Obesity impacts hypoxia adaptation of the lung.

Obesity is mostly associated with adverse health consequences, but may also elicit favorable effects under chronic conditions. This "obesity paradox" is under debate for pulmonary diseases. As confounding factors complicate conclusions from human studies, this study used a controlled animal model combining diet-induced obesity and chronic hypoxia as a model for pulmonary hypertension and chronic obstructive pulmonary disease. Male C57BL/6 mice were fed control or high-fat diet for 30 wk, and half of the animals were exposed to chronic hypoxia (13% O2) for 3 wk. Hypoxia induced right ventricular hypertrophy, thickening of pulmonary arterial and capillary walls, higher lung volumes, and increased hemoglobin concentrations irrespective of the body weight. In contrast, lung proteomes differed substantially between lean- and obese-hypoxic mice. Many of the observed changes were linked to vascular and extracellular matrix (ECM) proteins. In lean-hypoxic animals, circulating platelets were reduced and abundances of various clotting-related proteins were altered, indicating a hypercoagulable phenotype. Moreover, the septal ECM composition was changed, and airspaces were significantly distended pointing to lung hyperinflation. These differences were mostly absent in the obese-hypoxic group. However, the obesity-hypoxia combination induced the lowest blood CO2 concentrations, indicating hyperventilation for sufficient oxygen supply. Moreover, endothelial surface areas were increased in obese-hypoxic mice. Thus, obesity exerts differential effects on lung adaptation to hypoxia, which paradoxically include not only adverse but also rather protective changes. These differences have a molecular basis in the lung proteome and may influence the pathogenesis of lung diseases. This should be taken into account for future individualized prevention and therapy.NEW & NOTEWORTHY An "obesity paradox" is discussed for pulmonary diseases. By linking lung proteome analyses to pulmonary structure and function, we demonstrate that diet-induced obesity affects lung adaptation to chronic hypoxia in various ways. The observed changes include not only adverse but also protective effects and are associated with altered abundances of vascular and extracellular matrix proteins. These results highlight the existence of relevant differences in individuals with obesity that may influence the pathogenesis of lung diseases.

A double deletion prevents replication of the pestivirus bovine viral diarrhea virus in the placenta of pregnant heifers.

In contrast to wild type bovine viral diarhea virus (BVDV) specific double deletion mutants are not able to establish persistent infection upon infection of a pregnant heifer. Our data shows that this finding results from a defect in transfer of the virus from the mother animal to the fetus. Pregnant heifers were inoculated with such a double deletion mutant or the parental wild type virus and slaughtered pairwise on days 6, 9, 10 and 13 post infection. Viral RNA was detected via qRT-PCR and RNAscope analyses in maternal tissues for both viruses from day 6 p.i. on. However, the double deletion mutant was not detected in placenta and was only found in samples from animals infected with the wild type virus. Similarly, high levels of wild type viral RNA were present in fetal tissues whereas the genome of the double deletion mutant was not detected supporting the hypothesis of a specific inhibition of mutant virus replication in the placenta. We compared the induction of gene expression upon infection of placenta derived cell lines with wild type and mutant virus via gene array analysis. Genes important for the innate immune response were strongly upregulated by the mutant virus compared to the wild type in caruncle epithelial cells that establish the cell layer on the maternal side at the maternal-fetal interface in the placenta. Also, trophoblasts which can be found on the fetal side of the interface showed significant induction of gene expression upon infection with the mutant virus although with lower complexity. Growth curves recorded in both cell lines revealed a general reduction of virus replication in caruncular epithelial cells compared to the trophoblasts. Compared to the wild type virus this effect was dramtic for the mutant virus that reached only a TCID50 of 1.0 at 72 hours post infection.

Ischaemic postconditioning reduces apoptosis in experimental jejunal ischaemia in horses.

