Search

Knowledge of host associations of blood-feeding vectors may afford insights into managing disease systems and protecting public health. However, the ability of methods to distinguish bloodmeal sources varies widely. We used two methods—Sanger sequencing and amplicon deep sequencing—to target a 228 bp region of the vertebrate Cytochrome b gene and determine hosts fed upon by triatomines (n = 115) collected primarily in Texas, USA. Direct Sanger sequencing of PCR amplicons was successful for 36 samples (31%). Sanger sequencing revealed 15 distinct host species, which included humans, domestic animals (Canis lupus familiaris, Ovis aries, Gallus gallus, Bos taurus, Felis catus, and Capra hircus), wildlife (Rattus rattus, Incilius nebulifer, Sciurus carolinensis, Sciurus niger, and Odocoileus virginianus), and captive animals (Panthera tigris, Colobus spp., and Chelonoidis carbonaria). Samples sequenced by the Sanger method were also subjected to Illumina MiSeq amplicon deep sequencing. The amplicon deep sequencing results (average of 302,080 usable reads per sample) replicated the host community revealed using Sanger sequencing, and detected additional hosts in five triatomines (13.9%), including two additional blood sources (Procyon lotor and Bassariscus astutus). Up to four bloodmeal sources were detected in a single triatomine (I. nebulifer, Homo sapiens, C. lupus familiaris, and S. carolinensis). Enhanced understanding of vector-host-parasite networks may allow for integrated vector management programs focusing on highly-utilized and highly-infected host species.

The geographic expansion of chronic wasting disease (CWD) in U.S. white-tailed deer (Odocoileus virginianus) has been largely unabated by best management practices, diagnostic surveillance, and depopulation of positive herds. Using a custom Affymetrix Axiom single nucleotide polymorphism (SNP) array, we demonstrate that both differential susceptibility to CWD, and natural variation in disease progression, are moderately to highly heritable (h2=0.337±0.079─0.637±0.070) among farmed U.S. white-tailed deer, and that loci other than PRNP are involved. Genome-wide association analyses using 123,987 quality filtered SNPs for a geographically diverse cohort of 807 farmed U.S. white-tailed deer (n = 284 CWD positive; n = 523 CWD non-detect) confirmed the prion gene (PRNP; G96S) as a large-effect risk locus (P-value < 6.3E-11), as evidenced by the estimated proportion of phenotypic variance explained (PVE ≥ 0.05), but also demonstrated that more phenotypic variance was collectively explained by loci other than PRNP. Genomic best linear unbiased prediction (GBLUP; n = 123,987 SNPs) with k-fold cross validation (k = 3; k = 5) and random sampling (n = 50 iterations) for the same cohort of 807 farmed U.S. white-tailed deer produced mean genomic prediction accuracies ≥ 0.81; thereby providing the necessary foundation for exploring a genomically-estimated CWD eradication program.

The gray short-tailed opossum (Monodelphis domestica) is an established laboratory-bred marsupial model for biomedical research. It is a critical species for comparative genomics research, providing the pivotal phylogenetic outgroup for studies of derived vs ancestral states of genomic/epigenomic characteristics for eutherian mammal lineages. To characterize the current genetic profile of this laboratory marsupial, we examined 79 individuals from eight established laboratory strains. Double digest restriction site-associated DNA sequencing and whole-genome resequencing experiments were performed to investigate the genetic architecture in these strains. A total of 66,640 high-quality single nucleotide polymorphisms (SNPs) were identified. We analyzed SNP density, average heterozygosity, nucleotide diversity, and population differentiation parameter Fst within and between the eight strains. Principal component and population structure analysis clearly resolve the strains at the level of their ancestral founder populations, and the genetic architecture of these strains correctly reflects their breeding history. We confirmed the successful establishment of the first inbred laboratory opossum strain LSD (inbreeding coefficient F > 0.99) and a nearly inbred strain FD2M1 (0.98

The Odon Device™ has been described in the literature since 2013 and the World Health Organization supports its development through the Human Reproduction Program. This innovative device could be easier to use than usual instruments and could be an alternative to caesarean section during the second stage of labor, especially in countries where access to obstetric care is limited. The aim of the Odon Device™ is to position an air cuff over the fetal head, past its widest point (around the level of the fetal mouth anteriorly and the nape of the fetal neck posteriorly). Three mechanical principles favor the progression of the fetal head with the Odon Device™: partial propulsion, limited flexion and traction. Preliminary clinical studies on animals and simulators are reassuring and show that an appropriate use is no more at risk than the vaccum or forceps. A phase 1 study was conducted in Argentina and South Africa between 2011 and 2017. The reported failure rate was 29%, of which 77% was secondary to a mechanical failure of one of the components of the device. Improvements concerning the applicator, the handles and the inflatable air cuff have been made to the device. Phase II of the clinical research program began in 2018 and includes two studies in two different centers: The ASSIST Study in Bristol, England, and The BESANCON ASSIST Study, Besançon, France.

