Challenges and recommendations for magnetic hyperthermia characterization measurements.

PURPOSE: The localized heating of magnetic nanoparticles (MNPs) via the application of time-varying magnetic fields - a process known as magnetic field hyperthermia (MFH) - can greatly enhance existing options for cancer treatment; but for broad clinical uptake its optimization, reproducibility and safety must be comprehensively proven. As part of this effort, the quantification of MNP heating - characterized by the specific loss power (SLP), measured in W/g, or by the intrinsic loss power (ILP), in Hm2/kg - is frequently reported. However, in SLP/ILP measurements to date, the apparatus, the analysis techniques and the field conditions used by different researchers have varied greatly, leading to questions as to the reproducibility of the measurements. MATERIALS AND METHODS: An interlaboratory study (across N = 21 European sites) of calorimetry measurements that constitutes a snapshot of the current state-of-the-art within the MFH community has been undertaken. Identical samples of two stable nanoparticle systems were distributed to all participating laboratories. Raw measurement data as well as the results of in-house analysis techniques were collected along with details of the measurement apparatus used. Raw measurement data was further reanalyzed by universal application of the corrected-slope method to examine relative influences of apparatus and results processing. RESULTS: The data show that although there is very good intralaboratory repeatability, the overall interlaboratory measurement accuracy is poor, with the consolidated ILP data having standard deviations on the mean of ca. ± 30% to ± 40%. There is a strong systematic component to the uncertainties, and a clear rank correlation between the measuring laboratory and the ILP. Both of these are indications of a current lack of normalization in this field. A number of possible sources of systematic uncertainties are identified, and means determined to alleviate or minimize them. However, no single dominant factor was identified, and significant work remains to ascertain and remove the remaining uncertainty sources. CONCLUSION: We conclude that the study reveals a current lack of harmonization in MFH characterization of MNPs, and highlights the growing need for standardized, quantitative characterization techniques for this emerging medical technology.

γδ T cells compose a developmentally regulated intrauterine population and protect against vaginal candidiasis.

This most comprehensive analysis to date of γδ T cells in the murine uterus reveals them to compose a unique local T-cell compartment. Consistent with earlier reports, most cells expressed a canonical Vγ6Vδ1 TCR, and produced interleukin (IL)-17A upon stimulation. Nonetheless, contrasting with earlier reports, uterine γδ T cells were not obviously intraepithelial, being more akin to sub-epithelial Vγ6Vδ1+ T cells at several other anatomical sites. By contrast to other tissues however, the uterine compartment also included non-Vγ6+, IFN-γ-producing cells; was strikingly enriched in young mice; expressed genes hitherto associated with the uterus, including the progesterone receptor; and did not require microbes for development and/or maintenance. This notwithstanding, γδ T-cell deficiency severely impaired resistance to reproductive tract infection by Candida albicans, associated with decreased responses of IL-17-dependent neutrophils. These findings emphasise tissue-specific complexities of different mucosal γδ cell compartments, and their evident importance in lymphoid stress-surveillance against barrier infection.

Functional role of type I and type II interferons in antiviral defense.

Mice lacking the known subunit of the type I interferon (IFN) receptor were completely unresponsive to type I IFNs, suggesting that this receptor chain is essential for type I IFN-mediated signal transduction. These mice showed no overt anomalies but were unable to cope with viral infections, despite otherwise normal immune responses. Comparison of mice lacking either type I or type II IFN receptors showed that, at least in response to some viruses, both IFN systems are essential for antiviral defense and are functionally nonredundant.

Multi-color magnetic nanoparticle imaging using magnetorelaxometry.

Magnetorelaxometry (MRX) is a well-known measurement technique which allows the retrieval of magnetic nanoparticle (MNP) characteristics such as size distribution and clustering behavior. This technique also enables the non-invasive reconstruction of the spatial MNP distribution by solving an inverse problem, referred to as MRX imaging. Although MRX allows the imaging of a broad range of MNP types, little research has been done on imaging different MNP types simultaneously. Biomedical applications can benefit significantly from a measurement technique that allows the separation of the resulting measurement signal into its components originating from different MNP types. In this paper, we present a theoretical procedure and experimental validation to show the feasibility of MRX imaging in reconstructing multiple MNP types simultaneously. Because each particle type has its own characteristic MRX signal, it is possible to take this a priori information into account while solving the inverse problem. This way each particle type's signal can be separated and its spatial distribution reconstructed. By assigning a unique color code and intensity to each particle type's signal, an image can be obtained in which each spatial distribution is depicted in the resulting color and with the intensity measuring the amount of particles of that type, hence the name multi-color MNP imaging. The theoretical procedure is validated by reconstructing six phantoms, with different spatial arrangements of multiple MNP types, using MRX imaging. It is observed that MRX imaging easily allows up to four particle types to be separated simultaneously, meaning their quantitative spatial distributions can be obtained.