BACKGROUND: Ischaemic postconditioning (IPoC) refers to brief periods of reocclusion of blood supply following an ischaemic event. This has been shown to ameliorate ischaemia reperfusion injury in different tissues, and it may represent a feasible therapeutic strategy for ischaemia reperfusion injury following strangulating small intestinal lesions in horses. The objective of this study was to assess the degree cell death, inflammation, oxidative stress, and heat shock response in an equine experimental jejunal ischaemia model with and without IPoC. METHODS: In this randomized, controlled, experimental in vivo study, 14 horses were evenly assigned to a control group and a group subjected to IPoC. Under general anaesthesia, segmental ischaemia with arterial and venous occlusion was induced in 1.5 m jejunum. Following ischaemia, the mesenteric vessels were repeatedly re-occluded in group IPoC only. Full thickness intestinal samples and blood samples were taken at the end of the pre-ischaemia period, after ischaemia, and after 120 min of reperfusion. Immunohistochemical staining or enzymatic assays were performed to determine the selected variables. RESULTS: The mucosal cleaved-caspase-3 and TUNEL cell counts were significantly increased after reperfusion in the control group only. The cleaved-caspase-3 cell count was significantly lower in group IPoC after reperfusion compared to the control group. After reperfusion, the tissue myeloperoxidase activity and the calprotectin positive cell counts in the mucosa were increased in both groups, and only group IPoC showed a significant increase in the serosa. Tissue malondialdehyde and superoxide dismutase as well as blood lactate levels showed significant progression during ischaemia or reperfusion. The nuclear immunoreactivity of Heat shock protein-70 increased significantly during reperfusion. None of these variables differed between the groups. The neuronal cell counts in the myenteric plexus ganglia were not affected by the ischaemia model. CONCLUSIONS: A reduced apoptotic cell count was found in the group subjected to IPoC. None of the other tested variables were significantly affected by IPoC. Therefore, the clinical relevance and possible protective mechanism of IPoC in equine intestinal ischaemia remains unclear. Further research on the mechanism of action and its effect in clinical cases of strangulating colic is needed.

In vitro effects of Type I interferons (IFNτ and IFNα) on bovine hepatocytes cultured with or without Kupffer cells.

In cattle, maternal recognition of early pregnancy depends on the effects of the embryonic signal interferon (IFN)-τ. IFN-stimulated genes have been upregulated in the maternal liver during early pregnancy. In this study, primary hepatocyte cell culture models were evaluated for their suitability to test Type I IFN effects invitro. The expression of target genes (interferon-stimulated gene 15 (ISG-15), interferon-induced GTP-binding protein (MX-1), C-X-C motif chemokine 10 (CXCL-10), CXCL-5, insulin-like growth factor 1 (IGF-1), IGF binding protein 2 (IGFBP-2)) was measured using reverse transcription-quantitative polymerase chain reaction in hepatocytes from monoculture or in indirect coculture with Kupffer cells (HKCid) on Days 1, 2, 3 and 4 of culture (n=21 donor cows). Gene expression was also measured on Day 4 after challenging the cultures with recombinant IFNτ, IFNα, progesterone (P4), IFNτ+IFNα or IFNτ+P4 for 6h. A significant increase in the mRNA expression of target genes in hepatocytes was shown in response to stimulation with IFNτ. The Kupffer cells in coculture did not influence the effects of IFNτ in hepatocytes. In conclusion, primary bovine hepatocyte cultures are suitable for stimulation experiments with Type I IFNs and as an extrauterine model for embryo-maternal communication. The proposed endocrine action of IFNτ in the liver may affect maternal metabolism and immune function in the liver.

In vitro effects of lipopolysaccharides on bovine uterine contractility.

Metritis is an important disorder in dairy cows during the early postpartum period. Myometrial contractility is a prerequisite for uterine involution; however, very scanty literature is available about the effect of metritis on this process and endocrine responsiveness. This study was aimed to evaluate the effect of inflammation on uterine contractility in vitro, and the inflammation was induced by incubating myometrial strips with lipopolysaccharides (LPS). Myometrial samples were collected from 17 healthy Holstein Friesian cows during caesarean section. Eight longitudinal strips from each cow were incubated in organ baths with LPS concentrations of 0 (LPS0 ), 0.1 (LPS0.1 ), 1 (LPS1 ) and 10 µg/ml (LPS10 ). Spontaneous contractility and contractility induced by increasing concentrations of oxytocin (10-10 - 10-7  mol/L) were recorded during nine 30-min intervals (T1 to T9). The minimum amplitude (minA), maximum amplitude (maxA), mean amplitude (meanA) and area under the curve (AUC) were calculated for each time interval. LPS had an effect (p ≤ .05) on maxA, meanA and AUC. In T1, myometrial strips incubated with LPS0.1 and LPS1 had higher (p ≤ .05) maxA, meanA and AUC than the strips incubated with LPS0 . In T9 without oxytocin, LPS0 led to higher (p ≤ .05) maxA, meanA and AUC than LPS0.1 and LPS1 . In T8 and T9 with oxytocin, LPS1 had lower (p ≤ .05) maxA, meanA and AUC than the other LPS concentrations. Interestingly, the results show that LPS has a transient positive effect on myometrial contractility in vitro and that this effect is dependent on LPS concentration and duration of incubation.

