Effect of the local energy distribution of x-ray beams generated through inverse Compton scattering in dual-energy imaging applications.

X-ray sources based on the inverse Compton interaction between a laser and a relativistic electron beam are emerging as a promising compact alternative to synchrotron for the production of intense monochromatic and tunable radiation. The emission characteristics enable several innovative imaging techniques, including dual-energy K-edge subtraction (KES) imaging. The performance of these techniques is optimal in the case of perfectly monochromatic x-ray beams, and the implementation of KES was proven to be very effective with synchrotron radiation. Nonetheless, the features of inverse Compton scattering (ICS) sources make them good candidates for a more compact implementation of KES techniques. The energy and intensity distribution of the emitted radiation is related to the emission direction, which means different beam qualities in different spatial positions. In fact, as the polar angle increases, the average energy decreases, while the local energy bandwidth increases and the emission intensity decreases. The scope of this work is to describe the impact of the local energy distribution variations on KES imaging performance. By means of analytical simulations, the reconstructed signal, signal-to-noise ratio, and background contamination were evaluated as a function of the position of each detector pixel. The results show that KES imaging is possible with ICS x-ray beams, even if the image quality slightly degrades at the detector borders for a fixed collimation angle and, in general, as the beam divergence increases. Finally, an approach for the optimization of specific imaging tasks is proposed by considering the characteristics of a given source.

Evaluation of microcalcification contrast in clinical images for digital mammography and synthetic mammography.

PURPOSE: The aim of this work was to compare, in a clinical study, digital mammography and synthetic mammography imaging by evaluating the contrast in microcalcifications of different sizes. METHODS: A retrospective review of microcalcifications from 46 patients was undertaken. A Hologic 3-Dimensions mammography system and a HD Combo protocol was used for simultaneous acquisition of the digital and synthetic images. Microcalcifications were classified in accordance with their size, and patient breast images were classified in accordance with their density as adipose, moderately dense and dense. The contrast of the microcalcifications was measured and the contrast ratio between synthetic and digital images was compared. An additional qualitative assessment of the images was presented to correlate the conspicuity of the microcalcifications with the suppression of the structure noise. RESULTS: Microcalcifications in adipose background always exhibit a comparable or better contrast on synthetic images, regardless their size. For moderately dense background, synthetic images show a better contrast in 91.2 % of cases for small microcalcifications and in 90.9 % of cases for large microcalcifications. For a dense background, better contrast is seen in 89.5 % of cases for small microcalcifications, and in 85.7 % of cases for large microcalcifications. The contrast ratio increases with increasing breast glandularity. The suppression of structure noise also contributes to the enhancement of microcalcifications in the synthetic images. CONCLUSIONS: Synthetic mammography imaging is superior to digital mammography imaging in terms of microcalcification contrast, regardless their size and breast density.

Comprehensive data set to include interference effects in Monte Carlo models of x-ray coherent scattering inside biological tissues.

Interference effects are included in the x-ray coherent scattering models used in Monte Carlo codes by modifying each material form factor through a proper interference function, which is obtained directly from the measured scattering pattern. This approach is effective for non-biological materials, but it is impractical for biological tissues, due the wide composition variability they can feature. Instead, a given biological sample can be considered as a proper mixture of four basis materials: fat, water, collagen and calcium hydroxyapatite. The sample form factor can then be obtained through a weighted mean of the form factors of the basis materials, which include interference effects. Here, we fully demonstrate the validity of the proposed segmentation method by applying it to 31 biological tissue samples whose form factors are available in the literature. The segmentation, namely the determination of the optimal weight of the basis components, was carried out through a multiple linear regression or, in some cases, by using a controlled trial and error sequence. The form factors of the basis materials were extracted from previous works and elaborated to include more scattering features. In particular, they were interpolated at a denser grid. Furthermore, the data measured separately in wide angle and small angle regimes, for fat and collagen, were merged. In general, a very good agreement was obtained between the original sample and the calculated mixture, being the mean relative difference of their scattering profiles and their attenuation coefficients ∼10%. The segmentation method is fully supported by our extension to the Geant4 model of x-ray coherent scattering, which was used to compare simulated scatter distributions with known experimental data. The developed Geant4 code and a series of molecular form factors, including those of the basis materials, are freely downloadable from a dedicated web repository.