The molecular clock plays key roles in daily physiological functions, development and cancer. Period 2 (PER2) is a repressive element, which inhibits transcription activated by positive clock elements, resulting in diurnal cycling of genes. However, there are gaps in our understanding of the role of the clock in normal development outside of its time-keeping function. Here, we show that PER2 has a noncircadian function that is crucial to mammalian mammary gland development. Virgin Per2-deficient mice, Per2(−/−), have underdeveloped glands, containing fewer bifurcations and terminal ducts than glands of wild-type mice. Using a transplantation model, we show that these changes are intrinsic to the gland and further identify changes in cell fate commitment. Per2(−/−) mouse mammary glands have a dual luminal/basal phenotypic character in cells of the ductal epithelium. We identified colocalization of E-cadherin and keratin 14 in luminal cells. Similar results were demonstrated using MCF10A and shPER2 MCF10A human cell lines. Collectively this study reveals a crucial noncircadian function of PER2 in mammalian mammary gland development, validates the Per2(−/−) model, and describes a potential role for PER2 in breast cancer.

Northern bobwhite (Colinus virginianus; hereafter bobwhite) and scaled quail (Callipepla squamata) populations have suffered precipitous declines across most of their US ranges. Illumina-based first- (v1.0) and second- (v2.0) generation draft genome assemblies for the scaled quail and the bobwhite produced N50 scaffold sizes of 1.035 and 2.042 Mb, thereby producing a 45-fold improvement in contiguity over the existing bobwhite assembly, and ≥90% of the assembled genomes were captured within 1313 and 8990 scaffolds, respectively. The scaled quail assembly (v1.0 = 1.045 Gb) was ∼20% smaller than the bobwhite (v2.0 = 1.254 Gb), which was supported by kmer-based estimates of genome size. Nevertheless, estimates of GC content (41.72%; 42.66%), genome-wide repetitive content (10.40%; 10.43%), and MAKER-predicted protein coding genes (17,131; 17,165) were similar for the scaled quail (v1.0) and bobwhite (v2.0) assemblies, respectively. BUSCO analyses utilizing 3023 single-copy orthologs revealed a high level of assembly completeness for the scaled quail (v1.0; 84.8%) and the bobwhite (v2.0; 82.5%), as verified by comparison with well-established avian genomes. We also detected 273 putative segmental duplications in the scaled quail genome (v1.0), and 711 in the bobwhite genome (v2.0), including some that were shared among both species. Autosomal variant prediction revealed ∼2.48 and 4.17 heterozygous variants per kilobase within the scaled quail (v1.0) and bobwhite (v2.0) genomes, respectively, and estimates of historic effective population size were uniformly higher for the bobwhite across all time points in a coalescent model. However, large-scale declines were predicted for both species beginning ∼15–20 KYA.

In Marfan Syndrome (MFS), development of thoracic aortic aneurysms (TAAs) is characterized by degeneration of the medial layer of the aorta, including fragmentation and loss of elastic fibers, phenotypic changes in the smooth muscle cells, and an increase in the active form of transforming growth factor-β (TGFβ), which is thought to play a major role in development and progression of the aneurysm. We hypothesized that regional difference in elastic fiber fragmentation contributes to TGFβ activation and hence the localization of aneurysm formation. The fibrillin-1-deficient mgR/mgR mouse model of MFS was used to investigate regional changes in elastin fiber fragmentation, TGFβ activation and changes in gene expression as compared to wild-type littermates. Knockdown of Smad 2 and Smad 3 with shRNA was used to determine the role of the specific transcription factors in gene regulation in aortic smooth muscle cells. We show increased elastin fiber fragmentation in the regions associated with aneurysm formation and altered TGFβ signaling in these regions. Differential effects of Smad 2 and Smad 3 were observed in cultured smooth muscle cells by shRNA-mediated knockdown of expression of these transcription factors. Differential signaling through Smad 2 and Smad 3 in regions of active vascular remodeling likely contribute to aneurysm formation in the mgR/mgR model of MFS. Increased elastin fiber fragmentation in these regions is associated with these changes as compared to other regions of the thoracic aorta and may contribute to the changes in TGFβ signaling in these regions.