Quantification of magnetic nanoparticles by magnetorelaxometry and comparison to histology after magnetic drug targeting.

Magnetic nanoparticles can be used in medicine in vivo as contrast agents and as a drug carrier system for chemotherapeutics. Thus local cancer therapy is performed with Magnetic Drug Targeting (MDT) and allows a specific delivery of therapeutic agents to desired targets, i.e., tumors, by using a chemotherapeutic substance bound to magnetic nanoparticles and focused with an external magnetic field to the tumor after intraarterial application. Important for this therapeutic principle is the distribution of the particles in the whole organism and especially in the tumor. Therefore we used magnetorelaxometry to quantify ferrofluids delivered after MDT. Tissue samples of some mm3 volume of a VX2 squamous cell carcinoma were measured by magnetic relaxation and the amount of iron was determined using the original ferrofluid suspension as a reference. From this the distribution of the magnetic particles within the slice of tumor was reconstructed. Histological cross-sections of the respective tumor offer the opportunity to map quantitatively the particle distribution and the vascularisation in the targeted tumor on a microscopic scale. Our data show that the integral method magnetorelaxometry and microscopic histological methods can complete each other efficiently.

Transcription factor c-Rel plays a crucial role in driving anti-CD40-mediated innate colitis.

Genetic and environmental factors, including the commensal microbiota, have a crucial role in the development of inflammatory bowel disease. Aberrant activation of the transcription factor NF-κB is associated with chronic intestinal inflammation in mice and humans. Recently, an emerging family of innate lymphoid cells (ILCs) has been identified at mucosal sites contributing to the maintenance of gut homeostasis and intestinal immunopathology. Here, we show that the NF-κB protein c-Rel regulates the inflammatory potential of colonic IFN-γ(+)Thy1(+) ILCs to induce anti-CD40-mediated colitis in rag1(-/-) mice. Stimulation of dendritic cells (DCs) with anti-CD40 or CD40L led to translocation of c-Rel into the nucleus resulting in induction of expression of interleukin-12 (IL-12) and IL-23, key regulators of innate cell-induced colitis. While c-Rel deficiency completely abrogated anti-CD40-induced colitis, adoptively transferred wild-type DCs were able to induce pronounced colonic inflammation in rag1(-/-)rel(-/-) mice. In summary, these results suggest that the expression of c-Rel in DCs is essential for initiating anti-CD40-mediated intestinal pathogenesis.

Lysozyme-encoding bovine cDNAs from neutrophile granulocytes and mammary gland are derived from a different gene than stomach lysozymes.

cDNA copies of a bovine lysozyme (bLys)-encoding gene (Lys) were isolated from libraries specific for granulocytes, as well as the lactating mammary gland. Analysis of each of the longest Lys-specific cDNA inserts revealed nucleotide sequence identity over the entire overlap of 1418 bp. Incomplete at the 5' end, the combined sequence codes for 11 of the 18-amino-acid (aa) Lys leader peptide and 130 aa residues of the mature Lys. Similar to mouse and human Lys from blood cells, the encoded protein contains one aa residue more (Pro103) than any of the bLys derived from stomach. Furthermore, unlike any of the known bLys genes, our sequence reveals the copy of a bovine retroposon element in the 3' untranslated region (UTR) of the mRNA approximately at the same position where an Alu-retroposon element resides within the human copy of the gene. As a further distinction from bLys expressed in stomach, we identified a segment within the 3'UTR of the mRNA which is conserved between the bovine and human blood cell variants of the Lys, but does not have significant sequence homology to any of the bovine lysozyme genes known so far. By sequence comparisons, we present evidence that this segment has been deleted during evolutionary divergence of the stomach Lys. Hence, we describe the sequence of a heretofore unknown bLys, being expressed in granulocytes. Bearings of our observations on the understanding of Lys evolution are discussed, as well as the possibility that the product of this gene may be responsible for the functional Lys activity in bovine milk.

Localization of T helper cell epitopes in the vesicular stomatitis virus: the nucleoprotein is responsible for serotype cross-reactive T help.

