Longitudinal In Vivo Assessment of Host-Microbe Interactions in a Murine Model of Pulmonary Aspergillosis.

The fungus Aspergillus fumigatus is ubiquitous in nature and the most common cause of invasive pulmonary aspergillosis (IPA) in patients with a compromised immune system. The development of IPA in patients under immunosuppressive treatment or in patients with primary immunodeficiency demonstrates the importance of the host immune response in controlling aspergillosis. However, study of the host-microbe interaction has been hampered by the lack of tools for their non-invasive assessment. We developed a methodology to study the response of the host's immune system against IPA longitudinally in vivo by using fluorine-19 magnetic resonance imaging (19F MRI). We showed the advantage of a perfluorocarbon-based contrast agent for the in vivo labeling of macrophages and dendritic cells, permitting quantification of pulmonary inflammation in different murine IPA models. Our findings reveal the potential of 19F MRI for the assessment of rapid kinetics of innate immune response against IPA and the permissive niche generated through immunosuppression.

Baseline normal values and phylogenetic class of the electrocardiogram of anesthetized free-ranging brown bears (Ursus arctos).

Electrocardiographic (ECG) variables were measured in 22 healthy, free-ranging brown bears (Ursus arctos) anesthetized with a combination of medetomidine, tiletamine, and zolazepam during research and management operations in south-central Sweden. Six-limb lead ECGs and the base-apex lead were recorded. Morphologies, amplitudes, rhythms, and durations of P waves, PQ intervals, QRS complexes, T waves, QT intervals, and QT intervals corrected for heart rate (QTc) were calculated from the base-apex lead. The mean electrical axis (MEA) for each individual was approximated in the frontal plane from the heights of R waves in leads I (X axis) and aVF (Y axis). All 22 bears had sinus rhythms and 10 of them had respiratory sinus arrhythmia. Heart rates ranged from 43 to 103 beats per minute and were independent of body mass. The MEA was 78.6 (+ or - 5.5) degrees. Some bears had strikingly peaked T waves, and many had pronounced notching (high frequency components) to the QRS complex. This information contributes to the limited data base of electrocardiography for brown bears and elucidates ECG similarities with other species in the order Carnivora.

Regulation of inflammation by Rac2 in immune complex-mediated acute lung injury.

Acute lung injury (ALI) is an inflammatory disorder associated with recruitment and activation of neutrophils in lungs. Rac2, a member of the Rho GTPase subfamily, is an essential regulator of neutrophil degranulation, superoxide release, and chemotaxis. Here, we hypothesized that Rac2 is important in mediating lung injury. Using a model of IgG immune complex-mediated ALI, we showed that injury was attenuated in rac2(-/-) mice compared with wild-type (WT) mice undergoing ALI, with significant decreases in alveolar leukocyte numbers, vascular leakage, and the inflammatory mediators, myeloperoxidase (MPO) and matrix metalloproteinases (MMPs). Reduced injury in rac2(-/-) mice was not associated with diminished cytokine and chemokine production, since bronchoalveolar lavage (BAL) levels of IL-17, TNF, CCL3, CXCL1, and CXCL2 were similarly increased in WT and rac2(-/-) mice with ALI compared with sham-treated mice (no ALI). BAL levels of MMP-2 and MMP-9 were significantly decreased in the airways of rac2(-/-) mice with ALI. Immunohistochemical analysis revealed that MMP-2 and MMP-9 expression was evident in alveolar macrophages and interstitial neutrophils in WT ALI. In contrast, MMP-positive cells were less prominent in rac2(-/-) mice with ALI. Chimeric mice showed that Rac2-mediated lung injury was dependent on hematopoietic cells derived from bone marrow. We propose that lung injury in response to immune complex deposition is dependent on Rac2 in alveolar macrophages and neutrophils.

Serologic response to West Nile virus vaccination in the greater one-horned rhinoceros (Rhinoceros unicornis).

