Micro-CT based experimental liver imaging using a nanoparticulate contrast agent: a longitudinal study in mice

Background

Micro-CT imaging of liver disease in mice relies on high soft tissue contrast to detect small lesions like liver metastases. Purpose of this study was to characterize the localization and time course of contrast enhancement of a nanoparticular alkaline earth metal-based contrast agent (VISCOVER ExiTron nano) developed for small animal liver CT imaging.

Methodology

ExiTron nano 6000 and ExiTron nano 12000, formulated for liver/spleen imaging and angiography, respectively, were intravenously injected in C57BL/6J-mice. The distribution and time course of contrast enhancement were analysed by repeated micro-CT up to 6 months. Finally, mice developing liver metastases after intrasplenic injection of colon carcinoma cells underwent longitudinal micro-CT imaging after a single injection of ExiTron nano.

Principal Findings

After a single injection of ExiTron nano the contrast of liver and spleen peaked after 4–8 hours, lasted up to several months and was tolerated well by all mice. In addition, strong contrast enhancement of abdominal and mediastinal lymph nodes and the adrenal glands was observed. Within the first two hours after injection, particularly ExiTron nano 12000 provided pronounced contrast for imaging of vascular structures. ExiTron nano facilitated detection of liver metastases and provided sufficient contrast for longitudinal observation of tumor development over weeks.

Conclusions

The nanoparticulate contrast agents ExiTron nano 6000 and 12000 provide strong contrast of the liver, spleen, lymph nodes and adrenal glands up to weeks, hereby allowing longitudinal monitoring of pathological processes of these organs in small animals, with ExiTron nano 12000 being particularly optimized for angiography due to its very high initial vessel contrast.     

Enhanced matrix metalloproteinase activity in skeletal muscles of rats with congestive heart failure

Patients with congestive heart failure (CHF) are prone to increased skeletal muscle fatigue. Elevated circulatory concentrations of tumor necrosis factor (TNF)-alpha and monocyte chemoattractant protein-1, which may stimulate matrix metalloproteinase (MMP) activity and, thereby, contribute to skeletal muscle dysfunction, are frequently found in CHF. However, whether skeletal muscle MMP activity is altered in CHF is unknown. Hence, we have used a gelatinase assay to assess the activity of MMP and tissue inhibitors of MMP in single skeletal muscles of rats with CHF 6 wk after induction of myocardial infarction. Sham-operated (Sham) rats were used as controls. We also measured the gene expression and protein contents of MMP-2 and MMP-9 in skeletal muscles of these rats. Plasma MMP activity was nearly seven times higher (P < 0.05) in CHF than in Sham rats. Concomitantly, the MMP activity within single slow- and fast-twitch skeletal muscles of CHF rats increased two- to fourfold compared with Sham animals, whereas tissue inhibitor of MMP activity did not differ (P > 0.05). Preformed MMP-2 and MMP-9 were probably activated in CHF, because neither their gene expression nor protein levels were altered (P > 0.05). Serum concentrations of TNF-alpha and monocyte chemoattractant protein-1 remained unchanged (P > 0.05) between CHF and Sham rats during the 6-wk observation period. We conclude that development of CHF in rats enhances MMP activity, which in turn may distort the normal contractile function of skeletal muscle, thereby contributing to increased skeletal muscle fatigue.     

Helicostatins: brain-gut peptides of the moth, Helicoverpa armigera (Lepidoptera: Noctuidae)

