DNA methylation profiling in human B cells reveals immune regulatory elements and epigenetic plasticity at Alu elements during B-cell activation.

Memory is a hallmark of adaptive immunity, wherein lymphocytes mount a superior response to a previously encountered antigen. It has been speculated that epigenetic alterations in memory lymphocytes contribute to their functional distinction from their naive counterparts. However, the nature and extent of epigenetic alterations in memory compartments remain poorly characterized. Here we profile the DNA methylome and the transcriptome of B-lymphocyte subsets representing stages of the humoral immune response before and after antigen exposure in vivo from multiple humans. A significant percentage of activation-induced losses of DNA methylation mapped to transcription factor binding sites. An additional class of demethylated loci mapped to Alu elements across the genome and accompanied repression of DNA methyltransferase 3A. The activation-dependent DNA methylation changes were largely retained in the progeny of activated B cells, generating a similar epigenetic signature in downstream memory B cells and plasma cells with distinct transcriptional programs. These findings provide insights into the methylation dynamics of the genome during cellular differentiation in an immune response.

Direct fluorochrome labeling of phage display library clones for studying binding specificities: applications in flow cytometry and fluorescence microscopy.

Phage display technology is increasingly employed to identify high-affinity peptides and single-chain antibodies with binding specificities for a diversity of target types. The analysis of phage-binding sensitivity and specificity typically employs directly labeled secondary antiphage antibodies and potentially tertiary labels, such as fluorochromes and enzymes, when biotinylated antibodies are used. However, secondary or tertiary reagents may not be feasible or desirable for some target types and applications. Here, we present a simple approach for directly labeling phage clones with two common amine-reactive fluorochromes. We show that these fluorochromes label the pVIII major coat protein and that the binding selectivity of peptides displayed on the pIII protein of several well-characterized phage clones is maintained in flow cytometric analysis and immunofluorescence microscopy. Uniquely, such labeled phage, in part, represent self-propagating reagents because conjugation does not impair the ability to efficiently reproduce in bacteria, although relabeling with fluorochrome would be necessary. Our data suggest that primary labeled phage clones may be used similarly to primary antibody conjugates.

Immune status of physically active women during lactation.

PURPOSE: The purpose of this study was to provide baseline data on immune status of exercising and sedentary exclusively lactating women. Dietary intake and body composition were also investigated to determine whether they related to immune function. METHODS: Healthy, exclusively breastfeeding women with a body mass index between 20 and 30 kg x m were studied at 3 months postpartum. Participants in the exercise group (EG; N = 27) exercised aerobically at least 30 min x d, 3x wk, and women in the sedentary group (SG; N = 23) exercised once a week or less during the previous 6 wk. Immune status while at rest was determined by measuring: 1) a complete blood cell count and differential leukocyte count; 2) percentages and absolute counts of peripheral blood T cells (CD3+), cytotoxic T cells (CD3+CD8+), helper T cells (CD3+CD4+), B cells (CD19+), and natural killer cells (CD56+); 3) neutrophil bacterial killing and oxidative burst activity; and 4) in vitro mitogenic responsiveness of lymphocytes. Cardiorespiratory fitness, body composition, and dietary intake were also measured. RESULTS: Participants in the EG had a significantly higher level of mean predicted cardiorespiratory fitness than women in the SG (39.5 +/- 1.1 vs 32.5 +/- 1.0 mL O2 x min x kg; P < 0.05); however, there were no significant differences in body composition or dietary intake. There were no significant differences in any of the indicators of immune status between groups. In addition, there were no significant relationships between body composition or dietary intake and immune status. CONCLUSIONS: The results of this study suggest that women may exercise moderately during lactation and increase their fitness level without impairing their immune function.

Enrichment of LDL with EPA and DHA decreased oxidized LDL-induced apoptosis in U937 cells.

Oxidized LDL (oxLDL) may contribute to the accumulation of apoptotic cells in atherosclerotic plaques. Although it is well established in monophasic chemical systems that the highly unsaturated EPA and DHA will oxidize more readily than FA that contain fewer double bonds, our previous studies showed that enrichment of LDL, which has discrete polar and nonpolar phases, with these FA did not increase oxidation. The objective of this study was to compare the extent of apoptosis induced by EPA/DHA-rich oxLDL to that induced by EPA/DHA-non-rich oxLDL in U937 cells. LDL was obtained from one healthy subject three times before and after supplementation for 5 wk with 15 g/d of fish oil (FO), an amount easily obtainable from a diet that contains fatty fish. After supplementation, an EPA/DHA-rich LDL was obtained. Oxidative susceptibility of LDL, as determined by measuring the formation of conjugated dienes and the accumulation of cholesteryl ester hydroperoxides, was not higher in EPA/DHA-rich LDL. The oxLDL-induced cell apoptosis was detected by the activation of caspase-3, the translocation of PS to the outer surface of the plasma membrane using the Annexin V-fluorescein isothiocyanate binding assay, and the presence of chromatin condensation and nuclear fragmentation using the 4,6-diamidino-2-phenylindole staining assay. All three measures showed that after FO supplementation, EPA/DHA-rich oxLDL-induced cell apoptosis decreased. The decrease was not related to the concentration of lipid hydroperoxides. This study suggests that a possible protective effect of EPA/DHA-rich diets on atherosclerosis may be through lessening cell apoptosis in the arterial wall.

