The relationship between dietary trophic level, parasites and the microbiome of Pacific walrus (Odobenus rosmarus divergens).

Arctic species are likely to experience rapid shifts in prey availability under climate change, which may alter their exposure to microbes and parasites. Here, we describe fecal bacterial and macroparasite communities and assess correlations with diet trophic level in Pacific walruses harvested during subsistence hunts by members of the Native Villages of Gambell and Savoonga on St Lawrence Island, Alaska. Fecal bacterial communities were dominated by relatively few taxa, mostly belonging to phyla Fusobacteriota and Firmicutes. Members of parasite-associated phyla Nematoda, Acanthocephala and Platyhelminthes were prevalent in our study population. We hypothesized that high versus low prey trophic level (e.g. fish versus bivalves) would result in different gut bacterial and macroparasite communities. We found that bacterial community structure correlated to diet, with nine clades enriched in walruses consuming higher-trophic-level prey. While no parasite compositional differences were found at the phylum level, the cestode genus Diphyllobothrium was more prevalent and abundant in walruses consuming higher-trophic-level prey, probably because fish are the intermediate hosts for this genus. This study suggests that diet is important for structuring both parasite and microbial communities of this culturally and ecologically important species, with potential implications for population health under climate change.

BRIDGING GAPS BETWEEN ZOO AND WILDLIFE MEDICINE: ESTABLISHING REFERENCE INTERVALS FOR FREE-RANGING AFRICAN LIONS (PANTHERA LEO).

The International Species Information System has set forth an extensive database of reference intervals for zoologic species, allowing veterinarians and game park officials to distinguish normal health parameters from underlying disease processes in captive wildlife. However, several recent studies comparing reference values from captive and free-ranging animals have found significant variation between populations, necessitating the development of separate reference intervals in free-ranging wildlife to aid in the interpretation of health data. Thus, this study characterizes reference intervals for six biochemical analytes, eleven hematologic or immune parameters, and three hormones using samples from 219 free-ranging African lions ( Panthera leo ) captured in Kruger National Park, South Africa. Using the original sample population, exclusion criteria based on physical examination were applied to yield a final reference population of 52 clinically normal lions. Reference intervals were then generated via 90% confidence intervals on log-transformed data using parametric bootstrapping techniques. In addition to the generation of reference intervals, linear mixed-effect models and generalized linear mixed-effect models were used to model associations of each focal parameter with the following independent variables: age, sex, and body condition score. Age and sex were statistically significant drivers for changes in hepatic enzymes, renal values, hematologic parameters, and leptin, a hormone related to body fat stores. Body condition was positively correlated with changes in monocyte counts. Given the large variation in reference values taken from captive versus free-ranging lions, it is our hope that this study will serve as a baseline for future clinical evaluations and biomedical research targeting free-ranging African lions.

MRI tracking of transplanted iron-labeled mesenchymal stromal cells in an immune-compromised mouse model of critical limb ischemia.

Peripheral arterial disease is a clinical problem in which mesenchymal stromal cell (MSC) transplantation may offer substantial benefit by promoting the generation of new blood vessels and improving limb ischemia and wound healing via their potent paracrine activities. MRI allows for the noninvasive tracking of cells over time using iron oxide contrast agents to label cells before they are injected or transplanted. However, a major limitation of the tracking of iron oxide-labeled cells with MRI is the possibility that dead or dying cells will transfer the iron oxide label to local bystander macrophages, making it very difficult to distinguish between viable transplanted cells and endogenous macrophages in the images. In this study, a severely immune-compromised mouse, with limited macrophage activity, was investigated to examine cell tracking in a system in which bystander cell uptake of dead, iron-labeled cells or free iron particles was minimized. MRI was used to track the fate of MSCs over 21 days after their intramuscular transplantation in mice with a femoral artery ligation. In all mice, a region of signal loss was observed at the injection site and the volume of signal hypointensity diminished over time. Fluorescence and light microscopy showed that iron-positive MSCs persisted at the transplant site and often appeared to be integrated in perivascular niches. This was compared with MSC transplantation in immune-competent mice with femoral artery ligation. In these mice, the regions of signal loss caused by iron-labeled MSC cleared more slowly, and histology revealed iron particles trapped at the site of cell transplantation and associated with areas of inflammation.