The use of statins in the prevention and treatment of cardiovascular diseases is well established. Their use as anti-inflammatory and immunomodulatory agents in the treatment of rheumatoid arthritis (RA) has also been investigated, with several clinical and experimental studies indicating an anti-inflammatory effect of statins for RA, but other studies showing no effect or even the opposite. The current study was designed to examine the effect of simvastatin in an in vivo murine model of arthritis using intravital microscopy.We assigned four groups (n = 7, female C57Bl6 mice), two with and two without antigen-induced arthritis (AiA), from which one of the non-AiA groups and one of the AiA groups were treated with simvastatin 40 mg/kg i.p. daily for 14 consecutive days after induction of arthritis. Platelet- and leucocyte-endothelial cell interaction was assessed by measurement of rolling and adherent fluorescence-labelled platelets and leucocytes, functional capillary density (FCD) was evaluated, and knee joint diameter was determined as a clinical parameter.In arthritic mice treated with simvastatin, a significant reduction in platelet- and leucocyte-endothelial cell interaction was observed in comparison to arthritic mice treated with vehicle. In addition, a significant reduction in FCD was seen in arthritic mice treated with simvastatin, along with a reduction in knee joint swelling of the AiA mice.Treatment of AiA mice with simvastatin showed significant reductions in platelet- and leucocyte-endothelial cell interactions, in FCD, and in the swelling of the knee joint. These results support the hypothesis of the anti-inflammatory effects of statins in the treatment of RA.

IDO2 is implicated in tryptophan catabolism and immunity but its physiological functions are not well established. Here we report the characterization of mice genetically deficient in IDO2, which develop normally but exhibit defects in IDO-mediated T-cell regulation and inflammatory responses. Construction of this strain was prompted in part by our discovery that IDO2 function is attenuated in macrophages from Ido1−/− mice due to altered message splicing, generating a functional mosaic with implications for interpreting findings in Ido1–/– mice. No apparent defects were observed in Ido2–/– mice in embryonic development or hematopoietic differentiation, with wild-type profiles documented for kynurenine in blood serum and for immune cells in spleen, lymph nodes, peritoneum, thymus and bone marrow of naive mice. In contrast, upon immune stimulation we determined that IDO1-dependent T regulatory cell generation was defective in Ido2−/− mice, supporting Ido1–Ido2 genetic interaction and establishing a functional role for Ido2 in immune modulation. Pathophysiologically, both Ido1−/− and Ido2−/− mice displayed reduced skin contact hypersensitivity responses, but mechanistic distinctions were apparent, with only Ido2 deficiency associated with a suppression of immune regulatory cytokines that included GM-CSF, G-CSF, IFN-γ, TNF-α, IL-6 and MCP-1/CCL2. Different contributions to inflammation were likewise indicated by the finding that Ido2−/− mice did not phenocopy Ido1−/− mice in the reduced susceptibility of the latter to inflammatory skin cancer. Taken together, our results offer an initial glimpse into immune modulation by IDO2, revealing its genetic interaction with IDO1 and distinguishing its non-redundant contributions to inflammation.

