Radiation pneumonitis: correlation of toxicity with pulmonary metabolic radiation response.

PURPOSE: To characterize the relationship between radiation pneumonitis (RP) clinical symptoms and pulmonary metabolic activity on post-treatment [(18)F]-fluorodeoxyglucose positron emission tomography (FDG-PET). PATIENTS AND METHODS: We retrospectively studied 101 esophageal cancer patients who underwent restaging FDG-PET/computed tomography imaging 3-12 weeks after completing thoracic radiotherapy. The National Institutes of Health Common Toxicity Criteria, version 3, was used to score the RP clinical symptoms. Linear regression was applied to the FDG-PET/computed tomography images to determine the normalized FDG uptake vs. radiation dose. The pulmonary metabolic radiation response (PMRR) was quantified as this slope. Modeling was performed to determine the interaction of PMRR, mean lung dose (MLD), and the percentage of lung receiving >20 Gy with RP outcomes. RESULTS: Of the 101 patients, 25 had Grade 0, 10 had Grade 1, 60 had Grade 2, 5 had Grade 3, and 1 had Grade 5 RP symptoms. Logistic regression analysis demonstrated that increased values of both MLD and PMRR were associated with a greater probability of RP clinical symptoms (p = 0.032 and p = 0.033, respectively). Spearman's rank correlation found no association between the PMRR and the dosimetric parameters (planning target volume, MLD, percentage of lung receiving >5-30 Gy). Twofold cross-validation demonstrated that the combination of MLD and PMRR was superior to either alone for assessing the development of clinical RP symptoms. The combined MLD (or percentage of lung receiving >20 Gy) and PMRR had a greater sensitivity and accuracy (53.3% and 62.5%, respectively) than either alone. CONCLUSION: The results of this study have demonstrated a significant correlation between RP clinical symptoms and the PMRR measured by FDG-PET/computed tomography after thoracic radiotherapy.

Mast cell activators: a new class of highly effective vaccine adjuvants.

Mast cells (MCs) have recently received recognition as prominent effectors in the regulation of immune cell migration to draining lymph nodes and lymphocyte activation. However, their role in the development of humoral immune responses is not clear. Here, we demonstrate that subcutaneous or nasal administration of small-molecule MC activators with vaccine antigens evokes large increases in antigen-specific serum immunoglobulin G (IgG) responses. These responses were MC dependent and correlated with increased dendritic cell and lymphocyte recruitment to draining lymph nodes. Nasal instillation of these formulations also evoked antigen-specific secretory IgA and provided protection against anthrax lethal toxin challenge in vitro and against vaccinia virus infection in vivo. Collectively, these results define the MC as an integral sensory arm of the adaptive immune system. Moreover, they highlight MC activators as a new class of vaccine adjuvants, capable of inducing protective antigen-specific immune responses through needle-free routes of administration.

Cytokine profiling for prediction of symptomatic radiation-induced lung injury.

PURPOSE: To analyze plasma cytokine profiles before the initiation of radiation therapy to define a cytokine phenotype that correlates with risk of developing symptomatic radiation-induced lung injury (SRILI). METHODS AND MATERIALS: Symptomatic radiation-induced lung injury was evaluated in 55 patients (22 with SRILI and 33 without SRILI), according to modified National Cancer Institute common toxicity criteria. These plasma samples were analyzed by the multiplex suspension bead array system (Bio-Rad Laboratories; Hercules, CA), which included the following cytokines: interleukin (IL)-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17, granulocyte/macrophage colony-stimulating factor, interferon-gamma, monocyte chemotactic protein 1, macrophage inflammatory protein 1beta, tumor necrosis factor alpha, and granulocyte colony-stimulating factor. RESULTS: Significant differences in the median values of IL-8 were observed between patients with and without SRILI. Patients who did not develop SRILI had approximately fourfold elevated levels of IL-8 as compared with patients who did subsequently develop SRILI. Significant correlations were not found for any other cytokine in this study, including transforming growth factor beta1. CONCLUSIONS: Patients with lower levels of plasma IL-8 before radiation therapy might be at increased risk for developing SRILI. Further studies are necessary to determine whether IL-8 levels are predictive of SRILI in a prospective trial and whether this marker might be used to determine patient eligibility for dose escalation.

Soluble alpha2-macroglobulin receptor is increased in endotracheal aspirates from infants and children after cardiopulmonary bypass.

