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The decidual immunome is dynamic, dramatically changing its composition across gestation. Early pregnancy is dominated by decidual NK cells, with a shift towards T cells later in pregnancy. However, the degree, timing, and subsetspecific nature of leukocyte traffic between the decidua and systemic circulation during gestation remains poorly understood. Herein, we employed intravascular staining in pregnant C57BL/6J mice and cynomolgus macaques (Macaca fascicularis) to examine leukocyte traffic into the decidual basalis during pregnancy. Timed-mated or virgin mice were tail-vein injected with labelled αCD45 antibodies 24 hours and 5 minutes before sacrifice. Pregnant cynomolgus macaques (GD155) were infused with labelled αCD45 at 2 hours or 5 mins before necropsy. Decidual cells were isolated and resulting suspensions analyzed by flow cytometry. We found that the proportion of intravascular (IVAs)-negative leukocytes (cells labeled by the 24h infusion of αCD45 or unlabeled) decreased across murine gestation while recent immigrants (24h label only) increased in mid- to late-gestation. In the cynomolgus model our data confirmed differential labeling of decidual leukocytes by the infused antibody, with the 5 min infused animal having a higher proportion of IVAs+ cells compared to the 2hr infused animal. Decidual tissue sections from both macaques showed the presence of intravascularly labeled cells, either in proximity to blood vessels (5min infused animal) or deeper into decidual stroma (2hr infused animal). These results demonstrate the value of serial intravascular staining as a sensitive tool for defining decidual leukocyte traffic during pregnancy. [ABSTRACT FROM AUTHOR]

We previously reported that human keratinocytes express protease-activated receptor (PAR)-2 and play an important role in activated protein C (APC)-induced cutaneous wound healing. This study investigated the involvement of PAR-2 in the production of gelatinolytic matrix metalloproteinases (MMP)-2 and -9 by APC during cutaneous wound healing. Full-thickness excisional wounds were made on the dorsum of male C57BL/6 mice. Wounds were treated with APC on days 1, 2, and 3 post-wounding. Cultured neonatal foreskin keratinocytes were treated with APC with or without intact PAR-2 signalling to examine the effects on MMP-2 and MMP-9 production. Murine dermal fibroblasts from PAR-2 knock-out (KO) mice were also assessed. MMP-2 and -9 were measured via gelatin zymography, fluorometric assay, and immunohistochemistry. APC accelerated wound healing in WT mice, but had a negligible effect in PAR-2 KO mice. APC-stimulated murine cutaneous wound healing was associated with the differential and temporal production of MMP-2 and MMP-9, with the latter peaking on day 1 and the former on day 6. Inhibition of PAR-2 in human keratinocytes reduced APC-induced MMP-2 activity by 25~50%, but had little effect on MMP-9. Similarly, APC-induced MMP-2 activation was reduced by 40% in cultured dermal fibroblasts derived from PAR-2 KO mice. This study shows for the first time that PAR-2 is essential for APC-induced MMP-2 production. Considering the important role of MMP-2 in wound healing, this work helps explain the underlying mechanisms of action of APC to promote wound healing through PAR-2. [ABSTRACT FROM AUTHOR]

Megakaryocytes (MKs) are specialized precursor cells committed to producing and proliferating platelets. In a cytoskeletal-driven process, mature MKs generate platelets by releasing thin cytoplasmic extensions, named proplatelets, into the sinusoids. Due to knowledge gaps in this process and mounting clinical demand for non-donor-based platelet sources, investigators are successfully developing artificial culture systems to recreate the environment of platelet biogenesis. Nevertheless, drawbacks in current methods entail elaborate procedures for stem cell enrichment, extensive growth periods, low MK yield, and poor proplatelet production. We propose a simple, robust method of primary MK culture that utilizes fetal livers from pregnant mice. Our technique reduces expansion time to 4 days, and generates ~15,000-20,000 MKs per liver. Approximately, 20-50% of these MKs produce structurally dense, high-quality proplatelets. In this review, we outline our method of MK culture and isolation. [ABSTRACT FROM AUTHOR]

Abstract: Background: Gastric emptying is a complex physiological process regulating the division of a meal into smaller partitions for the small intestine. Disrupted gastric emptying contributes to digestive disease, yet current measures may not reflect different mechanisms by which the process can be altered. Methods: We have developed high temporal resolution solid and liquid gastric emptying breath tests in mice using [13C]‐octanoic acid and off axis‐ integrated cavity output spectroscopy (OA‐ICOS). Stretched gamma variate and 2‐component stretched gamma variate models fit measured breath excretion data. Key Results: These assays detect acceleration and delay using pharmacological (7.5 mg/kg atropine) or physiological (nutrients, cold exposure stress, diabetes) manipulations and remain stable over time. High temporal resolution resolved complex excretion curves with 2 components, which was more prevalent in mice with delayed gastric emptying following streptozotocin‐induced diabetes. There were differences in the gastric emptying of Balb/c vs C57Bl6 mice, with slower gastric emptying and a greater occurrence of two‐phase gastric emptying curves in the latter strain. Gastric emptying of C57Bl6 could be accelerated by halving the meal size, but with no effect on the occurrence of two‐phase gastric emptying curves. A greater proportion of two‐phase gastric emptying was induced in Balb/c mice with the administration of PYY (8‐80 nmol) 60 min following meal ingestion. Conclusions and Inferences: Collectively, these results demonstrate the utility of high temporal resolution gastric emptying assays. Two‐phase gastric emptying is more prevalent than previously reported, likely involves intestinal feedback, but contributes little to the overall rate of gastric emptying. [ABSTRACT FROM AUTHOR]