The circadian clock plays a role in many biologic processes, yet very little is known about its role in metabolism of drugs and carcinogens. The purpose of this study was to define the impact of circadian rhythms on benzo-a-pyrene (BaP) metabolism in the mouse mammary gland and develop a circadian in vitro model for investigating changes in BaP metabolism resulting from cross-talk between the molecular clock and aryl hydrocarbon receptor. Female 129sv mice (12 weeks old) received a single gavage dose of 50 mg/kg BaP at either noon or midnight, and mammary tissues were isolated 4 or 24 hours later. BaP-induced Cyp1a1 and Cyp1b1 mRNA levels were higher 4 hours after dosing at noon than at 4 hours after dosing at midnight, and this corresponded with parallel changes in Per gene expression. In our in vitro model, we dosed MCF10A mammary cells at different times after serum shock to study how time of day shifts drug metabolism in cells. Analysis of CYP1A1 and CYP1B1 gene expression showed the maximum enzyme-induced metabolism response 12 and 20 hours after shock, as determined by ethoxyresorufin-O-deethylase activity, metabolism of BaP, and formation of DNA-BaP adducts. The pattern of PER-, BMAL-, and aryl hydrocarbon receptor–induced P450 gene expression and BaP metabolism was similar to BaP-induced Cyp1A1 and Cyp1B1 and molecular clock gene expression in mouse mammary glands. These studies indicate time-of-day exposure influences BaP metabolism in mouse mammary glands and describe an in vitro model that can be used to investigate the circadian influence on the metabolism of carcinogens.

Enhanced amino acid catabolism is a common response to inflammation, but the immunologic significance of altered amino acid consumption remains unclear. The finding that tryptophan catabolism helped maintain fetal tolerance during pregnancy provided novel insights into the significance of amino acid metabolism in controlling immunity. Recent advances in identifying molecular pathways that enhance amino acid catabolism and downstream mechanisms that affect immune cells in response to inflammatory cues support the notion that amino acid catabolism regulates innate and adaptive immune cells in pathologic settings. Cells expressing enzymes that degrade amino acids modulate antigen-presenting cell and lymphocyte functions and reveal critical roles for amino acid- and catabolite-sensing pathways in controlling gene expression, functions, and survival of immune cells. Basal amino acid catabolism may contribute to immune homeostasis that prevents autoimmunity, whereas elevated amino acid catalytic activity may reinforce immune suppression to promote tumorigenesis and persistence of some pathogens that cause chronic infections. For these reasons, there is considerable interest in generating novel drugs that inhibit or induce amino acid consumption and target downstream molecular pathways that control immunity. In this review, we summarize recent developments and highlight novel concepts and key outstanding questions in this active research field.

Chronic inflammation underlies the basis for development and progression of cancers and a variety of other disorders, but what specifically defines its pathogenic nature remains largely undefined. Recent genetic and pharmacological studies in the mouse suggest that the immune modulatory enzyme indoleamine 2,3-dioxygenase (IDO), identified as an important mediator of immune escape in cancer, can also contribute to the development of pathology in the context of chronic inflammatory models of arthritis and allergic airway disease. IDO-deficient mice do not display spontaneous disorders of classical inflammation and small molecule inhibitors of IDO do not elicit generalized inflammatory reactions. Rather, in the context of a classical model of skin cancer that is promoted by chronic inflammation, or in models of inflammation-associated arthritis and allergic airway disease, IDO impairment can alleviate disease severity. Here we offer a survey of preclinical literature suggesting that IDO functions as a modifier of inflammatory states rather than simply as a suppressor of immune function. We propose that IDO induction in a chronically inflamed tissue may shape the inflammatory state to support, or in some cases retard, pathogenesis and disease severity.

Postlactational involution of the mammary gland provides a unique model to study breast cancer susceptibility and metastasis. We have shown that the short isoform of Singleminded-2s (Sim2s), a basic helix loop helix/PAS transcription factor, plays a role in promoting lactogenic differentiation, as well as maintaining mammary epithelial differentiation and malignancy. Sim2s is dynamically expressed during mammary gland development, with expression peaking during lactation, and decreasing in early involution. To determine the role of SIM2S in involution, we used transgenic mice expressing SIM2S under the mouse mammary tumor virus-Sim2s promoter. Overexpression of Sim2s in the mouse mammary gland resulted in delayed involution, indicated by a lower proportion of cleaved caspase-3-positive cells and slower reestablishment of the mammary fat pad. Immunohistochemical and quantitative RNA analysis showed a decrease in apoptotic markers and inflammatory response genes, and an increase in antiapoptotic genes, which were accompanied by inhibition of signal transducer and activator of transcription 3 activity. Microarray analysis confirmed that genes in the signal transducer and activator of transcription 3 signaling pathway were repressed by SIM2S expression, along with nuclear factor-κB and other key pathways involved in mammary gland development. Multiparous mouse mammary tumor virus-Sim2s females displayed a more differentiated phenotype compared with wild-type controls, characterized by enhanced β-casein expression and alveolar structures. Together, these results suggest a role for SIM2S in the normal involuting gland and identify potential downstream pathways regulated by SIM2S.