Umbilical cord blood-derived aldehyde dehydrogenase-expressing progenitor cells promote recovery from acute ischemic injury.

Umbilical cord blood (UCB) represents a readily available source of hematopoietic and endothelial precursors at early ontogeny. Understanding the proangiogenic functions of these somatic progenitor subtypes after transplantation is integral to the development of improved cell-based therapies to treat ischemic diseases. We used fluorescence-activated cell sorting to purify a rare (<0.5%) population of UCB cells with high aldehyde dehydrogenase (ALDH(hi) ) activity, a conserved stem/progenitor cell function. ALDH(hi) cells were depleted of mature monocytes and T- and B-lymphocytes and were enriched for early myeloid (CD33) and stem cell-associated (CD34, CD133, and CD117) phenotypes. Although these cells were primarily hematopoietic in origin, UCB ALDH(hi) cells demonstrated a proangiogenic transcription profile and were highly enriched for both multipotent myeloid and endothelial colony-forming cells in vitro. Coculture of ALDH(hi) cells in hanging transwells promoted the survival of human umbilical vein endothelial cells (HUVEC) under growth factor-free and serum-free conditions. On growth factor depleted matrigel, ALDH(hi) cells significantly increased tube-like cord formation by HUVEC. After induction of acute unilateral hind limb ischemia by femoral artery ligation, transplantation of ALDH(hi) cells significantly enhanced the recovery of perfusion in ischemic limbs. Despite transient engraftment in the ischemic hind limb, early recruitment of ALDH(hi) cells into ischemic muscle tissue correlated with increased murine von Willebrand factor blood vessel and CD31+ capillary densities. Thus, UCB ALDH(hi) cells represent a readily available population of proangiogenic progenitors that promote vascular regeneration. This work provides preclinical justification for the development of therapeutic strategies to treat ischemic diseases using UCB-derived ALDH(hi) mixed progenitor cells.

Equal contributions of feline immunodeficiency virus and coinfections to morbidity in African lions.

Feline immunodeficiency virus (FIV) is a pathogenic lentivirus related to human and simian immunodeficiency viruses that has been associated with AIDS-like pathologies in domestic and wild cats, as well as in hyenas. Despite known pathologies, progressive immunosuppression and ill health effects driven by these lentiviruses in association with other secondary infections remain understudied in free-ranging species. Here, the role of coinfections by gastrointestinal parasites and tick-borne hemoparasites for FIV disease progression was explored in 195 free-ranging African lions (Panthera leo) living in Kruger National Park (KNP), South Africa. Using statistical methodology, we evaluated the effects of FIV on a range of health indicators to explore how direct and indirect effects of FIV and associated coinfections align to determine lion health outcomes. Findings show direct negative effects of FIV on host immunity and nutritional status, and exacerbation of aggressive behaviors, conditions which may increase exposure/susceptibility to other secondary infections. When taken together, the contribution of coinfecting parasites to morbidity in lions is of similar magnitude as direct effects of FIV infection alone, suggesting that the particular coinfection assemblage may play a role in mediating disease progression within natural lion populations.

Fecal Methylmercury Correlates With Gut Microbiota Taxa in Pacific Walruses (Odobenus rosmarus divergens).