In this study, we report the genetic diversity and nucleotide mutation rates of five representative regions of the murine norovirus genome during in vitro passages. The mutation rates were similar in genomic regions encompassing partial coding sequences for non-structural (NS) 1-2, NS5, NS6, NS7 proteins within open reading frame (ORF) 1. In a region encoding a portion of the major capsid protein (VP1) within ORF2 (also including the ORF4 region) and a portion of the minor structural protein (VP2), the mutation rates were estimated to be at least one order of magnitude higher. The VP2 coding region was found to have the highest mutation rate. [ABSTRACT FROM AUTHOR]

The metabotropic glutamate (mGlu) receptor, mGlu5, is of particular relevance for hippocampal function. It is critically required for the expression of long-term potentiation (LTP) and long-term depression (LTD), regulates neuronal oscillations, maintains the stability of place fields and is required for hippocampus-dependent memory. MGlu5-dysfunctions are associated with profound cognitive deficits in humans, and mGlu5 has been targeted as a putative cognitive enhancer. Cognitive enhancement, by means of environmental enrichment (EE) in rodents, results in improved hippocampal synaptic plasticity and memory. Here, we explored whether mGlu5 contributes to these enhancements. MGlu5-antagonism dose-dependently impaired the early phase of LTP (>4 h) in the CA1 region of young(3–4 month old) mice. Late-LTP (>24 h) was also impaired. LTP (>24 h) elicited in old (10–14 month old) mice displayed reduced sensitivity to mGlu5 antagonism. Short-term potentiation (STP, < 2 h) that was elicited by weaker afferent stimulation was unaffected by mGlu5-antagonism in both age-groups. EE significantly amplified STP (<2 h) in old and young animals, but did not increase the duration of synaptic potentiation, or promote induction of LTP. The improvement in STP was prevented by mGlu5-antagonism, in both young and old animals. These results indicate that modifications of the synapse that underlie improvements of LTP by EE require the contribution of mGlu5. Strikingly, although LTP in old mice does not critically depend on mGlu5, improvements in synaptic potentiation resulting from EE are mGlu5-dependent in old mice. Regarded in light of the known role for mGlu5 in hippocampal function and pathophysiology, these data suggest that mGlu5 regulation of synaptic information storage is pivotal to optimal hippocampal function. This article is part of the Special Issue entitled ‘Metabotropic Glutamate Receptors, 5 years on’. [ABSTRACT FROM AUTHOR]

The infectivity of a recombinant murine norovirus (RecMNV) strain, previously isolated following in vitro coinfections, was evaluated in vivo in comparison with its parental strains (MNV-1-CW1 and WU20) in Balb/cByJ mice via measurement of weight loss and estimation of viral loads in faeces, tissues and organs 48 and 72 h post-infection. The presence of infectious virus in all analysed tissues and organs suggests that, similarly to its parental viruses, RecMNV can disseminate beyond organs associated with the digestive tract. Our results also suggest that recombination occurring in vitro between two homologous murine norovirus strains can give rise to a chimeric strain which, despite slight differences, shows similar biological properties to its parental strains. This study provides the first report on in vivo replication of a recombinant norovirus strain isolated following in vitro coinfection. These results have great significance for norovirus genetic evolution and future vaccine development. [ABSTRACT FROM AUTHOR]

• Mice are a valuable genetic and structural homologue for studying NIHL in humans, particularly at the level of traits governed by Mendelian genetics • GWAS and mapping studies in mice have characterized a broad range of candidate genes for NIHL and phenotype correlation • Mouse-to-human homology declines in higher-order regulatory mechanisms such as eQTL and network analysis • Transcriptomics and single cell analysis have the potential to identify a broad range of new therapeutic targets for modulation of NIHL Hearing loss is the most common sensory deficit worldwide, with the majority of preventable injury attributed to noise-induced hearing loss (NIHL). Highly conserved cochlear genetics between humans and mice have made this animal model a high-yield candidate for better characterizing the biologic and genetic underpinnings of human NIHL. This review aims to summarize advances in understanding the genetics of noise-induced hearing loss in mouse models dating from the early 1990s. We review the genetic mechanisms underpinning NIHL as understood in the mouse model, including histopathological and phenotypic associations, molecular and cellular mechanisms of changes in cochlear structures, synaptopathy and neuropathy, and transcriptomics. We describe variations in pathophysiology of hearing loss between mouse strains, with particular emphasis on susceptibility of different strains to different mechanisms of damage after acoustic trauma, and the potential of novel targeted therapeutic approaches for NIHL based on understanding of genetic mechanisms. Finally, we review the current state of research on the cochlear transcriptome after noise exposure in the mouse and implications for translation of these findings to humans. [ABSTRACT FROM AUTHOR]