The chronic inflammatory bowel diseases are characterized by aberrant innate and adaptive immune responses to commensal luminal bacteria. In both human inflammatory bowel disease and in experimental models of colitis, there is an increased expression of the enzyme IDO. IDO expression has the capacity to exert antimicrobial effects and dampen adaptive immune responses. In the murine trinitrobenzene sulfonic acid model of colitis, inhibition of this enzyme leads to worsened disease severity, suggesting that IDO acts as a natural break in limiting colitis. In this investigation, we show that induction of IDO-1 by a TLR-9 agonist, immunostimulatory (ISS) DNA, critically contributes to its colitis limiting capacities. ISS DNA induces intestinal expression of IDO-1 but not the recently described paralog enzyme IDO-2. This induction occurred in both epithelial cells and in subsets of CD11c+ and CD11b+ cells of the lamina propria, which also increase after ISS-oligodeoxynucleotide. Signaling required for intestinal IDO-1 induction involves IFN-dependent pathways, as IDO-1 was not induced in STAT-1 knockout mice. Using both the trinitrobenzene sulfonic acid and dextran sodium sulfate models of colitis, we show the importance of IDO-1s induction in limiting colitis severity. The clinical parameters and histological correlates of colitis in these models were improved by administration of the TLR-9 agonist; however, when the function of IDO is inhibited, the colitis limiting effects of ISS-oligodeoxynucleotide were abrogated. These findings support the possibility that targeted induction of IDO-1 is an approach deserving further investigation as a therapeutic strategy for diseases of intestinal inflammation.

We have previously demonstrated that the bHLH/PAS transcription factor, singleminded 2s (Sim2s), is required for proper mammary ductal morphogenesis and luminal epithelial differentiation. Furthermore, loss of Sim2s in breast cancer cells resulted in downregulation of epithelial markers and acquisition of a basal-like phenotype. The objective of this study was to further define the role of Sim2s in mammary differentiation. We found that Sim2s is developmentally regulated throughout mammary gland development with highest expression during lactation. Mammary glands from nulliparous mice expressing Sim2s driven by the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) promoter were morphologically indistinguishable from wild-type mice but displayed hallmarks of precocious lactogenic differentiation. These included elevated expression of the milk protein genes Wap and Csn2, and apical localization of the lactation marker Npt2b. Consistent with the in vivo results, Sim2s enhanced prolactin-mediated Csn2 expression in HC11 and CIT3 mouse mammary epithelial cells, and downregulation of Sim2s by shRNA in HC11 cells inhibited Csn2 expression. Chromatin immunoprecipitation (ChIP) analyses of the Csn2 gene found that Sim2s associates with the Csn2 promoter and re-ChIP experiments showed that Sim2s interacted with the RNA II polymerase (RNAPII) complex. Together, these data demonstrate, for the first time, that Sim2s is required for establishing and maintaining mammary gland differentiation.

The aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) are transcription factors that express Per-Arnt-Sim (PAS) DNA-binding motifs and mediate the metabolism of drugs and environmental toxins in the liver. Because these transcription factors interact with other PAS genes in molecular feedback loops forming the mammalian circadian clockworks, we determined whether targeted disruption or siRNA inhibition of Per1 and Per2 expression alters toxin-mediated regulation of the AhR signaling pathway in the mouse liver and Hepa1c1c7 hepatoma cells in vitro. Treatment with the prototypical Ahr ligand, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), had inductive effects on the primary targets of AhR signaling, Cyp1A1 and Cyp1B1, in the liver of all animals, but genotype-based differences were evident such that the toxin-mediated induction of Cyp1A1 expression was significantly greater (2-fold) in mice with targeted disruption of Per1 (Per1ldc and Per1ldc/Per2ldc). In vitro experiments yielded similar results demonstrating that siRNA inhibition of Per1 significantly increases the TCDD-induced expression of Cyp1A1 and Cyp1B1 in Hepa1c1c7 cells. Per2 inhibition in siRNA-infected Hepa1c1c7 cells had the opposite effect and significantly decreased both the induction of these p450 genes as well as AhR and Arnt expression in response to TCDD treatment. These findings suggest that Per1 may play a distinctive role in modulating AhR-regulated responses to TCDD in the liver.

