Consensus molecular subtypes of colorectal cancer are recapitulated in in vitro and in vivo models.

Colorectal cancer (CRC) is a highly heterogeneous disease both from a molecular and clinical perspective. Several distinct molecular entities, such as microsatellite instability (MSI), have been defined that make up biologically distinct subgroups with their own clinical course. Recent data indicated that CRC can be best segregated into four groups called consensus molecular subtypes (CMS1-4), each of which has a unique biology and gene expression pattern. In order to develop improved, subtype-specific therapies and to gain insight into the molecular wiring and origin of these subtypes, reliable models are needed. This study was designed to determine the heterogeneity and identify the presence of CMSs in a large panel of CRC cell lines, primary cultures and patient-derived xenografts (PDX). We provide a repository encompassing this heterogeneity and moreover describe that a large part of the models can be robustly assigned to one of the four CMSs, independent of the stromal contribution. We subsequently validate our CMS stratification by functional analysis which for instance shows mesenchymal enrichment in CMS4 and metabolic dysregulation in CMS3. Finally, we observe a clear difference in sensitivity to chemotherapy-induced apoptosis, specifically between CMS2 and CMS4. This relates to the in vivo efficacy of chemotherapy, which delays outgrowth of CMS2, but not CMS4 xenografts. Combined our data indicate that molecular subtypes are faithfully modelled in CRC cell cultures and PDXs, representing tumour cell intrinsic and stable features. This repository provides researchers with a platform to study CRC using the existing heterogeneity.

INFLAMMATORY MARKERS ASSOCIATED WITH TRAUMA AND INFECTION IN RED-TAILED HAWKS (BUTEO JAMAICENSIS) IN THE USA.

Changes in inflammatory marker concentrations or activity can be used to monitor health and disease condition of domestic animals but have not been applied with the same frequency to wildlife. We measured concentrations or activity of six inflammatory markers (ceruloplasmin, haptoglobin, mannan-binding lectin-dependent complement [MBL/complement], unsaturated iron-binding capacity (UIBC) and total iron-binding capacity (TIBC), and plasma iron) in apparently healthy and sick or injured Red-tailed Hawks (Buteo jamaicensis). Haptoglobin and ceruloplasmin activities were consistently elevated in sick or injured hawks (2.1 and 2.5 times higher, respectively), and plasma iron concentrations decreased (0.46 times lower), relative to those of healthy birds. There were no differences between healthy and unhealthy hawks in TIBC and UIBC concentrations or MBL/complement activity. Therefore, haptoglobin, ceruloplasmin, and plasma iron would be useful inclusions in a panel of inflammatory markers for monitoring health in raptors.

Inflammatory marker profiles in an avian experimental model of aspergillosis.

Aspergillosis is a common infection in avian species, but can be a challenge to diagnose. Inflammatory markers have been successfully used in mammals for diagnostic and monitoring purposes of various diseases. The aim of this study was to identify inflammatory markers that could aid in the diagnosis of aspergillosis in an avian species. Five-week-old Japanese quail were infected experimentally with Aspergillus fumigatus, and inflammatory markers were measured in plasma. In addition, lung tissues were cultured to quantify the fungal burden. Infected quail had higher plasma levels of ceruloplasmin, unsaturated iron-binding capacity (UIBC), iron, and total iron-binding capacity (TIBC), and lower levels of haptoglobin, compared with uninfected controls. There were positive linear relationships between A. fumigatus colony-forming units cultured from the lungs of infected quail, and levels of ceruloplasmin, UIBC, and TIBC. Quail that died prior to the end of the experiment (day 10 post-infection) had higher ceruloplasmin, UIBC, and TIBC, and lower haptoglobin levels than infected quail that survived. The inflammatory marker profile in quail infected with aspergillosis in this study differs from that seen in mammals, and from the pattern of inflammatory markers seen in birds with bacterial infections. Inflammatory markers could prove useful for diagnosing aspergillosis in birds, and for monitoring disease progression in infected avian species.

Variation with land use of immune function and prevalence of avian pox in Galapagos finches.

Introduced disease has been implicated in recent wildlife extinctions and population declines worldwide. Both anthropogenic-induced change and natural environmental features can affect pathogen spread. Furthermore, environmental disturbance can result in changes in stress physiology, nutrition, and social structure, which in turn can suppress immune system function. However, it remains unknown whether landscape variation results in heterogeneity in host resistance to pathogens. Avian pox virus, a pathogen implicated in avian declines and extinctions in Hawaii, was introduced to the Galapagos in the 1890 s, and prevalence (total number of current infections) has increased recently in finches. We tested whether prevalence and recovery trends in 7 species of Galapagos finches varied by elevation or human land use. To do so, we used infection data obtained from 545 wild-caught birds. In addition, we determined whether annual changes in 4 aspects of innate immune function (complement protein activity, natural antibody activity, concentration of PIT54 protein, and heterophil:lymphocyte ratio) varied by elevation or land use. Prevalence and recovery rates did not vary by elevation from 2008 to 2009. Avian pox prevalence and proportion of recovered individuals in undeveloped and urban areas did not change from 2008 to 2009. In agricultural areas, avian pox prevalence increased 8-fold (from 2% to 17% of 234 individuals sampled) and proportion of recovered individuals increased (11% to 19%) from 2008 to 2009. These results suggest high disease-related mortality. Variation in immune function across human land-use types correlated with variation in both increased prevalence and susceptibility, which indicates changes in innate immune function may underlie changes in disease susceptibility. Our results suggest anthropogenic disturbance, in particular agricultural practices, may underlie immunological changes in host species that themselves contribute to pathogen emergence.