Crosstalk between Neospora caninum and the bovine host at the maternal-foetal interface determines the outcome of infection.

Neospora caninum is an apicomplexan cyst-forming parasite that is considered one of the main causes of abortion. The pathogenic mechanisms associated with parasite virulence at the maternal-foetal interface that are responsible for the outcome of infection are largely unknown. Here, utilizing placentomes from cattle experimentally infected with high-virulence (Nc-Spain7) and low-virulence (Nc-Spain1H) isolates, we studied key elements of the innate and adaptive immune responses, as well as components of the extracellular matrix (ECM), at 10 and 20 days post-infection (dpi). The low-virulence isolate elicited a robust immune response characterized by upregulation of genes involved in pathogen recognition, chemokines and pro-inflammatory cytokines, crucial for its adequate control. In addition, Nc-Spain1H triggered the expression of anti-inflammatory cytokines and other mechanisms implicated in the maintenance of ECM integrity to ensure foetal survival. In contrast, local immune responses were initially (10 dpi) impaired by Nc-Spain7, allowing parasite multiplication. Subsequently (20 dpi), a predominantly pro-inflammatory Th1-based response and an increase in leucocyte infiltration were observed. Moreover, Nc-Spain7-infected placentomes from animals carrying non-viable foetuses exhibited higher expression of the IL-8, TNF-α, iNOS and SERP-1 genes and lower expression of the metalloproteases and their inhibitors than Nc-Spain7-infected placentomes from animals carrying viable foetuses. In addition, profound placental damage characterized by an alteration in the ECM organization in necrotic foci, which could contribute to foetal death, was found. Two different host-parasite interaction patterns were observed at the bovine placenta as representative examples of different evolutionary strategies used by this parasite for transmission to offspring.

Aging exacerbates acute lung injury-induced changes of the air-blood barrier, lung function, and inflammation in the mouse.

Acute lung injury (ALI) is characterized by hypoxemia, enhanced permeability of the air-blood barrier, and pulmonary edema. Particularly in the elderly, ALI is associated with increased morbidity and mortality. The reasons for this, however, are poorly understood. We hypothesized that age-related changes in pulmonary structure, function, and inflammation lead to a worse prognosis in ALI. ALI was induced in young (10 wk old) and old (18 mo old) male C57BL/6 mice by intranasal application of 2.5 mg lipopolysaccharide (LPS)/kg body wt or saline (control mice). After 24 h, lung function was assessed, and lungs were either processed for stereological or inflammatory analysis, such as bronchoalveolar lavage fluid (BALF) cytometry and qPCR. Both young and old mice developed severe signs of ALI, including alveolar and septal edema and enhanced inflammatory BALF cells. However, the pathology of ALI was more pronounced in old compared with young mice with nearly sixfold higher BALF protein concentration, twice the number of neutrophils, and significantly higher expression of neutrophil chemokine Cxcl1, adhesion molecule Icam-1, and metalloprotease-9, whereas the expression of tight junction protein occludin significantly decreased. The old LPS mice had thicker alveolar septa attributable to higher volumes of interstitial cells and extracellular matrix. Tissue resistance and elastance reflected observed changes at the ultrastructural level in the lung parenchyma in ALI of young and old mice. In summary, the pathology of ALI with advanced age in mice is characterized by a greater neutrophilic inflammation, leakier air-blood barrier, and altered lung function, which is in line with findings in elderly patients.