Vaccination has been an important component of preventative health care programs of North American zoologic institutions in their protection of valuable species against West Nile virus (WNV) infection since its detection in 1999. Although approved only for horses, commercial WNV vaccine has been used for the purpose of protection of nondomestic species, including avian, equid, and rhinoceros species. Currently, there are two commercial equine vaccines available, a killed vaccine and a recombinant viral-vectored vaccine. Both products have been used for the vaccination of Greater One-horned rhinoceroses (Rhinoceros unicornis) held in North American zoologic institutions. In this study, the efficacy of these vaccines was evaluated in Greater One-horned rhinoceroses based on the humoral immune response stimulated by vaccine administration. Five rhinoceroses were vaccinated in 2005 by using the killed equine vaccine and four received boosters in 2006 by using the recombinant vaccine. Rhinoceroses were evaluated for differences in pre- and postvaccination neutralizing antibody titer and gamma and beta globulins on serum protein electrophoresis. No changes were observed after administration of the killed vaccine; however, antibody titers were observed in two of four rhinoceroses after administration of the recombinant vaccine. No significant changes were observed in the serum protein electrophoresis after either vaccine. Based on these findings, the WNV recombinant vaccine appeared to induce a more measurable humoral immune response than the killed product in the Greater One-horned rhinoceros. However, further investigation of both vaccines is warranted to evaluate whether changes in the frequency of administration, dosage, or adjuvant might stimulate an improved humoral response in these animals.

Developmental regulation of Foxp3 expression during ontogeny.

Thymectomy of neonatal mice can result in the development of autoimmune pathology. It has been proposed that thymic output of regulatory T (T reg) cells is delayed during ontogeny and that the development of autoimmune disease in neonatally thymectomized mice is caused by the escape of self-reactive T cells before thymectomy without accompanying T reg cells. However, the kinetics of T reg cell production within the thymus during ontogeny has not been assessed. We demonstrate that the development of Foxp3-expressing T reg cells is substantially delayed relative to nonregulatory thymocytes during ontogeny. Based on our data, we speculate that induction of Foxp3 in developing thymocytes and, thus, commitment to the T reg cell lineage is facilitated by a signal largely associated with the thymic medulla.

Regulatory T cell lineage specification by the forkhead transcription factor foxp3.

Regulatory T cell-mediated dominant tolerance has been demonstrated to play an important role in the prevention of autoimmunity. Here, we present data arguing that the forkhead transcription factor Foxp3 acts as the regulatory T cell lineage specification factor and mediator of the genetic mechanism of dominant tolerance. We show that expression of Foxp3 is highly restricted to the subset alphabeta of T cells and, irrespective of CD25 expression, correlates with suppressor activity. Induction of Foxp3 expression in nonregulatory T cells does not occur during pathogen-driven immune responses, and Foxp3 deficiency does not impact the functional responses of nonregulatory T cells. Furthermore, T cell-specific ablation of Foxp3 is sufficient to induce the identical early onset lymphoproliferative syndrome observed in Foxp3-deficient mice. Analysis of Foxp3 expression during thymic development suggests that this mechanism is not hard-wired but is dependent on TCR/MHC ligand interactions.

Organization of thymic medullary epithelial heterogeneity: implications for mechanisms of epithelial differentiation.

There are accumulating data to show that thymic epithelium expresses a remarkable array of molecules previously considered to be tissue-specific antigens, such as parathyroid hormone, thyroglobulin, insulin, and C-reactive protein. From an immunological perspective, this property of thymic epithelium would provide an ideal mechanism to effect central tolerance of epithelial-restricted antigens. However, from a mechanistic perspective, this phenomenon remains mysterious. Two explanations have been proposed. One invokes promiscuous gene expression by medullary thymic epithelial cells that would allow transient derepression of selected gene expression. The other proposes that the expression of tissue-restricted genes by thymic epithelium reflects alternate pathways of epithelial development by small numbers of cells to form a mosaic of different epithelial types within the thymus. Here we show thymic expression of lung-associated gene products by an organized epithelial 'organoid' with ultrastructural features of respiratory epithelium and present data suggesting that the thymus also contains structures that ultrastructurally and phenotypically resemble solitary thyroid follicles. Based on these data, it is proposed that some thymic epithelial progenitor cells resemble pharyngeal endoderm in terms of their developmental potential and that alternative differentiation fates taken by these cells serve to maintain the spectrum of epithelial 'self' in the thymus.

Experimental tests of nest site competition in two Peromyscus species.