OBJECTIVES: Methylmercury metabolism was investigated in Pacific walruses (Odobenus rosmarus divergens) from St. Lawrence Island, Alaska, United States. METHODS: Total mercury and methylmercury concentrations were measured in fecal samples and paired colon samples (n = 16 walruses). Gut microbiota composition and diversity were determined using 16S rRNA gene sequencing. Associations between fecal and colon mercury and the 24 most prevalent gut microbiota taxa were investigated using linear models. RESULTS: In fecal samples, the median values for total mercury, methylmercury, and %methylmercury (of total mercury) were 200 ng/g, 4.7 ng/g, and 2.5%, respectively, while in colon samples, the median values for the same parameters were 28 ng/g, 7.8 ng/g, and 26%, respectively. In fecal samples, methylmercury was negatively correlated with one Bacteroides genus, while members of the Oscillospirales order were positively correlated with both methylmercury and %methylmercury (of total mercury). In colon samples, %methylmercury (of total mercury) was negatively correlated with members of two genera, Romboutsia and Paeniclostridium. CONCLUSIONS: Median %methylmercury (of total mercury) was 10 times higher in the colon compared to the fecal samples, suggesting that methylmercury was able to pass through the colon into systemic circulation. Fecal total mercury and/or methylmercury concentrations in walruses were comparable to some human studies despite differences in seafood consumption rates, suggesting that walruses excreted less mercury. There are no members (at this time) of the Oscillospirales order which are known to contain the genes to methylate mercury, suggesting the source of methylmercury in the gut was from diet and not in vivo methylation.

Innate immunity in free-ranging African buffalo (Syncerus caffer): associations with parasite infection and white blood cell counts.

Mammalian immunology has been studied in great detail in laboratory animals, but few of the tools and less of the insight derived from these studies have been placed in the context of natural, outbred wildlife populations subject to variable environments. We investigated patterns of innate immunity in free-ranging African buffalo in relation to host traits (age, reproductive status, body condition, white blood cell counts) and disease status (bovine tuberculosis [BTB], gastrointestinal nematodes, coccidia, ticks). We evaluated and used an in vitro assay measuring bactericidal competence of blood to assess a component of innate immunity in 200 female buffalo captured at Kruger National Park, South Africa, in June/July and October 2008. Animals with BTB had higher bactericidal competence of blood. Animals with higher neutrophil counts had higher bactericidal competence, whereas animals with lower lymphocyte counts had higher bactericidal competence. This pattern was driven by animals captured at the end of the dry season (October) and may be evidence of immune polarization, whereby individuals are unable to upregulate multiple components of immunity simultaneously. Bactericidal competence did not vary with host pregnancy status, body condition, age, lactation, tick infestation, nematode egg count, or coccidia oocyst count. Overall, we demonstrate that the bactericidal competence assay is practical and informative for field-based studies in wild bovids. Our results also show a correlation between bactericidal competence and bovine tuberculosis infection and reveal possible functional polarizations between different types of immune response in a free-ranging mammal.

Transplanted human bone marrow progenitor subtypes stimulate endogenous islet regeneration and revascularization.

Transplanted murine bone marrow (BM) progenitor cells recruit to the injured pancreas and induce endogenous beta cell proliferation to improve islet function. To enrich for analogous human progenitor cell types that stimulate islet regeneration, we purified human BM based on high-aldehyde dehydrogenase activity (ALDH(hi)), an enzymatic function conserved in hematopoietic, endothelial, and mesenchymal progenitor lineages. We investigated the contributions of ALDH(hi) mixed progenitor cells or culture-expanded, ALDH-purified multipotent stromal cell (MSC) subsets to activate endogenous programs for islet regeneration after transplantation into streptozotocin-treated NOD/SCID mice. Intravenous injection of uncultured BM ALDH(hi) cells improved systemic hyperglycemia and augmented insulin secretion by increasing islet size and vascularization, without increasing total islet number. Augmented proliferation within regenerated endogenous islets and associated vascular endothelium indicated the induction of islet-specific proliferative and pro-angiogenic programs. Although cultured MSC from independent human BM samples showed variable capacity to improve islet function, and prolonged expansion diminished hyperglycemic recovery, transplantation of ALDH-purified regenerative MSC reduced hyperglycemia and augmented total beta cell mass by stimulating the formation of small beta cell clusters associated with the ductal epithelium, without evidence of increased islet vascularization or Ngn3(+) endocrine precursor activation. Thus, endogenous islet recovery after progenitor cell transplantation can occur via distinct regenerative mechanisms modulated by subtypes of progenitor cells administered. Further, understanding of how these islet regenerative and pro-angiogenic programs are activated by specific progenitor subsets may provide new approaches for combination cellular therapies to combat diabetes.