The short splice variant of the basic helix-loop-helix Per-Arnt-Sim transcription factor Singleminded-2, SIM2s, has been implicated in development and is frequently lost or reduced in primary breast tumors. Here, we show that loss of Sim2s causes aberrant mouse mammary gland ductal development with features suggestive of malignant transformation, including increased proliferation, loss of polarity, down-regulation of E-cadherin, and invasion of the surrounding stroma. Additionally, knockdown of SIM2s in MCF-7 breast cancer cells contributed to an epithelial-mesenchymal transition (EMT) and increased tumorigenesis. In both Sim2(-/-) mammary glands and SIM2s-depleted MCF7 cells, these changes were associated with increased SLUG and MMP2 levels. SIM2s protein was detectable on the SLUG promoter, and overexpression of SIM2s repressed expression from a SLUG-controlled reporter in a dose-dependent manner. To our knowledge, SIM2s is the first protein shown to bind and repress the SLUG promoter, providing a plausible explanation for the development role and breast tumor-suppressive activity of SIM2s. Together, our results suggest that SIM2s is a key regulator of mammary-ductal development and that loss of SIM2s expression is associated with an invasive, EMT-like phenotype.

Since an increase of platelet-endothelial cell interactions has been observed in mice with Antigen- induced-Arthritis (AiA) as well as an increase of NO expression, the aim of our study was to investigate in vivo the influence of NO, especially the platelet and endothelial inducible NO Synthase, on the platelet- and leukocyte endothelial cell interaction.C57/Bl6 mice and iNOS deficient mice were disposed in 6 groups (each=7). After induction of AiA, rolling and adherent fluorescence labelled platelets and leukocytes were investigated by intravital microscopy (IVM) on day 8 after AiA. Rank SUM Test and ANOVA on ranks have been performed regarding the data.All arthritic mice presented an increase in platelet and leukocyte interaction with the endothelium compared to control groups. The arthritic iNOS deficient mice showed a more intense interaction of platelets and leukocytes with the endothelium in comparison with the wild-type arthritic mice. The group using arthritic wild-type recipient and iNOS deficient donor mice showed an increase in cell-interactions, leading to an endothelial effect, compared to the group using iNOS deficient arthritic recipient and wild-type donor mice.The IVM data lead to an anti-inflammatory effect of NO, since NO followed an increase in platelet- and leukocyte- endothelial cell interaction in iNOS deficient mice with AiA. In addition, we have shown for the first time in vivo that platelet NO produced by iNOS seems to have a minor influence on the leukocyte induced tissue damage in contrast to endothelial iNOS. Therefore, selective platelet inhibition would not interfere with the protective effect of NO.

Platelets are thought to participate in the pathogenesis of chronic inflammatory diseases such as rheumatoid arthritis (RA). We showed recently an in vivo increase in platelet-endothelial cell interactions in mice with antigen-induced arthritis (AiA). The underlying mechanisms are not yet clear. The aim of this study was to investigate the impact of P-selectin in AiA by means of intravital fluorescence microscopy (IVM).C57/Bl6 mice and P-selectin-deficient mice were divided into four groups (n = 7; control/AiA per strain). The extent of AiA was assessed by measuring knee joint swelling and by histological scoring. Rolling and adherent fluorescence-labelled platelets and leucocytes were investigated by IVM.In arthritic P-selectin-deficient mice (rolling: 0.05+/-0.01; adherent: 130+/-20 mm(-2)), compared to arthritic C57/Bl6 mice (rolling: 0.20+/-0.04; adherent: 1910+/-200 mm(-2)), platelet interaction was significantly reduced (p0.05) and reached the level of both control groups without AiA. In addition, interaction of leucocytes in P-selectin-deficient arthritic animals (rolling: 0.12+/-0.06; adherent: 387+/-37 mm(-2)) was significantly decreased in comparison to arthritic C57/Bl6 animals (rolling: 0.21+/-0.06; adherent: 1492+/-284 mm(-2); p0.05). Swelling of the knee joint and histological scoring were reduced in arthritic P-selectin-deficient mice compared to arthritic C57/Bl6 mice.We have demonstrated for the first time in vivo a significant decrease in the interaction of platelets and leucocytes with the endothelium in P-selectin-deficient mice with AiA and a reduction in clinical and histological symptoms of arthritis. These findings suggest that leucocyte-endothelial cell interactions depend at least partially on platelet P-selectin and therefore platelets may be responsible for the leucocyte tissue damage in AiA.