DNA methylation prevents CTCF-mediated silencing of the oncogene BCL6 in B cell lymphomas.

Aberrant DNA methylation commonly occurs in cancer cells where it has been implicated in the epigenetic silencing of tumor suppressor genes. Additional roles for DNA methylation, such as transcriptional activation, have been predicted but have yet to be clearly demonstrated. The BCL6 oncogene is implicated in the pathogenesis of germinal center-derived B cell lymphomas. We demonstrate that the intragenic CpG islands within the first intron of the human BCL6 locus were hypermethylated in lymphoma cells that expressed high amounts of BCL6 messenger RNA (mRNA). Inhibition of DNA methyltransferases decreased BCL6 mRNA abundance, suggesting a role for these methylated CpGs in positively regulating BCL6 transcription. The enhancer-blocking transcription factor CTCF bound to this intronic region in a methylation-sensitive manner. Depletion of CTCF by short hairpin RNA in neoplastic plasma cells that do not express BCL6 resulted in up-regulation of BCL6 transcription. These data indicate that BCL6 expression is maintained during lymphomagenesis in part through DNA methylation that prevents CTCF-mediated silencing.

Selective targeting of nanocarriers to neutrophils and monocytes.

We previously identified and characterized cell-type selective binding peptides from random peptide phage display libraries. Here, we used one of these peptides (GGP) to target liposomal nanocarriers to leukocyte subsets. To profile the binding selectivity of GGP-coated liposomes to human blood cells, we performed flow cytometric analysis with whole anti-coagulated blood. It is shown that when liposomal nanocarriers present these peptides on their surface, they facilitated cell-type specific targeting of liposomes to neutrophils and monocytes in contrast to nontargeted liposomes. Our data suggest that engineering the appropriate number of targeting peptide ligands on the nanocarrier surface is a factor in cell-binding selectivity, as is dose. Increasing the peptide density on the surface of the liposomes from 250 to 500 molecules resulted in more binding to neutrophils and monocytes. Fluorescence confocal microscopy corroborated the flow cytometry data revealing that liposomes coated with targeting GGP peptides decorated the surface of targeting cells and facilitate cell uptake of payload as evidenced by nuclear localization of tracer. These data suggest that small peptides identified by phage display techniques can be used to target nanocarriers that potentially carry therapeutic or imaging agents to leukocyte subsets. This ability has important implications for diseases where neutrophils and monocytes play a major role such as arthritis, inflammatory bowel disease, chronic obstructive pulmonary disease, and glomerulonephritis.

Expression of the plasmacytoid dendritic cell marker BDCA-2 supports a spectrum of maturation among CD4+ CD56+ hematodermic neoplasms.

CD4+CD56+ hematodermic neoplasms are rare, aggressive hematopoietic malignancies usually presenting with cutaneous masses followed by a leukemic phase. The blastic morphology, CD56 expression and lack of definitive myeloid or T-cell markers initially resulted in assignment of this tumor to the NK-cell lineage. Accumulating evidence now suggests that these neoplasms represent malignant counterparts to the plasmacytoid dendritic cell. BDCA-2 is a cell surface protein whose expression is restricted to human plasmacytoid dendritic cells, in a differentiation stage-specific manner. In the current study, we assessed expression of BDCA-2 in CD4+CD56+ hematodermic neoplasms using a new antibody reagent we developed for use in fixed tissue sections. In 10 of 19 cases of CD4+CD56+ hematodermic neoplasm, BDCA-2 immunoreactivity was detected, whereas no expression was observed in NK-lineage tumors (0 of six). Interestingly, expression of terminal deoxynucleotidyl transferase, a marker of immaturity/blast stage, was significantly and negatively correlated with BDCA-2 in CD4+CD56+ hematodermic neoplasms whereas a positive correlation was observed between BDCA-2 and CD7. These findings demonstrate that BDCA-2 is expressed predominantly in the CD7+ subset of hematodermic neoplasms, and similar to non-neoplastic plasmacytoid dendritic cells, expression indicates a relatively more mature differentiation state. Clinical follow-up data confirm the aggressiveness of these tumors and suggests that BDCA-2 immunoreactivity, as identified here, may herald a significant reduction in survival.

Effect of exercise on immunologic factors in breast milk.