The glycoprotein (G) of vesicular stomatitis virus (VSV) is known to contain the biologically relevant sites for virus-neutralizing antibodies as well as T helper (Th) cell epitopes. The capacity of other VSV proteins to elicit potent Th cell responses has not yet been investigated. Additionally, a short-lived cross-reactivity between the two serologically distinct VSV serotypes Indiana (IND) and New Jersey (NJ) on the T helper cell level has been reported. Here we address the question of whether the VSV nucleoprotein (N) or matrix protein (M) can elicit T help to VSV-G-specific B cells and which of the VSV proteins contains the elements responsible for the IND/NJ cross-reactivity. The N, G, and M of the VSV Indiana serotype produced in a recombinant baculovirus system were assayed for the ability to activate VSV-specific Th cells to induce immunoglobulin class switch of neutralizing antibody responses in vivo in C57BL/6 (H-2b) mice. All three VSV-IND proteins helped in the production of neutralizing IgG antibodies to the homologous VSV-Indiana serotype but only VSV-IND N was able to trigger an IgG response to the heterologous VSV-New Jersey serotype. This data suggest that Th epitope(s) in the VSV-IND N are responsible for the observed cross-reactivity of T helper cells.

Magnetometry of evoked fields from human peripheral nerve, brachial plexus and primary somatosensory cortex using a liquid nitrogen cooled superconducting quantum interference device.

Superconducting Quantum Interference Devices (SQUIDs) can be used to detect neuromagnetic fields evoked in the peripheral and central nervous system. Up to now, such measurements had to be based on SQUIDs with a low critical temperature (Tc) requiring liquid helium cooling. Recent improvements in high-Tc SQUID technology relying on liquid nitrogen cooling led to a significant reduction in the system's noise level. Hare, first high-Tc recordings of weak neuromagnetic fields are demonstrated. In particular, along the entire somatosensory afferent pathway including peripheral nerves, brachial plexus and primary somatosensory neocortex evoked neuromagnetic activities were detected using conventional recording parameters for bandwidth and number of averages. This opens up a wide perspective for cost-effective high-Tc magnetometry in clinical neuroscience.

Magnetocardiographic analysis of the two-dimensional distribution of intra-QRS fractionated activation.

The spatial distribution of high-frequency components in magnetic signals during the QRS complex of the human heartbeat was investigated. Cardiomagnetic signals were recorded simultaneously using 49 first-order magnetogradiometer channels of a multi-SQUID system with a low noise power density. The QRS fragmentation score S, as a measure of the fragmentation of the bandpass-filtered QRS complex, was examined for its sensitivity and specificity to discriminate 34 healthy volunteers, 42 post-myocardial infarction patients and 43 patients with coronary heart disease and with a history of malignant sustained ventricular tachycardia or ventricular fibrillation. The multichannel information was visualized by two-dimensional mapping of the score values of the single channels. By averaging the score values for the seven central channels, S7, the score values of all 49 channels, S49, and calculating the standard deviation for all 49 channels, D49, a higher sensitivity and specificity for detecting patients with ventricular tachycardia (VT) or ventricular fibrillation (VF) was reached than by analysis of a single channel. Combination of these parameters furnishes a sensitivity of 90% and a specificity of 70% for identifying patients prone to VT/VF. The results were compared with diagnostic information obtained from the QRS duration of the signal as well as with results obtained by modified QRS integral mapping.

Magnetocardiographic mapping of QRS fragmentation in patients with a history of malignant tachyarrhythmias.

BACKGROUND: The identification of patients at increased risk for ventricular tachycardia or ventricular fibrillation (VT/VF) and sudden cardiac death has consequences for therapeutic options and thus may reduce mortality in patients with coronary artery disease (CAD). HYPOTHESIS: We hypothesized that the intra-QRS fragmentation in magnetocardiographic recordings is increased in patients with CAD and with a history of VT/VF. METHODS: Multichannel magnetocardiography (MCG) was carried out in 34 healthy controls, 42 patients with CAD without a history of VT/VF, and 43 patients with CAD and with a history of VT/VF. The intra-QRS fragmentation was quantified by a new fragmentation score. Its spatial distribution was investigated using two-dimensional (2-D) contour maps according to the sensor position of the 49-channel magnetogradiometer. RESULTS: Patients with CAD and with a history of VT/VF had significantly increased QRS fragmentation compared with patients with CAD without VT/VF or controls (72.9+/-37.5, 48.5+/-14.3, and 42.5+/-7.8, respectively: p <0.05). The area of high fragmentation in 2-D contour maps was twice as large in patients with than in those without a history of VT/VF (represented by the number of MCG channels with high fragmentation: 26.3+/-15.5 vs. 12.4+/-9.9, p<0.0001). Patients prone to VT/VF could be identified with a sensitivity of 64% and a specificity of 90%. CONCLUSION: In patients with CAD and with a history of VT/VF, intra-QRS fragmentation is increased and the area of high fragmentation in 2-D contour maps is enlarged. These findings may be helpful in identifying patients with CAD at risk for malignant tachyarrhythmias.