Inflammatory markers following acute fuel oil exposure or bacterial lipopolysaccharide in mallard ducks (Anas platyrhynchos).

Adult mallard ducks (Anas platyrhynchos) were orally dosed with bunker C fuel oil for 5 days, and five different inflammatory markers (haptoglobin, mannan-binding lectin, ceruloplasmin, unsaturated iron-binding capacity, and plasma iron) were measured in blood plasma prior to and 8, 24, 48, and 72 hr following exposure. In order to contrast the response to fuel oil with that of a systemic inflammatory response, an additional five ducks were injected intramuscularly with bacterial lipopolysaccharide (LPS). Oil-treated birds had an inflammatory marker profile that was significantly different from control and LPS-treated birds, showing decreases in mannan-binding lectin-dependent hemolysis and unsaturated iron-binding capacity, but no changes in any of the other inflammatory markers. Birds treated with oil also exhibited increased liver weights, decreased body and splenic weights, and decreased packed cell volume.

Corticosterone, testosterone and life-history strategies of birds.

Steroid hormones have similar functions across vertebrates, but circulating concentrations can vary dramatically among species. We examined the hypothesis that variation in titres of corticosterone (Cort) and testosterone (T) is related to life-history traits of avian species. We predicted that Cort would reach higher levels under stress in species with higher annual adult survival rates since Cort is thought to promote physiological and behavioural responses that reduce risk to the individual. Conversely, we predicted that peak T during the breeding season would be higher in short-lived species with high mating effort as this hormone is known to promote male fecundity traits. We quantified circulating hormone concentrations and key life-history traits (annual adult survival rate, breeding season length, body mass) in males of free-living bird species during the breeding season at a temperate site (northern USA) and a tropical site (central Panama). We analysed our original data by themselves, and also combined with published data on passerine birds to enhance sample size. In both approaches, variation in baseline Cort (Cort0) among species was inversely related to breeding season length and body mass. Stress-induced corticosterone (MaxCort) also varied inversely with body mass and, as predicted, also varied positively with annual adult survival rates. Furthermore, species from drier and colder environments exhibited lower MaxCort than mesic and tropical species; T was lowest in species from tropical environments. These findings suggest that Cort0, MaxCort and T modulate key vertebrate life-history responses to the environment, with Cort0 supporting energetically demanding processes, MaxCort promoting survival and T being related to mating success.

Quantifying and comparing constitutive immunity across avian species.

Studies that blend a comparative approach to immunology with an appreciation for physiological ecology are defining an important new field in biology--ecological immunology. However, a panel of assays that permits a comparative approach to immunology is not yet available. In this paper, we describe several assays of innate immunity that do not require species-specific reagents and therefore ideal for use in comparative immunology studies. We optimized the assays for use in small birds, where sample volumes are limiting. The bactericidal assay measures the capacity of whole blood to kill microorganisms, and integrates many important components of constitutive immunity. The phagocytosis assay measures the phagocytic capacity of macrophages in whole blood. Bioassays for mannan binding protein and lysozyme can be used to measure inflammation-induced levels of these acute phase proteins in the plasma. Species differences in bactericidal and phagocytic activities against Staphylococcus aureus and Escherichia coli were observed in populations of captive and in free-living birds, demonstrating the assays' utility for multi-species comparisons. However, clay-colored thrushes (Turdus grayi) that were stressed by prolonged capture and handling had diminished phagocytic and antibacterial activities, indicating the need to conduct these assays soon after capture. When birds were challenged with lipopolysaccharide (LPS), levels of mannan-binding protein, lysozyme, and haptoglobin were elevated and bactericidal and phagocytic activities of blood were altered, indicating that these measurements are sensitive to the current infection status of the animal. All assays could be done on as little as 10 microL of blood or plasma and should be useful in field studies of comparative immunity.

Contrasting adaptive immune defenses and blood parasite prevalence in closely related Passer sparrows.