OBJECTIVE: Cytokine dysregulation contributes to the systemic inflammatory response after cardiopulmonary bypass. Clearance of cytokine binding proteins may be important in the resolution of inflammation. Our aim was to determine whether the cytokine binding protein alpha 2 -macroglobulin and its soluble receptor were upregulated in endotracheal aspirates from infants and children undergoing cardiopulmonary bypass. METHODS: Seventy tracheal aspirates were collected before and after cardiopulmonary bypass from 35 infants and children undergoing surgical correction of congenital heart defects. alpha 2 -Macroglobulin and the soluble alpha 2 -macroglobulin receptor were identified by Western blot. With the use of multi-analyte cytokine profiling, pro-inflammatory and anti-inflammatory cytokines were quantified, normalized to total protein, and expressed as ratios. Paired t tests and Wilcoxon signed-rank tests were performed between prebypass and postbypass samples. Correlations were examined among alpha 2 -macroglobulin, soluble alpha 2 -macroglobulin receptor, cytokine ratios, and the clinical variables of cardiopulmonary bypass, aortic crossclamp, and circulatory arrest times. RESULTS: alpha 2 -Macroglobulin increased by 50% (mean densitometry increase 82,683 +/- 184,594, P = .012), and soluble alpha 2 -macroglobulin receptor increased by 17% (mean densitometry increase 506,148 +/- 687,037, P = .0001) after cardiopulmonary bypass. The ratio of interleukin-8/interleukin-4 increased by 136% ( P = .0001), and interleukin-8/interleukin-10 increased by 102% ( P = .001). The increase in soluble alpha 2 -macroglobulin receptor was positively correlated with the ratios of interleukin-8/interleukin-4 and interleukin-8/interleukin-10. There were no statistically significant positive correlations between the increase in alpha 2 -macroglobulin or soluble alpha 2 -macroglobulin receptor and measured clinical variables. CONCLUSIONS: We report for the first time the upregulation of alpha 2 -macroglobulin and soluble alpha 2 -macroglobulin receptor in tracheal aspirates after cardiopulmonary bypass in infants and children. Soluble alpha 2 -macroglobulin receptor correlates with increased alpha 2 -macroglobulin and a disproportionate increase in pro-inflammatory to anti-inflammatory cytokine ratios.

A CD91-positive subset of CD11c+ blood dendritic cells: characterization of the APC that functions to enhance adaptive immune responses against CD91-targeted antigens.

Dendritic cells (DC) and other APCs rely on a number of specialized receptors to facilitate the uptake and intracellular accumulation of Ags. In this capacity, APCs use receptor-mediated endocytosis to enhance Ag presentation and the stimulation of Ag-specific T cells. Studies have demonstrated that the targeted delivery of Ags in vivo to CD91/the low-density lipoprotein receptor-related protein (CD91/LRP) induces enhanced activation of the adaptive immune system. However, the APC that mediates these augmented, Ag-specific responses remains to be characterized. In this study, we show that a subset of CD11c(+) lineage-negative (lin(-)) DC expresses the scavenger receptor CD91/LRP and that these rare APC are primarily responsible for the T cell activation that occurs following CD91/LRP-mediated Ag uptake in whole blood. The targeting of Ags to CD91/LRP results in enhanced receptor-mediated uptake within both lin(-) DCs and monocytes, and this uptake results in markedly increased T cell activation. Finally, purified cellular populations were used to demonstrate that CD11c(+) lin(-) DC, but not monocytes, are capable of stimulating T cell activation following CD91/LRP-mediated Ag uptake. Therefore, CD11c(+) lin(-) DC use CD91/LRP to facilitate the uptake and subsequent presentation of an array of Ags complexed within the CD91/LRP ligand, the activated form of alpha2-macroglobulin (alpha2M*).

Scavenger receptor-A mediates gp96/GRP94 and calreticulin internalization by antigen-presenting cells.

gp96 (GRP94) elicits antigen-presenting cell (APC) activation and can direct peptides into the cross- presentation pathways of APC. These responses arise through interactions of gp96 with Toll-like (APC activation) and endocytic (cross-presentation) receptors of APC. Previously, CD91, the alpha2-macroglobulin receptor, was identified as the heat shock/chaperone protein receptor of APC. Recent data indicates, however, that inhibition of CD91 ligand binding does not alter gp96 recognition and uptake. Furthermore, CD91 expression is not itself sufficient for gp96 binding and internalization. We now report that scavenger receptor class-A (SR-A), a prominent scavenger receptor of macrophages and dendritic cells, serves a primary role in gp96 and calreticulin recognition and internalization. gp96 internalization and peptide re-presentation are inhibited by the SR-A inhibitory ligand fucoidin, although fucoidin was without effect on alpha2-macroglobulin binding or uptake. Ectopic expression of SR-A in HEK 293 cells yielded gp96 recognition and uptake activity. In addition, macrophages derived from SR-A-/- mice were substantially impaired in gp96 binding and uptake. These data identify new roles for SR-A in the regulation of cellular responses to heat shock proteins.