OBJECTIVE: Although it is well documented that breast milk provides optimal nutrition and immune benefits to the infant, factors that influence the immunologic composition of breast milk are less understood. A recent study reported that immunoglobulin A (IgA) levels in breast milk are lower after exercise compared with resting concentrations. However, the women exercised until exhaustion. The effect of moderate exercise on immunologic components in breast milk has not been reported. Therefore, the purpose of this study was to 1) compare the levels of immunologic compounds in breast milk of exercising women with the milk of sedentary women and 2) determine whether 30 minutes of moderate exercise affects immunologic properties of breast milk. METHODS: Exclusively lactating women were studied at 3 months' postpartum. Women in the exercise group (EG; n = 29) reported exercising aerobically at least 30 minutes/d for 3 days/wk, and women in the sedentary group (SG; n = 24) had exercised once a week or less during the previous 6 weeks. Cardiovascular fitness levels and concentrations of IgA, lactoferrin, and lysozyme in milk were measured. A subsample of the EG (n = 17) participated in a 30-minute exercise session at 75% of maximum heart rate and a rest session of 30 minutes of sitting rest on 2 separate days. Breast milk samples were collected before and 10 and 60 minutes after exercise and rest sessions. IgA, lactoferrin, and lysozyme concentrations were measured. RESULTS: Women in the EG had a higher level of cardiovascular fitness than women in the SG (39.7 +/- 1.0 vs 32.4 +/- 1.0 mL O(2)/kg/min). Milk concentrations of IgA, lactoferrin, or lysozyme were not significantly different between groups. In addition, there were no significant differences in the concentrations of IgA, lactoferrin, or lysozyme after moderate exercise compared with sitting rest. CONCLUSION: Moderate exercise during lactation improves cardiovascular fitness without affecting levels of IgA, lactoferrin, or lysozyme in breast milk.

MTA3 and the Mi-2/NuRD complex regulate cell fate during B lymphocyte differentiation.

The transcriptional repressor BCL-6 regulates B lymphocyte cell fate during the germinal center reaction by preventing terminal differentiation of B lymphocytes into plasma cells until appropriate signals are received. Here, we report a cofactor, MTA3, a cell type-specific subunit of the corepressor complex Mi-2/NuRD, for BCL-6-dependent cell fate determination. MTA3 is expressed in the same pattern in germinal centers as BCL-6. BCL-6 physically interacts with Mi-2/NuRD and this interaction is sensitive to BCL-6 acetylation status. Depletion of MTA3 by RNAi impairs BCL-6-dependent repression and alters the cell-specific transcriptional pattern characteristic of the B lymphocyte. Remarkably, exogenous expression of BCL-6 in a plasma cell line leads, in an MTA3-dependent manner, to repression of plasma cell-specific transcripts, reactivation of the B cell transcriptional program, expression of B lymphocyte cell surface markers, and reprogramming of cell fate.

MTA3, a Mi-2/NuRD complex subunit, regulates an invasive growth pathway in breast cancer.

Estrogen receptor is a key regulator of proliferation and differentiation in mammary epithelia and represents a crucial prognostic indicator and therapeutic target in breast cancer. Mechanistically, estrogen receptor induces changes in gene expression through direct gene activation and also through the biological functions of target loci. Here, we identify the product of human MTA3 as an estrogen-dependent component of the Mi-2/NuRD transcriptional corepressor in breast epithelial cells and demonstrate that MTA3 constitutes a key component of an estrogen-dependent pathway regulating growth and differentiation. The absence of estrogen receptor or of MTA3 leads to aberrant expression of the transcriptional repressor Snail, a master regulator of epithelial to mesenchymal transitions. Aberrant Snail expression results in loss of expression of the cell adhesion molecule E-cadherin, an event associated with changes in epithelial architecture and invasive growth. These results establish a mechanistic link between estrogen receptor status and invasive growth of breast cancers.

Use of real-time polymerase chain reaction to identify cell- and tissue-type-selective peptides by phage display.

Phage display approaches are used increasingly in efforts to identify cancer-specific binding peptides and antibodies. Phage-derived reagents are likely to have broad applications in diagnostic and research pathology. A critical element in the identification of cell or tissue-type-specific phage is the ability to reproducibly quantify bound or eluted phage at various stages of panning and screening procedures. Traditional biological assays of phage numbers such as plaque counting are commonly applied but are time-consuming, labor-intensive, and poorly reproducible. Moreover, enzyme immunoassays only support a subset of target types. Here, we report on the use of real-time polymerase chain reaction (PCR) (M13qPCR) in developing methods for identification of cell- and tissue-type-specific binding peptides. With M13qPCR, we demonstrate a >/=5 log(10) dynamic linear range with high reproducibility and significantly lower coefficients of variation (10 to 20%) than conventional methodology. Using M13qPCR in phage-panning experiments on live leukemia and prostate cancer cells, cancer-binding phage were identified. Similar results were obtained with conventional methodology such as flow cytometry. These results were extended to specific application of M13qPCR in panning phage libraries on tissue sections of prostate and breast cancer. With the PCR-based method, direct quantification of phage bound to tissue sections correlated well with staining intensity and yielded phage that bound to neoplastic and nonneoplastic epithelium. Thus, real-time PCR-based methodology significantly improves a number of aspects of conventional phage-panning protocols. Furthermore, identification of phage that bind specifically to diseased or cancerous tissue sections will likely be facilitated by this PCR-based approach.