Gene expression and immunolocalisation studies have determined that the helicostatins are brain-gut peptides in larvae of the lepidopteran, Helicoverpa armigera. Mapping of the distribution of these peptides in the nervous system and alimentary canal has provided evidence for multifunctional regulatory roles. In situ hybridisation studies have shown that the helicostatin precursor gene is expressed in neurones of the central and stomatogastric nervous systems, and endocrine cells of the midgut demonstrating that the helicostatins are true brain-gut peptides. Antisera raised against Leu-callatostatin 3 (ANRYGFGL-NH(2)), a peptide isolated from the blowfly, Calliphora vomitoria was used to map the distribution of allatostatin-like immunoreactive (Ast-ir) material in H. armigera to elucidate possible functions of the helicostatins. In situ hybridisation studies verified that the helicostatin precursor gene is expressed in neurones shown to contain Ast-ir, providing strong evidence that the Ast-ir material is helicostatins. Extensive immunoreactive axonal projections into complex regions of neuropile indicate that the helicostatins may have a neuromodulatory role in the brain and segmental ganglia of the ventral nerve cord. The presence of large amounts of immunoreactive material in axons within the corpora cardiaca (CC) and transverse nerves of the perisympathetic nervous system, two known neurohaemal organs, provides evidence for a neurohormonal role. The corpora allata (CA) were innervated only sparsely by Ast-ir axons suggesting that the CA are not a neurohaemal release site or a target. Thus, it is unlikely that the helicostatins regulate juvenile hormone (JH) biosynthesis or release. Ast-ir axons extended from the frontal ganglion through the recurrent nerve and many branches were closely associated with muscles of the foregut, stomodeal valve, and anterior midgut, implicating helicostatins in regulation of foregut motility. Ast-ir material was also present in nerves associated with muscles of the pyloric valve and rectum, and in endocrine cells of the midgut.     

Marine landscapes and population genetic structure of herring (Clupea harengus L.) in the Baltic Sea

Numerically small but statistically significant genetic differentiation has been found in many marine fish species despite very large census population sizes and absence of obvious barriers to migrating individuals. Analyses of morphological traits have previously identified local spawning groups of herring (Clupea harengus L.) in the environmentally heterogeneous Baltic Sea, whereas allozyme markers have not revealed differentiation. We analysed variation at nine microsatellite loci in 24 samples of spring-spawning herring collected at 11 spawning locations throughout the Baltic Sea. Significant temporal differentiation was observed at two locations, which we ascribe to sympatrically spawning but genetically divergent 'spawning waves'. Significant differentiation was also present on a geographical scale, though pairwise F(ST) values were generally low, not exceeding 0.027. Partial Mantel tests showed no isolation by geographical distance, but significant associations were observed between genetic differentiation and environmental parameters (salinity and surface temperature) (0.001 < P < or = 0.099), though these outcomes were driven mainly by populations in the southwestern Baltic Sea, which also exhibits the steepest environmental gradients. Application of a novel method for detecting barriers to gene flow by combining geographical coordinates and genetic differentiation allowed us to identify two zones of lowered gene flow. These zones were concordant with the separation of the Baltic Sea into major basins, with environmental gradients and with differences in migration behaviour. We suggest that similar use of landscape genetics approaches may increase the understanding of the biological significance of genetic differentiation in other marine fishes.     

A high-density screen for linkage in multiple sclerosis

To provide a definitive linkage map for multiple sclerosis, we have genotyped the Illumina BeadArray linkage mapping panel (version 4) in a data set of 730 multiplex families of Northern European descent. After the application of stringent quality thresholds, data from 4,506 markers in 2,692 individuals were included in the analysis. Multipoint nonparametric linkage analysis revealed highly significant linkage in the major histocompatibility complex (MHC) on chromosome 6p21 (maximum LOD score [MLS] 11.66) and suggestive linkage on chromosomes 17q23 (MLS 2.45) and 5q33 (MLS 2.18). This set of markers achieved a mean information extraction of 79.3% across the genome, with a Mendelian inconsistency rate of only 0.002%. Stratification based on carriage of the multiple sclerosis–associated DRB1*1501 allele failed to identify any other region of linkage with genomewide significance. However, ordered-subset analysis suggested that there may be an additional locus on chromosome 19p13 that acts independent of the main MHC locus. These data illustrate the substantial increase in power that can be achieved with use of the latest tools emerging from the Human Genome Project and indicate that future attempts to systematically identify susceptibility genes for multiple sclerosis will have to involve large sample sizes and an association-based methodology.     