Permissiveness of bovine epithelial cells from lung, intestine, placenta and udder for infection with Coxiella burnetii.

Ruminants are the main source of human infections with the obligate intracellular bacterium Coxiella (C.) burnetii. Infected animals shed high numbers of C. burnetii by milk, feces, and birth products. In goats, shedding by the latter route coincides with C. burnetii replication in epithelial (trophoblast) cells of the placenta, which led us to hypothesize that epithelial cells are generally implicated in replication and shedding of C. burnetii. We therefore aimed at analyzing the interactions of C. burnetii with epithelial cells of the bovine host (1) at the entry site (lung epithelium) which govern host immune responses and (2) in epithelial cells of gut, udder and placenta decisive for the quantity of pathogen excretion. Epithelial cell lines [PS (udder), FKD-R 971 (small intestine), BCEC (maternal placenta), F3 (fetal placenta), BEL-26 (lung)] were inoculated with C. burnetii strains Nine Mile I (NMI) and NMII at different cultivation conditions. The cell lines exhibited different permissiveness for C. burnetii. While maintaining cell viability, udder cells allowed the highest replication rates with formation of large cell-filling Coxiella containing vacuoles. Intestinal cells showed an enhanced susceptibility to invasion but supported C. burnetii replication only at intermediate levels. Lung and placental cells also internalized the bacteria but in strikingly smaller numbers. In any of the epithelial cells, both Coxiella strains failed to trigger a substantial IL-1ß, IL-6 and TNF-α response. Epithelial cells, with mammary epithelial cells in particular, may therefore serve as a niche for C. burnetii replication in vivo without alerting the host's immune response.

Generation of Multicellular Breast Cancer Tumor Spheroids: Comparison of Different Protocols.

Multicellular tumor spheroids are widely used models in tumor research. Because of their three dimensional organization they can simulate avascular tumor areas comprising proliferative and necrotic cells. Nonetheless, protocols for spheroid generation are still inconsistent. Therefore, in this study the breast cancer cell lines MCF-7, MDA-MB-231 and SK-BR-3 have been used to compare different spheroid generation models including hanging drop, liquid overlay and suspension culture techniques, each under several conditions. Experimental approaches differed in cell numbers (400-10,000), media and additives (25 % methocel, 25 % methocel plus 1 % Matrigel, 3.5 % Matrigel). In total, 42 different experimental setups have been tested. Generation of spheroids was evaluated by light microscopy and the structural composition was assessed immunohistochemically by means of Ki-67, cleaved poly (ADP-ribose) polymerase (cPARP) and mucin-1 (MUC-1) expression. Although the tested cell lines diverged widely in their capacity of forming spheroids we recommend hanging drops supplemented with 25 % methocel as the most reliable and efficient method with regard to success of generation of uniform spheroids, costs, experimental complexity and time expenditure in the different cell lines. MCF-7 cells formed spheroids under almost all analyzed conditions, and MDA-MB-231 cells under only one protocol (liquid overlay technique, 3.5 % Matrigel), while SK-BR-3 did not under neither condition. Therefore, we outline specific methods and recommend the use of adapted and standardized spheroid generation protocols for each cell line.

Clinical and subclinical endometritis induced alterations in bovine endometrial transcriptome and miRNome profile.