BriXS, a new X-ray inverse Compton source for medical applications.

MariX is a research infrastructure conceived for multi-disciplinary studies, based on a cutting-edge system of combined electron accelerators at the forefront of the world-wide scenario of X-ray sources. The generation of X-rays over a large photon energy range will be enabled by two unique X-ray sources: a Free Electron Laser and an inverse Compton source, called BriXS (Bright compact X-ray Source). The X-ray beam provided by BriXS is expected to have an average energy tunable in the range 20-180 keV and intensities between 1011 and 1013 photon/s within a relative bandwidth ΔE/E=1-10%. These characteristics, together with a very small source size (~20 µm) and a good transverse coherence, will enable a wide range of applications in the bio-medical field. An additional unique feature of BriXS will be the possibility to make a quick switch of the X-ray energy between two values for dual-energy and K-edge subtraction imaging. In this paper, the expected characteristics of BriXS will be presented, with a particular focus on the features of interest to its possible medical applications.

Identification of fibronectin receptors on T lymphocytes.

We report the identification of fibronectin receptors on thymocytes and T lymphoma cells. Affinity chromatography of extracts of the T cell lymphoma, WR16.1, on a fibronectin-Sepharose column combined with specific elution using a synthetic peptide containing the cell attachment-promoting sequence, arginine-glycine-aspartic acid, yielded two polypeptide components having apparent molecular masses of approximately 160 kD reduced and 175 and 150 kD nonreduced. Immunoprecipitations from surface-iodinated WR16.1 cells or fibronectin-adherent thymocytes using a rabbit antiserum raised against the fibronectin receptor that is present on human fibroblasts revealed, in each case, the same two radiolabeled components. In contrast, immunoprecipitation from fibronectin-nonadherent T lymphoma cells, designated WR2.3, revealed the presence of only the smaller subunit. Although the lymphocyte receptor and the fibronectin receptor identified on fibroblasts share immunologic determinants, they differ in that the molecular mass of the lymphocyte protein is larger. Moreover, trypsinization of either thymocytes or the WR16.1 T lymphoma cells resulted in a subsequent loss of their ability to adhere to fibronectin-coated substrates and a reduction in the electrophoretic mobility of each of the polypeptide chains of the fibronectin receptor present on their surfaces. These changes, however, were not observed with normal rat kidney fibroblasts or mouse 3T3 fibroblasts in response to trypsinization. The data establish the existence on normal lymphocytes of fibronectin receptors that are quite similar to those found on fibroblasts. The possible function of this molecule on thymocytes is discussed.

Preferential expression of fibronectin receptors on immature thymocytes.

Fibronectin-adherent (FNR+) thymocytes are enriched for immature (CD4-8-) and large (CD4+8+) cells, and depleted of mature (CD4-8+ and CD4+8-) and nonmature small (CD4+8+) cells. Among purified CD4-8- thymocytes, cells with the surface marker J11d and the IL-2 receptor, which can give rise to all other thymocyte subsets, showed selective attachment to fibronectin. Analysis of FNR+ thymocytes showed that such cells are greatly enriched for cells in cycle. Additionally, FNR+ cells expressed low levels of T cell receptor. These results suggest a role for the fibronectin receptor during the early, proliferative phase of thymocyte differentiation. The data suggest that loss of the fibronectin receptor is a hallmark of cells that have become committed either to functional maturation or to programmed cell death.

Inverse Compton radiation: a novel x-ray source for K-edge subtraction angiography?

