from age‐matched controls. However, approximately 75% MuSK− samples showed lymph node–type infiltrates similar to those in AChR+ patients, but with fewer germinal centers. These variations may explain some apparent differences in responses to thymectomy in SNMG. Ann Neurol 2005;57:444–448

The unusual distribution of the neuronal/lymphoid cell surface CD200 (OX2) glycoprotein is conserved in humans

OX2 (CD200) is a type‐1 membrane glycoprotein that contains two immunoglobulin superfamily domains and which is expressed on a variety of lymphoid and non‐lymphoid cells in the rat. The recent characterization of a receptor for OX2 (OX2R) on myeloid cells, and the phenotype of an OX2‐deficient mouse, suggests that OX2 may regulate myeloid cell activity in anatomically diverse locations. Here we report the tissue distribution of the human homologue of the rat OX2 glycoprotein using a new monoclonal antibody (mAb), OX104, raised against recombinant human OX2. Human OX2 was expressed at the cell surface of thymocytes, B cells, T cells, neurons, kidney glomeruli, tonsil follicles, the syncytiotrophoblast and endothelial cells. This broad, but not ubiquitous, distribution pattern is very similar to that observed in rats, suggesting that OX2 may regulate myeloid cell activity in a variety of tissues in humans.

Co-expression of CD79a (JCB117) and CD3 by lymphoblastic lymphoma

Acute lymphoblastic leukaemia/lymphoma is a malignant disorder derived from the clonal proliferation of lymphoid precursor cells. Whether the tumour cells are of B‐ or T‐cell type is an important criterion for prognosis which has not been available previously to pathologists, due to the lack of a reliable early B‐cell marker functioning on routinely processed material. This has changed with the production of monoclonal antibodies against the B‐cell signalling molecule CD79a. CD79a is expressed on normal and neoplastic B cells from the early stages of B‐cell maturation and has been considered to be B‐cell‐specific. Currently available antibodies against CD79a, in particular JCB117, allow the identification of B cells, and hence B lymphoblastic disease, in paraffin‐embedded material. In this study, the expression of CD79a (JCB117) and CD3 has been investigated in 149 cases of T and 68 cases of B lymphoblastic leukaemia/ lymphoma. For the first time, co‐expression of CD79a (JCB117) and CD3 is reported in 10 per cent of cases of T lymphoblastic leukaemia/lymphoma. This finding raises questions about the co‐expression of T‐ and B‐cell markers in the development of lymphocytes, benign as well as malignant, and alerts pathologists to a potential problem in diagnosis. Copyright

Kupffer cell staining by an HFE-specific monoclonal antibody : implications for hereditary haemochromatosis

Hereditary haemochromatosis is an inherited disorder of iron absorption that leads to excessive iron storage in the liver and other organs. A candidate disease gene HFE has been identified that encodes a novel MHC class I like protein. We report the development of a monoclonal antibody (HFE‐JB1) specific for recombinant refolded HFE protein. The antibody immunoprecipitates a 49 kD protein from the cell line U937, a histiocytic lymphoma. It binds HFE but does not recognize other recombinant non‐classic MHC class I proteins (HLA‐E, F and G), nor does it react with a variety of recombinant classic class I MHC molecules. COS cells transfected with HFE in culture are stained specifically. The immunohistochemical staining pattern in human tissues is unique and can be defined as a subset of the transferrin receptor positive cells. In the liver HFE protein was shown to be present on Kupffer cells and endothelium (sinusoidal lining cells), but absent from the parenchyma. Kupffer cells from an untreated C282Y HH patient failed to stain with the antibody. In the normal gut scattered cells in the crypts are stained. HFE was also present on capillary endothelium in the brain (a site of high levels of transferrin receptor) and on scattered cells in the cerebellum and cortex. These results raise interesting questions concerning the function of HFE in the control of body iron content and distribution.

Immunohistochemical detection of p53 and bcl-2 proteins in non-Hodgkin's lymphoma.

We have investigated the immunohistochemical expression of p53 protein in 96 cases of non‐Hodgkins lymphoma using a panel of five antibodies. Positive neoplastic cells were found in 30 (31.2%) cases, which could be divided into two groups according to their patterns of reactivity with the different antibodies; i.e. those positive with both polyclonal and monoclonal antibodies, and those which were stained only by monoclonal antibodies PAb1801 and/or PAb240. Positivity was nuclear in all but six cases in which cytoplasmic staining was found. In view of the hypothesis recently raised that p53 protein induces apoptosis we have compared our results with parallel staining for bcl‐2 protein since bcl‐2 is believed to be important, at least in lymphomas, in suppression of apoptosis. Staining for bcl‐2 protein was performed on 83 cases and it was shown that p53‐positive cases accounted for 10 out of 17 (59%) of the bcl‐2‐negative lymphomas but only for 15 out of the 66 (23%) bcl‐2‐positive cases, suggesting a possible relationship between the expression of these two proteins. Thus, our data show that p53 protein is abnormally expressed in a substantial proportion of non‐Hodgkins lymphomas and bears a significant inverse relationship to bcl‐2 protein expression. However the molecular basis of this expression remains to be elucidated.

Double immunofluorescence labelling of routinely processed paraffin sections

Double immunoenzymatic labelling of routinely processed human tissues has been used in many histopathology laboratories to compare the expression patterns of pairs of antigenic markers. However, these techniques are time‐consuming, prone to background staining, and rarely suitable for detecting two antigens present at the same site, since one label tends to obscure the other. This paper reports the use of immmunofluorescence for double labelling of pairs of molecular markers in routinely processed tissue. The primary antibodies are either monoclonal reagents of differing isotype/subclass, or antibodies from different species, and labelling is visualized on a conventional fluorescence microscope equipped with a cooled CCD camera. Images can be captured and adjusted using personal computer hardware and software. This approach could be used for a wide range of tissue markers and only minimal tissue autofluorescence was observed. The procedure is more rapid than enzyme‐based techniques and avoids the problems of interpreting two antigens present at the same site. Its establishment involves relatively minor expenditure and it may represent the optimal technical approach to the co‐localization of pairs of antigens in routinely processed tissue samples. Copyright

Inducible nitric oxide synthase expression is increased in the brain in fatal cerebral malaria

Aims