Bacterial LPS is a pluripotent agonist for PMNs. Although it does not activate the NADPH-dependent oxidase directly, LPS renders PMNs more responsive to other stimuli, a phenomenon known as "priming." Since the mechanism of LPS-dependent priming is incompletely understood, we investigated its effects on assembly and activation of the NADPH oxidase. LPS pretreatment increased superoxide (O2-) generation nearly 10-fold in response to N-formyl methionyl leucyl phenylalanine (fMLP). In a broken-cell O2--generating system, activity was increased in plasma membrane-rich fractions and concomitantly decreased in specific granule-rich fractions from LPS-treated cells. Oxidation-reduction spectroscopy and flow cytometry indicated LPS increased plasma membrane association of flavocytochrome b558. Immunoblots of plasma membrane vesicles from LPS-treated PMNs demonstrated translocation of p47-phox but not of p67-phox or Rac2. However, PMNs treated sequentially with LPS and fMLP showed a three- to sixfold increase (compared with either agent alone) in plasma membrane-associated p47-phox, p67-phox, and Rac2, and translocation paralleled augmented O2- generation by intact PMNs. LPS treatment caused limited phosphorylation of p47-phox, and plasma membrane-enriched fractions from LPS- and/or fMLP-treated cells contained fewer acidic species of p47-phox than did those from cells treated with PMA. Taken together, these studies suggest that redistribution of NADPH oxidase components may underlie LPS priming of the respiratory burst.

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The respiratory burst of human neutrophils is primed by a number of pro-inflammatory stimuli, including tumor necrosis factor-␣ (TNF␣) and lipopolysaccharide (LPS); however, the mechanism of priming remains unknown. LPS has been shown previously to increase membrane expression of flavocytochrome b 558 , a component of the NADPH oxidase. This study shows that TNF␣ also increases membrane expression of flavocytochrome b 558 . Mitogen-activated protein kinase (MAPK) modules have been implicated in the action of priming agents. Pharmacologic inhibitors of MAPKs, SB203580 and PD098059, revealed that priming of the respiratory burst and up-regulation of flavocytochrome b 558 are dependent on p38 MAPK but not on extracellular-signal regulated kinase (ERK). TNF␣ and LPS primed respiratory burst activity and increased membrane expression of CD35 and CD66b, specific markers of secretory vesicles and specific granules that contain flavocytochrome b 558 , with similar time courses and concentration dependences. These processes also required p38 MAPK but were independent of ERK. TNF␣ failed to prime respiratory burst activity or to increase membrane CD35 expression in enucleated neutrophil cytoplasts. These data suggest that one mechanism by which TNF␣ and LPS prime neutrophil respiratory burst activity is by increasing membrane expression of flavocytochrome b 558 through exocytosis of intracellular granules in a process regulated by p38 MAPK.

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activation involves assembly of membrane-integrated cytochrome b558 comprising gp91(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains, and the small G protein Rac. In the present study, we show that the N-terminal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail of p22(phox) with high affinity (KD = 0.34 microM). The binding is specific to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro156-containing proline-rich sequence but not other putative SH3 domain-binding sites of p22(phox). Replacement of Trp193 by Arg in the N-terminal SH3 domain completely abrogates the association with p22(phox). A mutant p47(phox) with this substitution is incapable of supporting superoxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal SH3 domain of p47(phox) and the proline-rich region of p22(phox) is essential for activation of the NADPH oxidase.

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We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91 phox ) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91 phox gene revealed a single-base mutation (C 3 T) at position ؊53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position ؊53 of the gp91 phox promoter, and the mutation at position ؊53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position ؊50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position ؊56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions ؊53 and ؊50 significantly reduced the gp91 phox promoter activity; however, the mutation at position ؊56 did not affect the promoter activity. In transient cotransfection study, PU

Interferon (IFN)-␥ induces the expression of the gp91phox gene both during myeloid differentiation and also in mature phagocytes through several cis-elements and their binding proteins. To find new cis-elements for this induction, transient expression assays were performed using a reporter gene driven by serially truncated gp91 phox promoters in U937 cells. The results suggest that a critical cis-element for induction exists in the region from bp ؊115 to ؊96 of the promoter. Site-directed mutagenesis showed that a ␥-activated sequence (GAS) element at bp ؊100 (؊100GAS) of the gp91 phox promoter plays a pivotal role for the IFN-␥-dependent activity of the bp ؊115 to ؉12 region of the gp91 phox promoter. Electrophoretic mobility shift assays using several GAS competitors and specific antibodies indicated that phosphorylated STAT-1␣ specifically binds to the ؊100GAS. Site-directed mutagenesis showed that an interferon-stimulated response element (ISRE) at bp ؊88 (؊88ISRE) mediates the induction of the gene by IFN-␥ in cooperation with ؊100GAS. Electrophoretic mobility shift assay showed that IRF-1 dominantly binds to ؊88ISRE in an IFN-␥-dependent fashion. These results demonstrate a new mechanism for IFN-␥-induced transcription of the gp91 phox gene by the cooperation of STAT-1␣ and IRF-1 binding to ؊100GAS and ؊88ISRE, respectively.

Aim: We planned a randomized, open-label trial to evaluate differences between pre-emptive and reactive skin treatment for panitumumab (Pmab)-associated skin toxicities in Japanese patients with metastatic colorectal cancer. Patients & methods:Patients receiving third-line Pmab-containing regimens were randomized to pre-emptive or reactive treatment. The primary end point was the cumulative incidence of ≥grade 2 skin toxicities during 6 weeks. Retrospectively, a dermatologist reviewed skin toxicities, in a blinded manner. Results: A total of 95 patients were enrolled (pre-emptive: 47, reactive: 48). The primary end point was achieved (21.3 and 62.5% [risk ratio: 0.34; p < 0.001], for preemptive and reactive treatment, respectively). A similar trend was observed in central review. Conclusion: Pre-emptive skin treatment could reduce the severity of Pmab-associated skin toxicities in Japanese metastatic colorectal cancer patients. KEYWORDS• colorectal cancerThe EGF receptor (EGFR) has been validated as a therapeutic target in metastatic colorectal cancer (mCRC). Panitumumab (Pmab) [1,2], a fully human immunoglobulin G2 monoclonal antibody, has demonstrated efficacy and safety for KRAS wild-type mCRC in pivotal global Phase III studies [3][4][5][6], and is used widely around the world. Some clinical trials have also revealed that the characteristic For reprint orders, please contact: reprints@futuremedicine.com