Identification and expression of two novel cytochrome P450 genes, belonging to CYP4 and a new CYP331 family, in the polychaete Capitella capitata sp.I

The polychaete Capitella capitata sp.I has a high capacity to metabolize polycyclic aromatic hydrocarbons (PAHs) which are among the most hazardous environmental pollutants with significant biological effects. In the present study, two novel cytochrome P450 (CYP) genes were identified in this species. One was named CYP331A1, the first member of a new family of CYP331, and the other CYP4AT1 is the first member of a new subfamily CYP4AT. Both of these genes are constitutively expressed in the worms and detectable by RT-PCR. The expression of CYP331A1 mRNA was observed to be more sensitive to PAH exposure than CYP4AT1, which indicated that CYP331A1 should play a more important role than CYP4AT1 in PAH metabolism in this species. Considering the importance of C. capitata sp.I in taking up PAH and other organic pollutants from contaminated marine sediments with the potential for subsequent food-chain transfer, our results are important for understanding the molecular basis of biotransformation and detoxification in this invertebrate, and also have evolutionary significance for understanding the diversity and history of the CYP superfamily.     

Newcastle Disease Virus-Vectored Vaccines Expressing the Hemagglutinin or Neuraminidase Protein of H5N1 Highly Pathogenic Avian Influenza Virus Protect against Virus Challenge in Monkeys