Combinatorial human progenitor cell transplantation optimizes islet regeneration through secretion of paracrine factors.

Transplanted human bone marrow (BM) and umbilical cord blood (UCB) progenitor cells activate islet-regenerative or revascularization programs depending on the progenitor subtypes administered. Using purification of multiple progenitor subtypes based on a conserved stem cell function, high aldehyde dehydrogenase (ALDH) activity (ALDH(hi)), we have recently shown that transplantation of BM-derived ALDH(hi) progenitors improved systemic hyperglycemia and augmented insulin secretion by increasing islet-associated proliferation and vascularization, without increasing islet number. Conversely, transplantation of culture-expanded multipotent-stromal cells (MSCs) derived from BM ALDH(hi) cells augmented total beta cell mass via formation of beta cell clusters associated with the ductal epithelium, without sustained islet vascularization. To identify paracrine effectors produced by islet-regenerative MSCs, culture-expanded BM ALDH(hi) MSCs were transplanted into streptozotocin-treated nonobese diabetic/severe combine immune deficient (SCID) mice and segregated into islet-regenerative versus nonregenerative cohorts based on hyperglycemia reduction, and subsequently compared for differential production of mRNA and secreted proteins. Regenerative MSCs showed increased expression of matrix metalloproteases, epidermal growth factor receptor (EGFR)-activating ligands, and downstream effectors of Wnt signaling. Regenerative MSC supernatant also contained increased levels of pro-angiogenic versus pro-inflammatory cytokines, and augmented the expansion of ductal epithelial but not beta cells in vitro. Conversely, co-culture with UCB ALDH(hi) cells induced beta cell but not ductal epithelial cell proliferation. Sequential transplantation of MSCs followed by UCB ALDH(hi) cells improved hyperglycemia and glucose tolerance by increasing beta cell mass associated with the ductal epithelium and by augmenting intra-islet capillary densities. Thus, combinatorial human progenitor cell transplantation stimulated both islet-regenerative and revascularization programs. Understanding the progenitor-specific pathways that modulate islet-regenerative and revascularization processes may provide new approaches for diabetes therapy.

Embryonic morphogen nodal promotes breast cancer growth and progression.

Breast cancers expressing human embryonic stem cell (hESC)-associated genes are more likely to progress than well-differentiated cancers and are thus associated with poor patient prognosis. Elevated proliferation and evasion of growth control are similarly associated with disease progression, and are classical hallmarks of cancer. In the current study we demonstrate that the hESC-associated factor Nodal promotes breast cancer growth. Specifically, we show that Nodal is elevated in aggressive MDA-MB-231, MDA-MB-468 and Hs578t human breast cancer cell lines, compared to poorly aggressive MCF-7 and T47D breast cancer cell lines. Nodal knockdown in aggressive breast cancer cells via shRNA reduces tumour incidence and significantly blunts tumour growth at primary sites. In vitro, using Trypan Blue exclusion assays, Western blot analysis of phosphorylated histone H3 and cleaved caspase-9, and real time RT-PCR analysis of BAX and BCL2 gene expression, we demonstrate that Nodal promotes expansion of breast cancer cells, likely via a combinatorial mechanism involving increased proliferation and decreased apopotosis. In an experimental model of metastasis using beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII (MPSVII) mice, we show that although Nodal is not required for the formation of small (<100 cells) micrometastases at secondary sites, it supports an elevated proliferation:apoptosis ratio (Ki67:TUNEL) in micrometastatic lesions. Indeed, at longer time points (8 weeks), we determined that Nodal is necessary for the subsequent development of macrometastatic lesions. Our findings demonstrate that Nodal supports tumour growth at primary and secondary sites by increasing the ratio of proliferation:apoptosis in breast cancer cells. As Nodal expression is relatively limited to embryonic systems and cancer, this study establishes Nodal as a potential tumour-specific target for the treatment of breast cancer.