Coronary angiography is clinically used worldwide to diagnose diseases of coronary arteries. Despite its effectiveness, this technique is quite invasive and it is associated with significant risks due to the arterial catheterisation needed to inject the contrast agent. A valid alternative is using the K-edge subtraction (KES) method, which is based on the subtraction of two images acquired at energies bracketing the K-edge of the contrast element. The enhanced sensitivity of KES allows the intravenous injection of the contrast agent, thus reducing the risks of catheterisation. This technique can be effectively implemented by using intense and quasi-monochromatic x-ray beams. Synchrotron radiation has been proven to work well for this purpose, but its cost and size prevent a widespread clinical application. Inverse Compton sources are among the most promising innovative sources of intense and quasi-monochromatic x-rays. These sources are intrinsically more compact than those based on synchrotron radiation. In this work, the potential application of inverse Compton radiation to KES angiography is investigated. To this purpose, after a short review of the physics behind the inverse Compton process, an analytical framework is described. The proposed model is based on the application of the KES algorithm to calculate the SNR of details inside a suitable mathematical phantom. That allowed us to identify the characteristics of an inverse Compton source required for KES imaging. In particular, it was estimated that a photon fluence of 108 ph mm-2 is necessary to detect signals of clinical interest. Novel sources based on inverse Compton promise to achieve this requirement with an acquisition time of few hundreds of ms. This feature, together with compactness, broad two-dimensional radiation field, absence of harmonic contamination and the ability to deliver high photon fluxes also at high energies, makes this kind of sources promising for KES angiography and other diagnostic applications.

Geant4 implementation of inter-atomic interference effect in small-angle coherent X-ray scattering for materials of medical interest.

An extension to Geant4 Monte Carlo code was developed to take into account inter-atomic (molecular) interference effects in X-ray coherent scattering. Based on our previous works, the developed code introduces a set of form factors including interference effects for a selected variety of amorphous materials useful for medical applications, namely various tissues and plastics used to build phantoms. The code is easily upgradable in order to include new materials and offers the possibility to model a generic tissue as a combination of a set of four basic components. A dedicated Geant4 application for the simulation of X-ray diffraction experiments was created to validate the proposed upgrade of Rayleigh scattering model. A preliminary validation of the code obtained through a comparison with EGS4 and an experiment is presented, showing a satisfactory agreement.

Soluble vascular cell adhesion molecule (VCAM)-Fc fusion protein induces leukotriene C4 secretion in platelet-activating factor-stimulated eosinophils.

Eosinophil adhesion to vascular cell adhesion molecule-1 (VCAM-1) is important for cellular recruitment into allergic inflammatory sites. To determine whether eosinophil adhesion to VCAM-1 affects cell function, leukotriene C4 (LTC4) was measured. Human eosinophils were incubated with platelet-activating factor (PAF) in the presence or absence of soluble VCAM-Fc fusion protein (sVCAM-Fc) or immobilized VCAM-Fc. sVCAM-Fc induced a concentration-dependent increase in LTC4 secretion, which was dependent on the presence of PAF and not blocked by cyclic peptides shown to inhibit alpha4beta1-dependent adhesion. Likewise, soluble ICAM-Fc induced a concentration-dependent LTC4 secretion. LTC4 secretion was induced by the calcium ionophore, A23187, and the combination of sVCAM-Fc and A23187 had synergistic properties. It is interesting to note that Mn2+ or anti-beta1 monoclonal antibody, TS2/16, inhibited LTC4 secretion induced by sVCAM-Fc and PAF. Eosinophil adhesion to VCAM-Fc or interleukin-1 beta-stimulated endothelial cells did not induce LTC4 secretion. These data suggest that sVCAM-Fc-induced LTC4 secretion depends on distinct signals from those of eosinophil adhesion.

Fibronectin-enhanced attachment of gelatin-coated erythrocytes to isolated hepatic Kupffer cells.