Immune system components differ in their functions and costs, and immune defense profiles are likely to vary among species with differing ecologies. We compared adaptive immune defenses in two closely related species that have contrasting inflammatory immune responses, the widespread and abundant house sparrow (Passer domesticus) and the less abundant tree sparrow (Passer montanus). We found that the house sparrow, which we have previously shown mounts weaker inflammatory responses, exhibits stronger adaptive immune defenses, including antibody responses, natural antibody titers, and specific T-cell memory, than the tree sparrow. Conversely, tree sparrows, which mount strong inflammatory responses, also mount stronger nonspecific inflammatory T-cell responses but weaker specific adaptive responses. Prevalence of avian malaria parasite infections, which are controlled by adaptive immune defenses, was higher in the geographically restricted tree sparrow than in the ubiquitous house sparrow. Together these data describe distinct immune defense profiles between two closely related species that differ greatly in numbers and distributions. We suggest that these immunological differences could affect fitness in ways that contribute to the contrasting abundances of the two species in North American and Western Europe.

Linking immune defenses and life history at the levels of the individual and the species.

Immune defenses have been suggested to play an important role in mediating life history trade-offs. Detecting and understanding such trade-offs, however, is complicated by the complexity of the immune system. The measurement of multiple immune indices in studies of "eco-immunology" has only recently become more common, but has great potential for furthering an understanding of the ecological and evolutionary forces driving immunological variation. Building on previous proposals, I create a framework integrating immunological and life history axes that can be used to formulate predictions and interpret variation in multiple types of immune defense at both the individual and species levels in vertebrates. In particular, this framework predicts that "fast-living" species (those with high reproductive and low survival rates) should rely more heavily on nonspecific and inflammatory immune defenses, while "slow-living" species should exhibit stronger specific and especially antibody-mediated immunity. At the level of individuals within species, nonspecific and inflammatory responses should be downregulated, and specific defenses upregulated (1) in individuals experiencing the greatest demands on their resources (for example, undertaking large reproductive efforts); (2) in the sex investing more in a particular activity (for example, females during reproduction); and (3) during the most demanding periods of the year (for example, the breeding season). A review of the literature reveals that incorporating multiple facets of the immune system into a model of the relationship between immune defense and life histories brings disparate questions and systems into a common context, and helps explain empirical results that are sometimes counterintuitive.

Responding to inflammatory challenges is less costly for a successful avian invader, the house sparrow (Passer domesticus), than its less-invasive congener.

When introduced into new regions, invading organisms leave many native pathogens behind and also encounter evolutionarily novel disease threats. In the presence of predominantly novel pathogens that have not co-evolved to avoid inducing a strong host immune response, costly and potentially dangerous defenses such as the systemic inflammatory response could become more harmful than protective to the host. We therefore hypothesized that introduced populations exhibiting dampened inflammatory responses will tend to be more invasive. To provide initial data to assess this hypothesis, we measured metabolic, locomotor, and reproductive responses to inflammatory challenges in North American populations of the highly invasive house sparrow (Passer domesticus) and its less-invasive relative, the tree sparrow (Passer montanus). In the house sparrow, there was no effect of phytohemagglutinin (PHA) challenge on metabolic rate, and there were no detectable differences in locomotor activity between lipopolysaccharide (LPS)-injected birds and saline-injected controls. In contrast, tree sparrows injected with PHA had metabolic rates 20-25% lower than controls, and LPS injection resulted in a 35% drop in locomotor activity. In a common garden captive breeding experiment, there was no effect of killed-bacteria injections on reproduction in the house sparrow, while tree sparrows challenged with bacteria decreased egg production by 40% compared to saline-injected controls. These results provide some of the first data correlating variation in immune defenses with invasion success in introduced-vertebrate populations.

A role for immunology in invasion biology.

Invasive species are of increasing conservation and economic concern, yet mechanisms underlying invasions remain poorly understood. We propose that variation in immune defences might help explain why only some introduced populations become invasive. Introduced species escape many of their native diseases, but also face novel pathogens that can induce costly, and sometimes deadly, immune responses in naïve hosts. Therefore, favouring less resource-demanding and dangerous defence mechanisms and allocating a greater proportion of resources to growth and reproduction should favour invasion. Specifically, we argue that successful invaders should reduce costly systemic inflammatory responses, which are associated with fever and metabolic and behavioural changes, and rely more heavily on less expensive antibody-mediated immunity. Here we provide supporting arguments for this hypothesis and generate predictions that are testable using tools from the growing field of ecological immunology.

Digestive response to restricted feeding in migratory yellow-rumped warblers.

Smaller guts and slow initial mass gains at stopover sites have led to the idea that digestive physiology limits refueling rates in migrating birds. We tested the digestive-limitation hypothesis in yellow-rumped warblers using food restriction to simulate infrequent feeding during migration, which may cause a reduction in alimentary tract mass. Restricted birds had small intestine, pancreas, and liver masses 18%-22% lower than ad lib.-fed controls. Total activities of sucrase, maltase, aminopeptidase, and amylase were significantly lower in restricted birds, while those of trypsin and chymotrypsin were not. Only aminopeptidase mass-specific activity was significantly lower in restricted birds. Previously restricted birds were able to feed and digest at a high rate immediately following return to ad lib. feeding. Digestive efficiency did not differ between groups. These results suggest that before migration yellow-rumped warblers have some spare digestive capacity to compensate for declines in their digestive organ masses during migration.