H5N1 highly pathogenic avian influenza virus (HPAIV) causes periodic outbreaks in humans, resulting in severe infections with a high (60%) incidence of mortality. The circulating strains have low human-to-human transmissibility; however, widespread concerns exist that enhanced transmission due to mutations could lead to a global pandemic. We previously engineered Newcastle disease virus (NDV), an avian paramyxovirus, as a vector to express the HPAIV hemagglutinin (HA) protein, and we showed that this vaccine (NDV/HA) induced a high level of HPAIV-specific mucosal and serum antibodies in primates when administered through the respiratory tract. Here we developed additional NDV-vectored vaccines expressing either HPAIV HA in which the polybasic cleavage site was replaced with that from a low-pathogenicity strain of influenza virus [HA(RV)], in order to address concerns of enhanced vector replication or genetic exchange, or HPAIV neuraminidase (NA). The three vaccine viruses [NDV/HA, NDV/HA(RV), and NDV/NA] were administered separately to groups of African green monkeys by the intranasal/intratracheal route. An additional group of animals received NDV/HA by aerosol administration. Each of the vaccine constructs was highly restricted for replication, with only low levels of virus shedding detected in respiratory secretions. All groups developed high levels of neutralizing antibodies against homologous and heterologous strains of HPAIV and were protected against challenge with 2 × 10⁷ PFU of homologous HPAIV. Thus, needle-free, highly attenuated NDV-vectored vaccines expressing either HPAIV HA, HA(RV), or NA have been developed and demonstrated to be individually immunogenic and protective in a primate model of HPAIV infection. The finding that HA(RV) was protective indicates that it would be preferred for inclusion in a vaccine. The study also identified NA as an independent protective HPAIV antigen in primates. Furthermore, we demonstrated the feasibility of aerosol delivery of NDV-vectored vaccines.H5N1 highly pathogenic avian influenza virus (HPAIV) was first detected in human infections in 1997; previously, it had been found only in birds (11, 50). To date, this virus has been identified in 436 confirmed cases of human infection in 15 countries, 262 (60%) of which were fatal (75). The currently circulating H5N1 strains are characterized by low human-to-human transmissibility. This has been attributed, in part, to a preference for binding to α-2,3-linked sialic acids that are present in high concentrations throughout the avian respiratory tract but were thought to be found primarily in the lower human respiratory tract (57), although this explanation has been questioned (48, 49). It has also been observed that mutations in the PB2 subunit of the viral polymerase are necessary to confer the ability for the virus to be spread by aerosolized nasal droplets in ferrets (72). Whatever factors may be involved, there is widespread concern that the avian virus could mutate to enhance its transmissibility among humans, possibly resulting in a global pandemic (28, 50). For the avian H9N2 virus, which also has pandemic potential, it has been demonstrated that only five amino acid changes were sufficient for the virus to gain the ability to be spread by aerosolized nasal droplets in a ferret model (60). Thus, there is an urgent need for vaccines against HPAIV.Several vaccine strategies for HPAIV have been evaluated (reviewed in references 32 and 41), including inactivated and live attenuated vaccines. These efforts have been hampered by several factors. HPAIV strains are highly virulent for embryonated chicken eggs, the most widely used substrate for vaccine manufacture, and their rapid death following inoculation renders eggs unsuitable for efficient virus propagation. In addition, the major protective antigen, hemagglutinin (HA), administered either as a purified protein or in inactivated HPAIV virions, appears to be poorly immunogenic (69, 70). An additional factor complicating the development of HPAIV vaccines based on inactivated virus is the high cost and biohazard associated with HPAIV propagation, which must be done under enhanced biosafety level 3 (BSL-3) containment, although this problem might be addressed by the use of live attenuated reassortant influenza virus vaccines that contain the HPAIV glycoproteins on the background of an avirulent human influenza virus strain (24, 37). In addition, such reassortant strains might serve directly as live attenuated vaccines. Unfortunately, the latter approach may be limited by subtle and unpredictable incompatibility between the avian-origin glycoproteins and human-origin vaccine backgrounds acceptable for human use, which can result in overattenuation in vivo (24). There are also lingering concerns about the significant potential, with a live HPAIV vaccine, for reassortment between gene segments of the vaccine virus and circulating influenza virus strains, which might result in novel strains with unpredictable biological properties (63).We and others have been evaluating Newcastle disease virus (NDV) as a general human vaccine vector for emerging pathogens, including H5N1 HPAIV (7, 18-20, 29). NDV is an avian paramyxovirus that is antigenically unrelated to common human pathogens; hence, its use in humans should not be affected by host immunity to common pathogens. The many naturally occurring strains of NDV can be categorized into three pathotypes based on virulence in chickens: velogenic strains, causing severe disease with high mortality; mesogenic strains, causing disease of intermediate severity with low mortality; and lentogenic strains, causing mild or inapparent infections (reviewed in reference 2). Lentogenic, and sometimes mesogenic, strains of NDV are in wide use as live attenuated vaccines against velogenic NDV in poultry (2). When mesogenic or lentogenic NDV was administered to the respiratory tracts of nonhuman primates as a model for the immunization of humans, the virus was highly attenuated for replication, was shed only at low titers, appeared to remain restricted to the respiratory tract, and was highly immunogenic for the expressed foreign antigen (7). We recently demonstrated that a mesogenic strain of NDV expressing the HA protein of H5N1 HPAIV (NDV/HA) elicited high titers of neutralizing antibodies in serum following combined intranasal (i.n.) and intratracheal (i.t.) delivery in a nonhuman primate model (20). Vaccination of mice with a similar NDV-vectored vaccine protected them from HPAIV challenge (29). However, results obtained with mice do not reliably predict the efficacy of an influenza virus vaccine for human use, due to the pathophysiological and phylogenetic differences between mice and humans (71). In particular, mice may produce a potent immune response to HPAIV vaccines (64) that may not be reproduced in clinical trials (38). These considerations are especially important for a vaccine based on a live viral vector platform, since its immunogenicity, and therefore its protective efficacy, is directly linked to replication, which can differ greatly in various experimental animals versus humans (reviewed in references 6 and 9). Therefore, the protective efficacy of NDV-based vaccines against HPAIV challenge in nonhuman primate models—the closest model to humans—has remained unknown.The protease recognition sequence of the HA protein is one of the major determinants of avian influenza virus pathogenicity (62). HPAIV strains have a “polybasic” cleavage site, containing multiple basic amino acids, that is readily cleaved by ubiquitous intracellular subtilisin-like proteases, facilitating the replication and spread of the virus. In contrast, the HA cleavage site of low-pathogenicity strains contains fewer basic amino acids and depends on secretory trypsin-like proteases found in the respiratory and enteric tracts, resulting in more-localized infections (30, 62). The presence of a polybasic cleavage site in the H5 HA of any live vaccine raises some concern about the possibility of genetic exchange with circulating strains of influenza virus. It should be noted that genetic exchange involving paramyxoviruses is a rare event (14) that has been documented only once (61). However, elimination of the polybasic HA cleavage site would mitigate the effects of even this rare possibility of genetic exchange. Another concern was based on our previous finding that the HPAIV H5 HA protein is incorporated into the NDV envelope as a trimer (20), consistent with its presence in a functional form. While we previously showed that this did not enhance the pathogenicity of the NDV/HA recombinant in chickens (20), we could not rule out the possibility that it might confer an altered tropism on the NDV/HA virus in other systems. For example, a recombinant parainfluenza virus type 3 expressing the Ebola virus glycoprotein incorporated the foreign protein into its envelope, allowing cellular attachment and fusion of the vaccine virus independently of the vector''s own envelope glycoproteins (10).In addition to the HA protein, the neuraminidase (NA) protein is also present on the surfaces of influenza virus-infected cells and virions. Antibodies specific for NA are not thought to interfere with the initial viral attachment and penetration of host cells (36, 40, 54). However, NA-specific antibodies prevent the release of virus from infected cells, thereby decreasing viral spread (35), and they increase resistance to viral infection in humans (40, 47, 54). They also provide at least some protection against viruses bearing homologous or heterologous NA proteins of the same subtype in a mouse model (12, 56). NA also appears to evolve at a lower rate than HA, suggesting that NA-specific antibodies may provide broader protection than a vaccine utilizing HA alone (39). Therefore, it was important to assess the immunogenicity and protective efficacy of the HPAIV NA independently of those of HA, which has not previously been done in a human or nonhuman primate model.     