The phagocytic process is a combination of a sequence of events which includes a recognition attachment phase and a subsequent internalization phase. The present study was designed to investigate the effect of plasma fibronectin on the attachment and ingestion of gelatinized sheep erythrocytes to isolated rat Kupffer cells in a monolayer assay. Kupffer cells were isolated by sequential collagenase-pronase digestion followed by metrizamide density gradient centrifugation and subsequent adherence to plastic. Classification as Kupffer cells was confirmed by the presence of functional Fc receptors, a positive peroxidase reaction, and phagocytic activity. Purified plasma fibronectin as well as rat serum containing fibronectin promoted attachment of gelatinized fixed sheep erythrocytes to Kupffer cells in a dose-dependent manner, whereas fibronectin-deficient serum did not. Heparin did not enhance the fibronectin-mediated attachment or ingestion of gelatinized sheep erythrocytes at lower particle doses, whereas at higher particle doses heparin augmented the response. These results indicate that fibronectin can enhance the binding and ingestion of foreign gelatin-coated particulates by Kupffer cells.

High-affinity binding of fibronectin to cultured Kupffer cells.

Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative "fibronectin receptors" per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

Mapping of MCP-1 functional domains by peptide analysis and site-directed mutagenesis.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the beta chemokine family which acts through specific seven transmembrane receptors to recruit monocytes, basophils, and T lymphocytes to sites of inflammation. To identify regions of the human MCP-1 protein which are important for its biological activity, we have synthesized domain-specific peptides and tested their ability to antagonize MCP-1 binding and chemotaxis in THP-1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13-35 inhibits MCP-1 binding and chemotactic activity, while peptides representing the amino-terminus (amino acids 1-10), second loop (amino acids 37-51), and carboxy-terminus (amino acids 56-71) of MCP-1 have no effect. In addition, we have found that cyclization of the first loop peptide by disulfide linkage and blocking the C-terminus of the peptide by amidation increases the activity of this peptide to block MCP-1 binding and chemotaxis. In order to specifically identify amino acid residues within the first loop that are crucial for MCP-1 functional activity, we have substituted alanine for tyrosine (Y13A) or arginine (R18A) in MCP-1 recombinant proteins. While baculovirus produced wild type and R18A MCP-1 proteins are indistinguishable in their ability to induce THP-1 chemotaxis and show modest effects in binding activity compared to commercially available recombinant MCP-1 protein, the Y13A point mutation causes a dramatic loss in function. The identification of functional domains of MCP-1 will assist in the design of MCP-1 receptor antagonists which may be clinically beneficial in a number of inflammatory diseases.

Reversal of opsonic deficiency in surgical, trauma, and burn patients by infusion of purified human plasma fibronectin. Correlation with experimental observations.

Plasma fibronectin deficiency has been documented in critically ill surgical, trauma, and burn patients. Human plasma fibronectin was isolated by gelatin-Sepharose affinity chromatography and evaluated with respect to its opsonic activity following pasteurization, its in vivo clearance kinetics, and its short-term influence on cardiovascular hemodynamics in postoperative septic sheep. Six patients with low plasma fibronectin levels were also evaluated with respect to temporal changes of immunoreactive fibronectin and opsonic activity following infusion of fibronectin at a dose calculated to elevate the plasma fibronectin level to 400 micrograms/ml. With utilization of three different in vitro radioisotopic phagocytic assays, i.e., liver slice assay, peritoneal macrophage monolayer assay, and Kupffer cell monolayer assay, retention of opsonic activity by fibronectin following pasteurization was documented. The normal biphasic kinetics associated with plasma clearance of fibronectin were also not altered by pasteurization. In postoperative septic sheep with hemodynamic instability, intravenous infusion of 500 mg of purified human fibronectin initiated no abnormal hemodynamic response. Indeed, as compared with placebo, the infusion of fibronectin into the postoperative septic sheep resulted in a more stable systemic vascular resistance and pulmonary vascular resistance with a higher arterial pressure. It also elevated immunoreactive fibronectin levels (p less than 0.05) and increased opsonic activity (p less than 0.05). Surgical, trauma, and burn patients (ages 18 to 80 years) with low plasma fibronectin levels (160 to 236 micrograms/ml) manifested no disturbance in cardiovascular, respiratory, or hematologic parameters following fibronectin infusion (590 to 988 mg per patient), but did display an early increase of opsonic activity. This standardized, pasteurized, and opsonically active preparation of purified human plasma fibronectin (5.0 mg/ml after reconstitution) has utility for future randomized clinical trials in injured patients with sepsis.