Fragmentation of bandpass-filtered QRS-complex of patients prone to malignant arrhythmia.

The structure of high-frequency components of electric and magnetic signals from the heart during the depolarisation phase is investigated. After averaging and broadband filtering with a binomial bandpass filter (37 Hz-90 Hz), the fragmentation of the QRS-complex is quantified. The number of extrema M and a new score value S are calculated from the signals of three electrical leads and one magnetic lead of 23 healthy subjects, 23 patients with coronary heart disease (CHD) without reported event of ventricular tachycardia or fibrillation at the time of measurement, and eight patients with CHD who have suffered from malignant tachycardia. For the parameter M, the sensitivity and specificity for healthy subjects against patients with CHD and ventricular tachycardia for the magnetic lead (the best electric lead) are 100% (75%) and 100% (100%). For the magnetic lead (best electric lead) and parameter S, the sensitivity and specificity are 100% (75%) and 95.6% (100%).

Identification of post-myocardial infarction patients with ventricular tachycardia by time-domain intra-QRS analysis of signal-averaged electrocardiogram and magnetocardiogram.

A new time-domain analysis method, which quantifies ECG/MCG intra-QRS fragmentation, is applied to parts of the QRS complex to identify post-myocardial infarction patients with ventricular tachycardia. Three leads of signal-averaged electrocardiograms and nine leads of magnetocardiograms were band-pass filtered (74 Hz to 180 Hz). The filtered signals showed fragmentation in the QRS region, which was quantified by the number of peaks M and a score S, that is the product of M and the sum of the peak amplitudes. Both parameters were determined for the first 80 ms of the QRS complex and the total QRS complex in each channel. For classification, the mean-values of the parameters M and S of the three electrical leads and the nine magnetic leads were calculated. Late potential and late field analyses were performed for the same signals. 31 myocardial infarction patients were included, 20 of them with a history of documented ventricular tachycardia (VT). Identification of VT patients using the SAECG led to better results (sensitivity 95%, specificity 91%) considering the entire QRS complex than with the standard late potential analysis suggested by Simson (sensitivity 90%, specificity 73%). For the SAMCG and the entire QRS complex results using the parameters S and M are also better (sensitivity 95%, specificity 100%) than for the late field analysis (sensitivity 90% and specificity 100%). For the first 80 ms, the performance of the parameters M and S is only slightly decreased.

Imaging characteristics of different multichannel magnetocardiographic systems.

In this study a comparison of multichannel magnetocardiographic systems is performed with respect to the "detectable" information content. We investigate the lead-field matrices, the slope of the singular values and the source spaces of three different devices: the VectorView (Neuromag: magnetometer-gradiometer mixed device) of the BioMag Laboratory, Helsinki University Central Hospital (HUCH), the arrangement of electronically coupled magnetometers of the Physikalisch-Technische Bundesanstalt Berlin (PTB) and a virtual sensor geometry which was optimized for an improved slope of the singular values at the Institute of Biomedical Engineering, Karlsruhe.

Spatial distribution of cardiac magnetic vector fields acquired from 3120 SQUID positions.

An extended measurement of the magnetic vector field of the human heart is presented. It is acquired by sequential recordings, shifting a 16 SQUID vector magnetometer across 195 positions over a healthy subject's thorax. The magnetocardiographic (MCG) signals were synchronized using a simultaneously measured ECG channel. The registration of the field extends over a volume of 1000 mm x 600 mm x 420 mm sampled at 3120 SQUID positions. We present diagrams of the vector amplitude of selected points in 6 planes at increasing distances from the frontal thorax. Each plane contains 76 vector points. Additionally, we measured the vector field at 126 points lateral to the chest. At the edge points of the measurement volume, the absolute value of the magnetic vector signal amplitude exceeds 0.3 pT in all measurement points. The dataset provides an excellent base to study dedicated MCG detection or rejection methods. Examples where rejection of the heart signal is necessary are magnetoencephalography, magnetoneurography and fetal MCG. The knowledge of the spatio-temporal distribution of the magnetic vector field of the heart supports the development and comparison of multi-SQUID systems and will be used to create new MCG interpretation and representation algorithms.

A sensor configuration for a 304 SQUID vector magnetometer.