The importance of interspecific competition for nest sites between the white-footed mouse (Peromyscus leucopus noveboracensis) and the cloudland deermouse (P. maniculatus nubiterrae) were investigated in the montane forests of southwestern Virginia over 3 years. Trials were conducted for both species using large, outdoor enclosures in order to examine: (i) nest site preference in isolation and (ii) nest site selection made in the presence of potential competitors. Both species demonstrated a strong preference for arboreal nest sites when tested without competitors present. After the introduction of a heterospecific intruder, P. leucopus often shifted to a non-arboreal nest while P. maniculatus continued to use arboreal nests. Intruding P. maniculatus displaced resident P. leucopus from P. leucopus' preferred nest sites in all 3 years while intruding P. leucopus never displaced P. maniculatus. Neither species was routinely displaced in conspecific trials. Resident P. maniculatus also excluded P. leucopus from access to preferred nesting sites in all three years while P. leucopus were only able to exclude P. maniculatus in the 3rd year. Both species exhibite relatively low frequencies of exclusion in conspecific trials with the exception of P. maniculatus which excluded high proportions of conspecific intruders in the second year. We suggest that the asymmetrical advantage enjoyed by P. maniculatus in nest site selection may result from both site-specific effects and a species-specific influence on P. leucopus. Nonetheless, the intensity of such influence varied between years, perhaps as a function of population density, and did not appear to drastically influence continued coexistence of these congeners.

Use of space and habitats by meadow voles at the home range, patch and landscape scales.

Using capture/recapture methods, we examined the spatial usage patterns of Microtus pennsylvanicus within and between experimentally created habitat patches of three sizes (1.0, 0.25 and 0.0625 ha) and between a 20-ha fragmented and a 20-ha continuous habitat landscape. We tested the prediction that home ranges near patch edges would be qualitatively different from those in patch interiors, and that the edge:interior habitat ratio could be used to make predictions concerning the dispersion and spatial use of individuals occupying different sized patches and between landscapes with different habitat structure. We found adult females on patch edges to have larger and more exclusive home ranges, larger body sizes, longer residence times, and to reproduce at a higher frequency than those in patch interiors. These "edge effects" also appeared to be largely responsible for the greater proportion of larger, reproductive females we found in small than larger patches and in the fragmented than in the continuous habitat (control) landscape. The selection of higher quality edge habitats by dominant females and the relegation of sub-dominants to patch interiors provides an explanation for the observed differences in the distribution and performance of females over patches and between landscapes.

Controlled experiments of habitat fragmentation: a simple computer simulation and a test using small mammals.

Habitat fragmentation involves a reduction in the effective area available to a population and the imposition of hard patch edges. Studies seeking to measure effects of habitat fragmentation have compared populations in fragments of different size to estimate and area effect but few have examined the effect of converting open populations to closed ones (an effect of edges). To do so requires a shift in spatial scope-from comparison of individual fragments to that of fragmented versus unfragmented landscapes. Here we note that large-scale, "controlled" studies of habitat fragmentation have rarely been performed and are needed. In making our case we develop a simple computer simulation model based on how individual animals with home ranges are affected by the imposition of habitat edges, and use it to predict population-level responses to habitat fragmentation. We then compare predictions of the model with results from a field experiment on Peromyscus and Microtus. Our model treats the case where home ranges/territories fall entirely within or partially overlap with that of sample areas in continuous landscapes, but are restricted to areas within habitat fragments in impacted landscapes. Results of the simulations demonstrate that the imposition of hard edges can produce different population abundances for similar-sized areas in continuous and fragmented landscapes. This edge effect is disproportionately greater in small than large fragments and for species with larger than smaller home ranges. These predictions were generally supported by our field experiment. We argue that large-scale studies of habitat fragmentation are sorely needed, and that control-experiment contrasts of fragmented and unfragmented microlandscapes provide a logical starting point.

Predation hazard and seed removal by small mammals: microhabitat versus patch scale effects.

Predator avoidance may involve response strategies of prey species that are time and space specific. Many studies have shown that foraging individuals avoid predators by altering microhabitat usage; alternatively, sites may be selected according to larger-scale features of the habitat mosaic. We measured seed removal by two small mammal species (Peromyscus leucopus, and Microtus pennsylvanicus) at 474 stations over an experimentally created landscape of 12 patches, and under conditions of relatively high (full moon) and low (new moon) predatory hazard. Our objective was to determine whether predator avoidance involved the selection of small-, medium-, or large-scale features of the landscape (i.e., at the scale of microhabitats, habitats, or habitat patches). We found rates of seed removal to vary more with features of whole patches than according to variation in structural microhabitats within patches. Specific responses included: under-utilization of patch edge habitats during full moon periods, and microhabitat effects that were only significant when considered in conjunction with larger-scale features of the landscape. Individuals residing on large patches altered use of microhabitats/habitats to a greater extent than those on smaller patches. Studies just focusing on patterns of microhabitat use will miss responses at the larger scales, and may underestimate the importance of predation to animal foraging behavior.