BACKGROUND: Clinical and subclinical endometritis are known to affect the fertility of dairy cows by inducing uterine inflammation. We hypothesized that clinical or subclinical endometritis could affect the fertility of cows by disturbing the molecular milieu of the uterine environment. Here we aimed to investigate the endometrial molecular signatures and pathways affected by clinical and subclinical endometritis. For this, Holstein Frisian cows at 42-60 days postpartum were classified as healthy (HE), subclinical endometritis (SE) or clinical endometritis (CE) based on veterinary clinical examination of the animals and histological evaluation the corresponding endometrial biopsies. Endometrial transcriptome and miRNome profile changes and associated molecular pathways induced by subclinical or clinical endometritis were then investigated using GeneChip® Bovine Genome Array and Exiqon microRNA PCR Human Panel arrays, respectively. The results were further validated in vitro using endometrial stromal and epithelial cells challenged with subclinical and clinical doses of lipopolysaccharide (LPS). RESULT: Transcriptome profile analysis revealed altered expression level of 203 genes in CE compared to HE animals. Of these, 92 genes including PTHLH, INHBA, DAPL1 and SERPINA1 were significantly upregulated, whereas the expression level of 111 genes including MAOB, CXCR4, HSD11B and, BOLA, were significantly downregulated in CE compared to the HE animal group. However, in SE group, the expression patterns of only 28 genes were found to be significantly altered, of which 26 genes including PTHLH, INHBA, DAPL1, MAOB, CXCR4 and TGIF1 were common to the CE group. Gene annotation analysis indicated the immune system processes; G-protein coupled receptor signaling pathway and chemotaxis to be among the affected functions in endometritis animal groups. In addition, miRNA expression analysis indicated the dysregulation of 35 miRNAs including miR-608, miR-526b* and miR-1265 in CE animals and 102 miRNAs including let-7 family (let-7a, let-7c, let-7d, let-7d*, let-7e, let-7f, let-7i) in SE animals. Interestingly, 14 miRNAs including let-7e, miR-92b, miR-337-3p, let-7f and miR-145 were affected in both SE and CE animal groups. Further in vitro analysis of selected differentially expressed genes and miRNAs in endometrial stroma and epithelial cells challenged with SE and CE doses of LPS showed similar results to that of the array data generated using samples collected from SE and CE animals. CONCLUSION: The results of this study unraveled endometrial transcriptome and miRNome profile alterations in cows affected by subclinical or clinical endometritis which may have a significant effect on the uterine homeostasis and uterine receptivity.

Impact of macrophages on tumor growth characteristics in a murine ocular tumor model.

Tumor associated macrophages (TAM), mean vascular density (MVD), PAS positive extravascular matrix patterns, and advanced patients' age are associated with a poor prognosis in uveal melanoma. These correlations may be influenced by M2 macrophages and their cytokine expression pattern. Thus, the effect of TAM and their characteristic cytokines on histologic tumor growth characteristics were studied under the influence of age. Ninety five CX3CR1(+/GFP) mice (young 8-12weeks, old 10-12months) received an intravitreal injection of 1 × 10(5) HCmel12 melanoma cells. Subgroups were either systemically macrophage-depleted by Clodronate liposomes (n = 23) or received melanoma cells, which were pre-incubated with the supernatant of M1- or M2-polarized macrophages (n = 26). Eyes were processed histologically/immunohistochemically (n = 75), or for flow cytometry (n = 20) to analyze tumor size, mean vascular density (MVD), extravascular matrix patterns, extracellular matrix (ECM) and the presence/polarization of TAM. Prognostically significant extravascular matrix patterns (parallels with cross-linkings, loops, networks) were found more frequently in tumors of untreated old compared to tumors of untreated young mice (p = 0.024); as well as in tumors of untreated mice compared to tumors of macrophage-depleted mice (p = 0.014). Independent from age, M2-conditioned tumors showed more TAM (p = 0.001), increased collagen IV levels (p = 0.024) and a higher MVD (p = 0.02) than M1-conditioned tumors. Flow cytometry revealed a larger proportion of M2-macrophages in old than in young mice. The results indicate that TAM and their cytokines appear to be responsible for a more aggressive tumor phenotype. Tumor favoring and pro-angiogenic effects can be directly attributed to a M2-dominated tumor microenvironment rather than to age-dependent factors alone. However, an aged immunoprofile with an increased number of M2-macrophages may provide a tumor-favoring basis. Further, old mice represent a more suitable tumor model instead of young mice since their histologic tumor pattern better resembles human tumors.

The effects of iodoacetic acid on the mouse retina.