Mast cell-derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection.

Palpable swelling of regional lymph nodes is a common sequela of microbial infections but the mechanism responsible for the sequestration and subsequent coordination of lymphocyte responses within these dynamic structures remains poorly understood. Here we show that draining lymph nodes of mast cell-deficient mice did not demonstrate swelling after intradermal bacterial challenge. Testing of individual mast cell-derived products in this model indicated that tumor necrosis factor was the main mediator of nodal hypertrophy, whereas tryptase and histamine had no effect. After peripheral mast cell activation, both tumor necrosis factor concentrations and the recruitment of circulating T cells were increased within draining nodes. These results show a critical function for peripheral mast cell-derived tumor necrosis factor in regulating the hypertrophy of draining lymph nodes during infection.

Protease-activated receptor-2 signaling triggers dendritic cell development.

Dendritic cells (DC) are potent antigen-presenting cells that govern the effector cell responses of the immune system. DC are thought to continuously develop from circulating progenitors in a process that is accelerated by inflammatory stimuli. However, the physiological signals that regulate the development of DC from precursor cells have not been well defined. Here we show that a serine protease acting via protease-activated receptor-2 (PAR-2) stimulates the development of DC from bone marrow progenitor cells cultured in granulocyte-macrophage colony-stimulating factor and IL-4. DC fail to develop in bone marrow cultures treated with soy bean trypsin inhibitor, a serine protease inhibitor, but this inhibition is overcome by a PAR-2 agonist peptide. DC do not spontaneously develop from the bone marrow of PAR-2-deficient mice, but can be stimulated to do so by inflammatory mediators. These results suggest that endogenous serine proteases stimulate DC development in vitro. Thus, serine proteases may help trigger adaptive immune responses in vivo.

The role of Grp 78 in alpha 2-macroglobulin-induced signal transduction. Evidence from RNA interference that the low density lipoprotein receptor-related protein is associated with, but not necessary for, GRP 78-mediated signal transduction.

The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds to many proteins, some of which trigger signal transduction. Receptor-recognized forms of alpha(2)-Macroglobulin (alpha(2)M*) bind to LRP, but the pattern of signal transduction differs significantly from that observed with other LRP ligands. For example, neither Ni(2+) nor the receptor-associated protein, which blocks binding of all known ligands to LRP, block alpha(2)M*-induced signal transduction. In the current study, we employed alpha(2)-macroglobulin (alpha(2)M)-agarose column chromatography to purify cell surface membrane binding proteins from 1-LN human prostate cancer cells and murine macrophages. The predominant binding protein purified from 1-LN prostate cancer cells was Grp 78 with small amounts of LRP, a fact that is consistent with our previous observations that there is little LRP present on the surface of these cells. The ratio of LRP:Grp 78 is much higher in macrophages. Flow cytometry was employed to demonstrate the presence of Grp 78 on the cell surface of 1-LN cells. Purified Grp 78 binds to alpha(2)M* with high affinity (K(d) approximately 150 pm). A monoclonal antibody directed against Grp 78 both abolished alpha(2)M*-induced signal transduction and co-precipitated LRP. Ligand blotting with alpha(2)M* showed binding to both Grp 78 and LRP heavy chains in these preparations. Use of RNA interference to silence LRP expression had no effect on alpha(2)M*-mediated signaling. We conclude that Grp 78 is essential for alpha(2)M*-induced signal transduction and that a "co-receptor" relationship exists with LRP like that seen with several other ligands and receptors such as the uPA/uPAR (urinary type plasminogen activator or urokinase/uPA receptor) system.

Cutting edge: CD91-independent cross-presentation of GRP94(gp96)-associated peptides.

GRP94(gp96) elicits CD8(+) T cell responses against its bound peptides, a process requiring access of its associated peptides into the MHC class I cross-presentation pathway of APCs. Entry into this pathway requires receptor-mediated endocytosis, and CD91 (low-density lipoprotein receptor-related protein) has been reported to be the receptor mediating GRP94 uptake into APC. However, a direct role for CD91 in chaperone-based peptide Ag re-presentation has not been demonstrated. We investigated the contribution of CD91 to GRP94 cell surface binding, internalization, and trafficking in APCs. Whereas a clear role for CD91 in alpha(2)-macroglobulin binding and uptake was readily obtained, the addition of excess CD91 ligand, activated alpha(2)-macroglobulin, or receptor-associated protein, an antagonist of all known CD91 ligands, did not affect GRP94 cell surface binding, receptor-mediated endocytosis, or peptide re-presentation. These data identify a CD91-independent, GRP94 internalization pathway that functions in peptide Ag re-presentation.