Experimental and Monte Carlo simulated spectra of a liquid-metal-jet x-ray source.

A prototype x-ray system based on a liquid-metal-jet anode was evaluated within the framework of the LABSYNC project. The generated spectrum was measured using a CZT-based spectrometer and was compared with spectra simulated by three Monte Carlo codes: MCNPX, PENELOPE and EGS5. Notable differences in the simulated spectra were found. These are mainly attributable to differences in the models adopted for the electron-impact ionization cross section. The simulation that more closely reproduces the experimentally measured spectrum was provided by PENELOPE.

Type I interferon correlates with serological and clinical manifestations of SLE.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease affecting multiple organ systems triggered by the production of autoantibodies. Previous clinical studies in humans and murine models suggest that type I interferons (IFNs) are important for the initiation and potentiation of SLE activity. METHODS: 65 consecutive patients with SLE were identified from the University of California, San Francisco Lupus Clinic with moderate-severe disease activity. 94 serological samples were collected. Type I IFN levels and the ability of plasma to induce expression of several surface markers of dendritic cell maturation were measured. RESULTS: Type I IFN levels correlated with the presence of cutaneous manifestations, and there was a trend towards correlation with renal disease. No correlation was found between type I IFN levels and neurological disease. Type I IFN levels correlated positively with the SLEDAI score and anti-dsDNA levels and inversely with C3 levels. Interestingly, type I IFN levels were highest in African American patients. SLE plasma also induced the expression of MHC class I, CD38, and CD123 on monocytes, and was blocked by the addition of a monoclonal antibody to IFNAR1. CONCLUSIONS: The pathogenic role of type I IFN is suggested by the induction of cell surface markers for dendritic cell maturation. The potential therapeutic utility of antibodies directed to either type I IFN or IFNAR1/IFNAR2 may be of interest in further studies.

T-lymphocyte differentiation and the extracellular matrix: identification of a thymocyte subset that attaches specifically to fibronectin.

A population of murine thymocytes adheres specifically to fibronectin but not to vitronectin, laminin, or collagen type I. The interaction of these thymocytes with fibronectin could be inhibited by the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which comprises the previously identified cell-attachment determinant of the molecule, suggesting that the cell attachment site on fibronectin is recognized by these cells. A similar peptide, in which the aspartate residue had been replaced with glutamate, had no effect on this adhesion. The fibronectin-adherent thymocytes were found to be cortisone-sensitive; to bind peanut agglutinin; to have a Thy-1.2+, Ia- surface phenotype; and to express H-2 antigen only weakly on their surface. In addition, approximately 80% of the fibronectin-adherent cells expressed L3T4 and 80% expressed Ly-1 on their surface, whereas greater than 95% were positive for Ly-2. The data suggest that these cells, which constitute 10% of all thymic lymphocytes, are cortical thymocytes. We propose that their adhesion to fibronectin may be important for their differentiation. The binding to fibronectin provides a means to selectively isolate these cells for study.

Fibronectin augments anti-CD3-mediated IL-2 receptor (CD25) expression on human peripheral blood lymphocytes.