IL-22RA2 associates with multiple sclerosis and macrophage effector mechanisms in experimental neuroinflammation

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the CNS. Recent advances in whole-genome screening tools have enabled discovery of several MS risk genes, the majority of which have known immune-related functions. However, disease heterogeneity and low tissue accessibility hinder functional studies of established MS risk genes. For this reason, the MS model experimental autoimmune encephalomyelitis (EAE) is often used to study neuroinflammatory disease mechanisms. In this study, we performed high-resolution linkage analysis in a rat advanced intercross line to identify an EAE-regulating quantitative trait locus, Eae29, on rat chromosome 1. Eae29 alleles from the resistant strain both conferred milder EAE and lower production of proinflammatory molecules in macrophages, as demonstrated by the congenic line, DA.PVG-Eae29 (Dc1P). The soluble IL-22R α2 gene (Il-22ra2) lies within the Eae29 locus, and its expression was reduced in Dc1P, both in activated macrophages and splenocytes from immunized rats. Moreover, a single nucleotide polymorphism located at the end of IL-22RA2 associated with MS risk in a combined Swedish and Norwegian cohort comprising 5019 subjects, displaying an odds ratio of 1.26 (p = 8.0 × 10(-4)). IL-22 and its receptors have been implicated in chronic inflammation, suggesting that IL-22RA2 regulates a central immune pathway. Through a combined approach including genetic and immunological investigation in an animal model and large-scale association studies of MS patients, we establish IL-22RA2 as an MS risk gene.     

Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain

Members of the family of adenosine deaminases acting on RNA (ADARs) can catalyze the hydrolytic deamination of adenosine to inosine and thereby change the sequence of specific mRNAs with highly double-stranded structures. The ADARs all contain one or more repeats of the double-stranded RNA binding motif (DRBM). By both in vitro and in vivo assays, we show that the DRBMs of rat ADAR2 are necessary and sufficient for dimerization of the enzyme. Bioluminescence resonance energy transfer (BRET) demonstrates that ADAR2 also exists as dimers in living mammalian cells and that mutation of DRBM1 lowers the dimerization affinity while mutation of DRBM2 does not. Nonetheless, the editing efficiency of the GluR2 Q/R site depends on a functional DRBM2. The ADAR2 DRBMs thus serve differential roles in RNA dimerization and GluR2 Q/R editing, and we propose a model for RNA editing that incorporates the new findings.