BACKGROUND: To characterize the effects of intravitreal injections of iodoacetic acid (IAA) in comparison to its systemic application as a measure to induce unilateral photoreceptor degeneration. METHODS: Seven-week-old C57BL/6 J mice received either intravitreal injections of IAA or systemic treatment (intraperitoneal vs intravenous) and were observed in the following 5 weeks using ERG, OCT, and histology. RESULTS: Systemic treatment with IAA induced high toxic effects and a high mortality in contrast to the intravitreal injection. Intraperitoneal application had no effect on the retina. Intravenous application of 2 × 30 mg/kg BW IAA (time between injections 3.5 h) resulted in an extinction of the ERG and a thinning of the retina, in particular of the outer nuclear layer (ONL) indicating photoreceptor degeneration. Animals receiving intravitreal injections developed cataracts already at low concentrations (up to 100% at 0.25 mg/kg BW). Higher intravitreal IAA doses led to extinguished ERGs. In histology, a thinning of the entire retina was observed that was most prominent in the inner part of the retina. CONCLUSIONS: In contrast to intraperitoneal administration, intravenous application of IAA led to a selective photoreceptor degeneration. After intravitreal injection, dense cataracts were already observed at concentrations lower than those needed to induce changes in the ERG. ERG results must be interpreted carefully. A thinning of all retinal layers rather than a specific outer retinal degeneration was observed upon intravitreal injection. IAA is not a useful model to induce outer retinal degeneration in mice.

Effect of epidermal growth factor (EGF) on the phosphorylation of mitogen-activated protein kinase (MAPK) in the bovine oviduct in vitro: Alteration by heat stress.

Epidermal growth factor (EGF) has been shown to be involved in control of the oviductal microenvironment. To elucidate the potential mechanisms responsible for the detrimental effect of heat stress and to identify the relation with the endocrine status, the effects of EGF on the level of phosphorylated mitogen-activated-protein kinase (MAPK) and proliferation of bovine oviductal epithelial cells (OECs) exposed to different cyclic ovarian steroidal environments (luteal phase (LP), follicular phase (FP) and postovulatory phase (PO)) and temperatures (mild heat stress (40 C) and severe heat stress (43 C)) were investigated. Western blot was performed to evaluate phosphorylated MAPK, while proliferation was analyzed by MTT assay. Stimulation of OECs with EGF alone or with EGF in the PO and FP environments significantly increased the amount of phosphorylated MAPK, with MAPK 44 phosphorylation being highest during exposure to PO conditions. These effects were not observed in the LP. Heat treatment completely blocked effects of EGF on phosphorylated MAPK. Additionally, severe heat stress led to a significantly lower basal level of phosphorylated MAPK. PD98059 (MAPK inhibitor) completely abolished EGF-stimulated MAPK phosphorylation and OECs proliferation. Overall the results indicate that EGF has the potential to increase the amount of phosphorylated MAPK in OECs and therefore could be involved in regulation of the bovine oviductal microenvironment. However, these regulatory mechanisms may be compromised in the presence of heat stress (high ambient temperature), leading to low fertility rates and impaired embryo survival.

The placenta in toxicology. Part III: Pathologic assessment of the placenta.

This short review is derived from the peer-reviewed literature and the experience and case materials of the authors. Brief illustrated summaries are presented on the gross and histologic normal anatomy of rodent and macaque placentas, including typical organ weights, with comments on differences from the human placenta. Common incidental findings, background lesions, and induced toxic lesions are addressed, and a recommended strategy for pathologic evaluation of placentas is provided.

The placenta in toxicology. Part IV: Battery of toxicological test systems based on human placenta.

This review summarizes the potential and also some limitations of using human placentas, or placental cells and structures for toxicology testing. The placenta contains a wide spectrum of cell types and tissues, such as trophoblast cells, immune cells, fibroblasts, stem cells, endothelial cells, vessels, glands, membranes, and many others. It may be expected that in many cases the relevance of results obtained from human placenta will be higher than those from animal models due to species specificity of metabolism and placental structure. For practical and economical reasons, we propose to apply a battery of sequential experiments for analysis of potential toxicants. This should start with using cell lines, followed by testing placenta tissue explants and isolated placenta cells, and finally by application of single and dual side ex vivo placenta perfusion. With each of these steps, the relative workload increases while the number of feasible repeats decreases. Simultaneously, the predictive power enhances by increasing similarity with in vivo human conditions. Toxic effects may be detected by performing proliferation, vitality and cell death assays, analysis of protein and hormone expression, immunohistochemistry or testing functionality of signaling pathways, gene expression, transport mechanisms, and so on. When toxic effects appear at any step, the subsequent assays may be cancelled. Such a system may be useful to reduce costs and increase specificity in testing questionable toxicants. Nonetheless, it requires further standardization and end point definitions for better comparability of results from different toxicants and to estimate the respective in vivo translatability and predictive value.

