Novel tools for functional analysis of CD11c: activation-specific, activation-independent, and activating antibodies.

Functions and binding properties of four CD11c-specific mAbs are described here. The mAb 496B stimulated, while 496K inhibited ligand binding of CD11c. The stimulatory mAb, 496B, as well as the inhibitory mAbs BU15 and 496 K appear to act allosterically, as they do not bind the CD11c I domain. The mAb 3.9 bound preferentially to activated forms of CD11c and the binding was divalent cation dependent. CD11c binding to 3.9 recapitulates many of the integrin-ligand interactions. Our data suggest that 3.9 is a competitive antagonist, BU15 and 496K are allosteric antagonists, and 496B is an allosteric agonist of CD11c. These mAbs provide a set of tools to study the functions of the dendritic cell marker, CD11c.

Various techniques of contouring the rectum and their impact on rectal dose-volume histograms.

Late rectal bleeding resulting from radiotherapy has been correlated with rectal dose-volume histograms (DVHs). The techniques of contouring the rectum have been inconsistent within the literature, making interpretations of DVHs difficult. This study was conducted to investigate the impact on rectal DVHs when using different ways of contouring. Ten prostate cancer patients were treated with a 4-field box-technique and received 70 Gy. Six different ways of contouring the rectum were implemented by using 3 different cross-sections and 2 different lengths. The 3 different cross sections were (1) anterior rectal wall (arw): only the anterior half of the rectal wall was contoured; (2) whole rectal wall (wrw): the entire rectal wall was contoured but excluding the rectal contents; and (3) the rectum (rec): including the rectal contents. Two different lengths were used for the above 3 volumes: (1) long (Lg): cranial border starting at where the rectum turned horizontally into the sigmoid and the caudal border 2 cm below the prostatic apex; (2) short (Sh): from 2 cm above to 2 cm below the prostate. Therefore, a total of 6 different volumes (Sh arw, Lg arw, Sh wrw, Lg wrw, Sh rec, and Lg rec) were generated. DVHs of all 6 volumes were compared with the y-axis being percentage volume as well as absolute volume (cc). When using percentage volume as the y-axis, Sh arw gave an impression that a large portion of rectum (median of 41.8%) received high dose (greater than 90% prescribed dose), while the Lg wrw and Lg rec revealed a smaller portion of rectum (median of 17.1% and 14.7%, respectively) received high dose. The other contours were somewhere in-between. When using absolute volume as the y-axis, the DVHs of the 4 rectal volumes, excluding the rectal content (Sh and Lg arw, Sh and Lg wrw), merged at doses greater than 80% to 85% prescription, therefore providing similar information within these high-dose regions. Configurations of rectal DVHs varied drastically with different techniques of contouring and may lead to different interpretations. By using absolute volume (cc) as the y-axis, the shape of the 4 rectal DVHs, excluding the rectal content, were similar in the high-dose region. Reporting rectal toxicities in relations to DVHs using absolute volume, as well as percentage volume, may eliminate inconsistencies secondary to different methods of contouring.

Selective role of PI3K delta in neutrophil inflammatory responses.

Although members of the class I phosphoinositide 3-kinases (PI3Ks) have been implicated in neutrophil inflammatory responses, the contribution of the individual PI3K isoforms in neutrophil activation has not been tractable with the non-selective inhibitors, LY294002 and wortmannin. We have developed a novel series of PI3K inhibitors that is selective for PI3K delta, an isoform expressed predominantly in hematopoietic cells. In addition to being selective between members of class I PI3Ks, representatives of these inhibitors such as IC980033 and IC87114 did not inhibit any protein kinases tested. Utilizing these inhibitors we report here a novel role for PI3K delta in neutrophil activation. Inhibition of PI3K delta with IC980033 and IC87114 blocked both fMLP- and TNF1 alpha-induced neutrophil superoxide generation and elastase exocytosis. The PI3K delta inhibitor IC87114 also blocked TNF1 alpha-stimulated elastase exocytosis from neutrophils in a mouse model of inflammation. To our knowledge, this is the first in vivo efficacy demonstration of a PI3K delta inhibitor in an animal model. Inhibition of PI3K delta, however, had no effect on in vitro neutrophil bactericidal activity and Fc gamma R-stimulated superoxide generation. Thus, PI3K delta plays an essential role in certain signaling pathways of neutrophil activation and appears to be an attractive target for the development of an anti-inflammatory therapeutic.

Essential role of phosphoinositide 3-kinase delta in neutrophil directional movement.

Neutrophil chemotaxis is a critical component of the innate immune response. Neutrophils can sense an extremely shallow gradient of chemoattractants and produce relatively robust chemotactic behavior. This directional migration requires cell polarization with actin polymerization occurring predominantly in the leading edge. Synthesis of phosphatidylinositol (3,4,5) trisphosphate (PIP3) by phosphoinositide 3-kinase (PI3K) contributes to asymmetric F-actin synthesis and cell polarization during neutrophil chemotaxis. To determine the contribution of the hemopoietic cell-restricted PI3K delta in neutrophil chemotaxis, we have developed a potent and selective PI3K delta inhibitor, IC87114. IC87114 inhibited polarized morphology of neutrophils, fMLP-stimulated PIP3 production and chemotaxis. Tracking analysis of IC87114-treated neutrophils indicated that PI3K delta activity was required for the directional component of chemotaxis, but not for random movement. Inhibition of PI3K delta, however, did not block F-actin synthesis or neutrophil adhesion. These results demonstrate that PI3K delta can play a selective role in the amplification of PIP3 levels that lead to neutrophil polarization and directional migration.