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.

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.