The placenta in toxicology. Part I: Animal models in toxicology: placental morphology and tolerance molecules in the cynomolgus monkey (Macaca fascicularis).

The immune system represents a key defense mechanism against potential pathogens and adverse non-self materials. During pregnancy, the placenta is the point of contact between the maternal organism and non-self proteins of the fetal allograft and hence undoubtedly fulfils immune functions. In the placenta bacteria, foreign (non-self) proteins and proteins that might be introduced in toxicological studies or by medication are barred from reaching the progeny, and the maternal immune system is primed for acceptance of non-maternal fetal protein. Both immunologic protection of the fetus and acceptance of the fetus by the mother require effective mechanisms to prevent an immunologic fetomaternal conflict and to keep both organisms in balance. This is why the placenta requires toxicological consideration in view of its immune organ function. The following articles deal with placenta immune-, control-, and tolerance mechanisms in view of both fetal and maternal aspects. Furthermore, models for experimental access to placental immune function are addressed and the pathological evaluation is elucidated. "The Placenta as an Immune Organ and Its Relevance in Toxicological Studies" was subject of a continuing education course at the 2012 Society of Toxicologic Pathology meeting held in Boston, MA.

The placenta in toxicology. Part II: Systemic and local immune adaptations in pregnancy.

During pregnancy, the maternal immune system is challenged by the semiallogeneic fetus, which must be tolerated without compromising fetal or maternal health. This review updates the systemic and local immune changes taking place during human pregnancy, including some examples in rodents. Systemic changes are induced by contact of maternal blood with placental factors and include enhanced innate immunity with increased activation of granulocytes and nonclassical monocytes. Although a bias toward T helper (Th2) and regulatory T cell (Treg) immunity has been associated with healthy pregnancy, the relationship between different circulating Th cell subsets is not straightforward. Instead, these adaptations appear most evidently at the fetal-maternal interface, where for instance Tregs are enriched and promote fetal tolerance. Also innate immune cells, that is, natural killer cells and macrophages, are enriched, constituting the majority of decidual leukocytes. These cells not only contribute to immune regulation but also aid in establishing the placenta by promoting trophoblast recruitment and angiogenesis. Thus, proper interaction between leukocytes and placental trophoblasts is necessary for normal placentation and immune adaptation. Consequently, spontaneous maladaptation or interference of the immune system with toxic substances may be important contributing factors for the development of pregnancy complications such as preeclampsia, preterm labor, and recurrent miscarriages.

An acute-phase protein as a regulator of sperm survival in the bovine oviduct: alpha 1-acid-glycoprotein impairs neutrophil phagocytosis of sperm in vitro.

We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E2 (PGE2) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE2 at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.

Correction: Gross anatomy, histology and blood vessel topography of the alimentary canal of the Inland Bearded Dragon (Pogona vitticeps).

[This corrects the article DOI: 10.1371/journal.pone.0234736.].

Blood Vessel Topography of the Feet in Selected Species of Birds of Prey and Owls.

Birds of prey and owls are susceptible to diseases of and traumatic injuries to their feet, which regularly require surgical intervention. A precise knowledge of the blood vessel topography is essential for a targeted therapy. Therefore, the metatarsal and digital vasculature was examined in eight species of birds of prey and owls. The study included contrast micro-computed tomography scans and anatomical dissections after intravascular injection of colored latex. In all examined species, the dorsal metatarsal arteries provided the main supply to the foot and their branching pattern and number differed between species. They continued distally as digital arteries. All examined species showed a basic pattern of four collaterally located digital blood vessels per toe: a prominent artery and small vein on one side and a small artery and prominent vein on the other side. Digital veins united to form common digital veins, most of which joined into a superficial, medially located metatarsal vein. This vein provided the main drainage of the foot. The detailed visualization of the topography of pedal blood vessels will help veterinary surgeons during surgical procedures. In addition, differences in the plantar arterial arch between hawks and falcons were discussed regarding their possible influence on the prevalence of pododermatitis (bumblefoot).