There is growing evidence that platelets play an important role in the development and maintenance of rheumatoid arthritis. Activation and adherence of platelets in the synovial microcirculation might be in part responsible for endothelial damage and activation of leukocytes. Recent findings show a direct influence of P-selectin on platelet- and leukocyte-endothelial cell interaction in mice with Antigen-induced Arthritis (AiA). P-selectin is only expressed by platelets and endothelial cells, not by leukocytes. Therefore, the aim of the present study was to investigate the differential influence of platelet and endothelial P-selectin on the extent of inflammation in AiA. AiA was induced in wild-type mice and in P-selectin-deficient mice from the same genetic background (four groups: each n = 7). Intravital fluorescence microscopy (IVM) was used to visualize platelets and leukocytes in the synovial microcirculation at day 8 after AiA. Platelets from either strain were fluorescence-labelled ex vivo and transferred into either strain. We were able to demonstrate a significant decrease of platelet- and leukocyte-endothelial cell interaction in P-selectin-deficient mice with AiA in comparison to wild-type mice with AiA. When wild-type platelets were donated into P-selectin-deficient AiA recipients, the leukocyte-endothelial cell interaction was significantly increased compared to the group consisting of P-selectin-deficient recipient and donor mice. These are the first in vivo results showing that the P-selectin stored in platelets is at least partly responsible for the leukocyte-endothelial cell interaction and the resulting tissue damage in AiA. In the future, a suppression of platelet P-selectin could potentially become a treatment option for reducing the effects of rheumatoid arthritis.

Cationic lipid complexes bind to angiogenic endothelial cells of solid tumours and microvessels of chronic inflammatory tissue. Methotrexate (MTX) is one of the drugs used in the therapy of rheumatoid arthritis (RA); it is applied systemically but can have serious side-effects. The aim of this study was to investigate the impact of MTX encapsulated in cationic liposomes (EndoMTX) in comparison to treatment with free MTX.We used an antigen-induced arthritis (AiA) model and investigated the leucocyte- and platelet-endothelial cell interaction in arthritic female C57/Bl6 mice and in healthy controls. The arthritic animals were divided into four different groups receiving either trehalose, free MTX, EndoMTX placebo, or EndoMTX. These parameters and functional capillary density (FCD) were measured and assessed by intravital microscopy (IVM). We controlled clinical parameters such as the knee joint diameter (KJD) throughout the observation period.Animals treated with EndoMTX showed a significant and superior reduction in leucocyte- and platelet-endothelial cell interaction, FCD, and KJD. Free MTX or empty liposomes also showed a reduction in these parameters but not to a significant level. FCD decreased in the EndoMTX group in comparison to using free drugs or empty carrier-like liposomes.This study demonstrates the advantage of using MTX encapsulated in cationic liposomes in contrast to free and generic MTX, with a higher efficacy in anti-inflammatory and anti-angiogenic abilities. Targeting with cationic liposomes may be a promising treatment option and should be elucidated in further experiments regarding dose reduction and side-effects due to MTX usage.

Objectives. Growing evidence supports the substantial pathophysiological impact of platelets on the development of rheumatoid arthritis. At present there are no methods for studying these cellular mechanisms in vivo. The aim of this study was to visualize and investigate platelet-endothelial cell interaction in the knee joint of mice with antigen-induced arthritis (AiA) by means of intravital microscopy. Methods. In 14 mice (Balbc) intravital microscopic assessment was performed on day 8 after AiA induction in two groups (controls, AiA). The severity of AiA was assessed by measuring knee joint swelling and by histological scoring. Ex vivo fluorescently labelled rolling and adherent platelets and leucocyte-endothelium interactions were investigated by intravital fluorescence microscopy. Results. Swelling of the knee joint as well as histological score was significantly enhanced in arthritic animals compared with controls. In control mice intravital microscopy revealed low baseline rolling and sticking of leucocytes and fluorescently labelled platelets. AiA induced a significant increase in the fraction of rolling leucocytes (3 times) and rolling platelets (6 times) compared to the control group. Furthermore, AiA induction resulted in a significantly enhanced number of adherent leucocytes (3-fold) and adherent platelets (12-fold) in comparison with control animals. Conclusions. Platelet kinetics were directly analysed using intravital microscopy in the arthritic microcirculation in vivo for the first time. We provide the first evidence that platelets accumulate in arthritic vessels, indicating platelet activation due to AiA. Platelet recruitment and subsequent activation might play an important role in the pathogenesis of rheumatoid arthritis.