The extracellular matrix (ECM) is composed of a number of macromolecules that promote cell adhesion, cell migration, and differentiation. Receptors for these molecules have been identified and belong to a superfamily of cell surface proteins, collectively known as the integrins. In this study, we show that the matrix protein fibronectin (FN) acts synergistically with immobilized anti-CD3 antibody to promote proliferation of total human peripheral blood lymphocytes (HPBL) in the absence of exogenous IL-2. Proliferation was inhibited by both the alpha 5 beta 1 and alpha 4 beta 1 recognition peptides. ARG-GLY-ASP (RGD), and GLU-ILE-LEU-ASP-VAL-PRO-SER-THR (EILDVPST), respectively. Expression of CD25 (IL-2 receptor) was significantly higher on cells cultured on anti-CD3 and FN, indicative of T-cell activation. Additionally, cells cultured on immobilized anti-CD3 and FN for 3 days showed increased adhesion to FN and increased forward light scatter/side scatter profile. Synthesis of both IL-1 and to a lesser extent IL-2 was elevated in supernatants from cultures containing both anti-CD3 and FN. These data are consistent with published reports which demonstrate that ECM proteins can act as costimulants of lymphocyte proliferation. Finally, our results show that cells cultured on anti-CD3 antibody and FN have an activated phenotype and that cytokines may be involved in this process.

Cytokine induction of monocyte chemoattractant protein-1 gene expression in human endothelial cells depends on the cooperative action of NF-kappa B and AP-1.

Chemokines are potent mediators of cell migration and activation and therefore play an essential role in early events of inflammation. In conjunction with cell adhesion molecules, chemokines help to localize cells to a specific site and enhance the inflammatory reaction at the site. Clinically, elevated levels of chemokines have been found in a variety of inflammatory diseases. The prototype C-C chemokine is monocyte chemoattractant protein-1 (MCP-1) which is synthesized by variety of cell types including endothelial cells in response to a variety of stimuli. MCP-1 is a major chemoattractant for monocytes, T lymphocytes, and basophils. In the present study, we investigated the factors involved in cytokine-induced MCP-1 gene expression in human endothelial cells. We present evidence that the nuclear factor (NF)-kappa B-like binding site and the AP-1 binding site located 90 and 68 base pairs upstream of the transcriptional start site, respectively, are required for maximal induction of the human MCP-1 promoter by interleukin-(IL)-1 beta. Site-directed mutagenesis or deletion of the NF-kappa B-like site decreased the cytokine-induced activity of the promoter. Site-directed mutagenesis of the AP-1 binding site also decreased the cytokine-induced activity of the promoter. We show that the NF-kappa B-like site located at-90 in the MCP-1 promoter binds to the p50/p65 heterodimer of the NF-kappa B/Rel family in IL-1 beta-stimulated human endothelial cells. Overexpression of p65 results in the transactivation of the MCP-1 promoter as well. The data presented in this study suggest that cytokine-induced MCP-1 gene expression in human endothelial cells depends on the cooperative action of NF-kappa B and AP-1.

The collagen receptor alpha 2 beta 1, from MG-63 and HT1080 cells, interacts with a cyclic RGD peptide.

Several receptors for the extracellular matrix protein collagen have been described which belong to the superfamily of receptors collectively known as integrins. Although several integrins have been shown to interact with extracellular matrix molecules via a common recognition site, arginine-glycine-aspartic Acid (RGD), within the beta 1 integrin subfamily, only the fibronectin receptor (alpha 5 beta 1) has been convincingly shown to interact with RGD. In the present study, we tested whether a collagen receptor could interact with RGD. Adhesion of an osteosarcoma cell line, MG-63, to immobilized collagen I was inhibited by the cyclic RGD-containing peptide, C*GRGDSPC* (where C* indicates that Cys participates in disulfide), and not by the linear GRGDSP or the non-RGD-containing cyclic peptide, C*GKGESPC*. Similarly, using collagen-Sepharose affinity chromatography, a heterodimeric protein could be specifically eluted from the column by the cyclic RGD peptide. Immunoprecipitations of the eluted material with monoclonal antibodies showed reactivity with the collagen receptor alpha 2 beta 1 and not alpha 3 beta 1. Our data demonstrate that RGD peptides can interact with the collagen receptor, and the differences seen with the linear and cyclic peptide suggest that the cyclic C*GRGDSPC* has a higher avidity for the receptor than the more flexible linear GRGDSP. In this paper, we provide supportive evidence that one possible mode of collagen interaction with alpha 2 beta 1 is via the RGD recognition sequence.