05-633 Sigma-AldrichAnti-BrdU Antibody, clone BU-1

The inflammatory infiltrate plays a pivotal role in classical Hodgkin lymphoma (cHL). Here, we focussed on the role of macrophages (MΦ) and dendritic cells (DC).MΦ and DC infiltration was investigated in 106 cHL specimens using immunohistochemistry and cytokine expression was analyzed in a subset by real-time PCR. Human peripheral blood-derived monocytes, DC, MΦ stimulated with GM-CSF (MΦGM-CSF, pro-inflammatory MΦ-1-model) or M-CSF (MΦM-CSF, immunomodulatory MΦ-2-model) were incubated with cHL cell line (L1236, HDLM2) supernatants (SN). DC maturation or MΦ polarization were investigated by flow cytometry. Furthermore, the impact of DC or MΦ on cHL cell proliferation was analyzed by BrdU/CFSE assay.In cHL tissues mature myeloid (m)DC and MΦ predominated. High numbers of CD83+ mDC and low numbers of CD163+ MΦ were associated with improved disease specific survival. In numerous cHL specimens increased levels of both pro- and anti-inflammatory cytokines and of IL13 and GM-CSF were observed compared to reactive lymphadenopathies. Maturation of DC and induction and maintenance of an immunomodulatory MΦ phenotype were promoted by SN derived from cHL cell lines. TNFα neutralization in SN resulted in a significant inhibition of mDC maturation. DC and pro-inflammatory MΦ inhibited the proliferation of cHL cells.Adopting an immunomodulatory phenotype is a potential mechanism for how MΦ promote immune evasion in cHL. Mature DC, in contrast, might participate in antitumoral immunity.

During early development in female mammals, most genes on one of the two X-chromosomes undergo transcriptional silencing. In the extraembryonic lineages of some eutherian species, imprinted X-inactivation of the paternal X-chromosome occurs. In the cells of the embryo proper, the choice of the future inactive X-chromosome is random. We mapped several genes on the X-chromosomes of five common vole species and compared their expression and methylation patterns in somatic and extraembryonic tissues, where random and imprinted X-inactivation occurs, respectively. In extraembryonic tissues, more genes were expressed on the inactive X-chromosome than in somatic tissues. We also found that the methylation status of the X-linked genes was always in accordance with their expression pattern in somatic, but not in extraembryonic tissues. The data provide new evidence that imprinted X-inactivation is less complete and/or stable than the random form and DNA methylation contributes less to its maintenance.

We measured plasma cell labeling indices (LI) in 52 patients with monoclonal gammopathies. Cytoplasmic reactivity with polyspecific or kappa- and lambda-specific light chain anti-Ig reagents identified monoclonal plasma cells, plasmablasts, and lymphocytoid plasma cells. Among newly diagnosed untreated patients, a high immunofluorescence LI distinguished those with multiple myeloma (MM) from those with stable monoclonal gammopathies (P less than 0.002). Among treated patients with MM, those in the plateau phase of the disease had low LI, whereas patients in the relapse phase or early in treatment had high LI. The immunofluorescence LI correctly classified three more patients with newly diagnosed MM than did the tritiated thymidine LI technique. LI specific for the neoplastic plasma cells resulted in excellent discrimination of patients with active disease. Because results are easily and rapidly obtained, this technique is useful clinically.

We have previously described a monoclonal antibody (BU-1) to 5-bromo-2-deoxyuridine (BrdUrd) that is useful for measurement of cell cycle S-phase. BU-1 hybridoma supernatant reacted with incorporated BrdUrd after the cells had been ethanol fixed; without a requirement for acid or base denaturation. We have found that this reactivity is lost if purified antibody is used, if the culture supernatants are heated, or if a mycoplasma-free hybridoma line is isolated. The supernatant contained endogenous DNase activity that was a result of mycoplasma infection of the cell line. This DNase activity was required for staining the cells with BU-1 in the absence of other denaturation steps. The endogenous DNase could be substituted for by the addition of bovine pancreatic DNase I. The disruption of the double stranded DNA structure with an enzyme rather than with harsh chemical or heat treatments does not affect protein structure or cellular morphology and allows the detection of incorporated BrdUrd of morphologic or antigenic cell subsets. DNase pre-treatment may also be useful for detection of other 'hidden' DNA antigens.

We describe a mouse monoclonal antibody (BU-1) reactive with 5-bromo-2-deoxyuridine (BrdUrd). The antibody is different from previously described BrdUrd monoclonal antibodies in that BU-1 does not require pretreatment of cells with strong DNA denaturants in order for the antibody to react with BrdUrd incorporated in the DNA. The antibody can be used in immunocytochemical and indirect immunofluorescent assays and can be used to identify cells that have incorporated BrdUrd. Double staining with BU-1 antibody and propidium iodide has been used to confirm S-phase measurements with the BU-1 antibody. Immunocytochemical stains using the BU-1 antibody do not destroy cell morphology and allow cell identification to be performed simultaneously with S-phase measurements. Flow cytometer two-color fluorescence analysis allows the simultaneous identification of cell surface or cytoplasmic markers and S-phase quantitation. The BU-1 antibody should broaden the application of cell kinetic measurements to individual elements of cell populations that are heterogeneous with respect to morphology, surface marker, and other biological features.