A novel SQUID vector magnetometer system is introduced which has been specially designed for the use inside the strongly magnetically shielded room BMSR-2 of PTB. The system is housed in a dewar with a flat bottom and an inner diameter of Ø 250 mm. The SQUIDs are arranged so that in addition to the usually measured Z-component of the field the horizontal magnetic fields are measured too. A total of 304 DC-SQUID magnetometers are divided up into 19 identical modules. The 16 low-Tc SQUIDs of each module are located in such a way that an estimation of the magnetic field in all three dimensions is possible at three points inside the module. The 57 SQUIDs of the lowest Z plane of all modules form a hexagonal grid with a base length of 29 mm. The design criteria and the physical principle behind the complex SQUID arrangement are explained.

Quantitative imaging of magnetic nanoparticles by magnetorelaxometry with multiple excitation coils.

New therapies against cancer based on magnetic nanoparticles (MNPs) require a quantitative spatially resolved imaging of MNPs inside a body. In magnetorelaxometry (MRX), a distribution of nanoparticles can be quantified non-invasively by measuring its relaxation after removal of an external magnetizing field. Conventionally, in MRX the sample is exposed to a homogeneous magnetizing field resulting in a quantitative reconstruction with rather poor spatial resolution. Theoretical work suggests an improvement of spatial resolution may be achieved by a sequential application of inhomogeneous fields magnetizing only parts of a sample. Here, we experimentally demonstrate the feasibility of this approach by reconstructing a nanoparticle distribution inside a compact three-dimensional volume phantom made of 54 gypsum cubes (1 cm(3) cube(-1)), of which 12 gypsum cubes were filled with MNPs. Using 48 small excitation coils surrounding the phantom, a sequence of MRX signals was obtained where only those MNPs near an individual coil contribute. By combined evaluation of these 48 MRX measurements, the positions and content of the 12 MNP-filled cubes could be determined accurately with a deviation below 4%, while by conventional homogeneous MRX only the MNP content was reconstructable with a deviation of about 9%. The results demonstrate the improvement of quantitative MRX imaging by using sequential activation of multiple magnetizing fields.

Genetic and pharmacological targeting of TPL-2 kinase ameliorates experimental colitis: a potential target for the treatment of Crohn's disease?

Inflammatory bowel disease is characterized by dysregulated immune responses against intestinal microflora leading to marked activation of nuclear factor-κB (NF-κB) with subsequent production of pro-inflammatory cytokines. Besides NF-κB, the tumor progression locus 2 (TPL-2)/extracellular signal-regulated kinase (ERK) pathway also regulates inflammatory cytokines such as interleukin-1ß and tumor necrosis factor-α, but its role during intestinal inflammation is incompletely understood. We analyzed the impact of TPL-2 in the dextran sulfate sodium-induced experimental colitis model. Despite normal activation of NF-κB, animals lacking TPL-2 developed only mild colitis with reduced synthesis of inflammatory cytokines. Further, pharmacological inhibition of the TPL-2 kinase was similarly effective in ameliorating colitis as TPL-2 deficiency without obvious side effects. Because increased TPL-2/ERK activation was seen in patients with Crohn's disease (CD) but not ulcerative colitis, our findings encourage further investigation of TPL-2 kinase as potential target for the treatment of CD patients.

Treatment with interleukin-18 binding protein ameliorates Toxoplasma gondii-induced small intestinal pathology that is induced by bone marrow cell-derived interleukin-18.

Peroral infection with Toxoplasma gondii results in a Th1-type immunopathology characterized by small intestinal necrosis and is dependent on IL-18. In the present study, we investigated whether treatment with IL-18 binding protein (IL-18bp) prevents ileal pathology. We observed increased expression of IL-18bp in intestinal biopsies of mice following infection. Whereas small intestines of control mice showed severe necrosis with complete destruction of the small intestinal architecture, mice treated with IL-18bp daily displayed only mild inflammatory changes including flattening of villi and edema in the space between the epithelium and lamina propria. Small intestinal parasite loads and concentrations of pro-inflammatory cytokines did not differ in control and IL-18bp-treated mice. Binding of IL-18 to immobilized IL-18bp revealed a remarkably slow dissociation rate, indicating high affinity. Using chimeric mice we observed that bone marrow-derived rather than stromal cells were the primary source of IL-18 that resulted in small intestinal pathology following peroral infection with T. gondii. In conclusion, the results presented here suggest that IL-18bp may be an effective and safe treatment for small intestinal inflammation. Antigen-presenting rather than epithelial cells appear to be the main source of IL-18 in T. gondii-induced small intestinal inflammation.