The L51S mutation in the D10.G4.1 TCR α-chain reduces the affinity of the TCR to its ligand by affecting the interactions among the TCR, the β-chain of I-Ak, and the bound peptide. We show that this mutation drives the generation of a pool of memory CD44highCD62LnegCD45RBneg CD4 TCR transgenic T cells. Their activation threshold is low, such that they proliferate in response to lower concentrations of agonist peptides than naive L51S CD4 T cells. Unlike effector memory CD4 T cells, however, they lack immediate effector function in response to TCR stimulation. These cells express IL-2Rα only after culture with specific peptide. Although they can be recovered from lymph nodes, the majority lack the expression of the lymph node homing receptor CCR7. When these cells receive a second TCR stimulation in vitro, they differentiate into potent Th2-like effector cells, producing high levels of IL-4 at doses of agonist peptide too low to stimulate cytokine release from similarly differentiated naive L51S CD4 T cells. Having these properties, the L51S TCR transgenic memory CD4 T cells cannot be classified as either strict central memory or effector memory, but, rather, as a pool of memory T cells containing effector memory precursors.

We tested the hypothesis that adolescent dietary vitamin A intake impacts mammary gland development and subsequent sensitivity to carcinogenesis. Sprague-Dawley rats were fed a purified diet that was vitamin A deficient, adequate (2.2 mg retinyl palmitate/kg diet), or supranutritional (16 mg retinyl palmitate/kg diet) from 21 to 63 days of age, the period of adolescent mammary gland development. At 73 days of age, rats were given 1-methyl-1-nitrosourea (25 mg/kg body wt i.p.) and monitored for mammary tumors. Tumors appeared earlier and more frequently in rats fed vitamin A-deficient or -supplemented diets. Vitamin A deficiency during adolescence was associated with alveolar mammary gland development and precocious milk protein expression, while supplementation was associated with ductal gland development and suppression of milk protein expression. Differences in circulating estradiol and mammary gland estrogen receptor-alpha, and estrogen-responsive progesterone receptor mRNA were not observed, suggesting that the effects of vitamin A on mammary gland development and carcinogenesis are estrogen independent. Mammary expression of another hormone receptor that regulates milk protein expression, the glucocorticoid receptor, was also unaffected. These results demonstrate that vitamin A intake during adolescence alters mammary gland differentiation and indicate that a narrow range of vitamin A intake during adolescence protects against carcinogenesis.

Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness. © 2014 Macmillan Publishers Limited. All rights reserved.

UDP-glucuronosyltransferase 1A7 (UGT1A7) is a major UGT contributing to the glucuronidation of xenobiotic phenols in rats. Its expression in rat liver is tightly regulated, with low constitutive and high inducible expression in response to aryl hydrocarbon receptor ligands and oltipraz. Previously, we reported the absence of 3-methylcholanthrene- or oltipraz-responsive elements in the 1.6-kbp region flanking the UGT1A7 promoter. However, potential binding sites were noted for several liver-enriched transcription factors. Here we show that deletion of the hepatic nuclear factor (HNF)3, HNF4, and CCAAT-enhancer binding protein-like binding sites had no effect on the expression of a UGT1A7 reporter plasmid, p(-965/+56)1A7-Luc, in primary rat hepatocytes. The full activity of the promoter was contained in the region between bases -157 and +76. Two sites of binding by rat liver nuclear proteins were detected in this region by DNase footprinting. PR-1 corresponded to the HNF1-like binding site between bases -52 and -38, whereas PR-2 was located between -30 to -6. Gel retardation studies supported the presence of HNF1alpha in the PR-1 DNA-liver nuclear protein complex. Mutation of PR-1 inhibited binding in the gel shift assay, prevented activation by overexpressed HNF1 in human embryonic kidney cells, and reduced by80% the maximal luciferase activities expressed from basal and 3-methylcholanthrene-responsive UGT1A7 gene reporter constructs in primary rat hepatocytes. These data provide evidence for an important stimulatory role of HNF1 in promoting UGT1A7 gene expression in rat liver.