Background Toll-like receptor 2 ( TLR2) is a widely expressed pattern recognition receptor critical for innate immunity. TLR2 is also a key regulator of mucosal immunity implicated in the development of allergic disease. TLR2 activators are found in many common foods, but the role of TLR2 in oral tolerance and allergic sensitization to foods is not well understood. Objective The purpose of this study was to evaluate the impacts of TLR2 expression and TLR2 activation on oral tolerance to food antigens in a murine model. Methods Mice were fed ovalbumin ( OVA) or peanut butter with or without the addition of low doses of TLR2 activators Pam3 CSK4 or FSL-1. Oral tolerance was assessed by analysing antibody responses after a systemic antigen challenge. OVA-specific Tregs were assessed in the Peyer's patches, mesenteric lymph nodes, and spleen in wild-type and TLR2−/− mice. Low-dose Pam3 CSK4 was also tested as an oral adjuvant. Results Oral tolerance was successfully induced in both wild-type and TLR2−/− recipient mice, with an associated regulatory T-cell response. Oral TLR2 activation, with low-dose Pam3 CSK4 or FSL-1, during oral antigen exposure was found to alter oral tolerance and was associated with the development of substantial IgE and IgA responses to foods upon systemic challenge. Low-dose oral Pam3 CSK4 treatment also selectively enhanced antigen-specific IgA responses to oral antigen exposure. Conclusions and Clinical Relevance TLR2 is not necessary for oral tolerance induction, but oral TLR2 activation modulates humoral IgE and IgA responses during tolerance development. Low-dose Pam3 CSK4 is also an effective oral adjuvant that selectively enhances IgA production. These observations are pertinent to the optimization of oral allergen immunotherapy and oral vaccine development. [ABSTRACT FROM AUTHOR]

Background: Inflammation forms an important part of the human innate immune system and is largely dependent on the activation of the "classical" NF-κB pathway through Toll-like receptors (TLRs). Understanding this has allowed researchers to explore roles of therapeutic targets in managing conditions such as sepsis. Recapitulating an inflammatory response using lipopolysaccharide (LPS), a "sterile" technique, can provide information that is dissimilar to the clinical condition. By examining NF-κB activation (through immunoblotting of the p65 subunit) in two separate cell lines (murine and human) and analyzing two murine models of sepsis (intraperitoneal [IP] LPS and IP stool inoculation), an evaluation of the translational disconnect between experimental and clinical sepsis can be made. Methods: THP-1 (human) cells and RAW 264.7 (murine) cells were dosed with concentrations of LPS (human, 1 pg/mL to 100 ng/mL; murine, 30 pg/mL to 1,000 ng/mL) and nuclear actin and p65 were immunoblotted to measure changes in nuclear density. In vivo, C57BL/6 mice received either IP injection of stool suspension (5 μL/g ) or LPS (25 mg/kg) or saline (1 mL/kg). Animals were culled at 6 hours and tissues were analyzed. Results: An increase in basal p65:actin density in THP-1 cells (mean 0.214, standard error of the mean 0.024) was seen at doses as small as 0.1 ng/mL (0.519±0.064). In contrast to RAW 264.7 cells, basal increases (0.170±0.025) were only seen when a dose of 3 ng/mL (0.387±0.078) was used. Dose-response analysis of p65:actin ratio showed that THP-1 cells respond to lower doses of LPS than RAW 264.7 cells and lower doses produce a greater fold increase in the nuclear p65 density. Both in vivo models showed evidence of neutrophil (NL) recruitment into tissues (which was more intense after LPS treatment). IP stool inoculation resulted in an acute suppurative peritonitis and more substantial evidence of NL recruitment into adipose tissue and skeletal muscle. Conclusion: Our results support previous observations that translation of murine models into the human clinical setting suffers from considerable limitations including species-associated differences in LPS response seen at a molecular level. Furthermore, the histopathological changes during clinical sepsis cannot be adequately reproduced by injection of LPS. Therefore, the so-called translational disconnect that exists between murine LPS models and human sepsis involves NF-κB activation at a molecular level and is further augmented by the use of LPS as a stimulus for infectious responses in vivo. [ABSTRACT FROM AUTHOR]

Genetically modified mesenchymal stem cells have been used in attempts to increase the expression of interleukin-1 receptor antagonist (IL-1Ra); however, the attempts thus far have been unsuccessful. The aim of the present study was to investigate whether the lentivirus transduced IL-1Ra gene was able to be stably expressed in murine bone marrow-derived mesenchymal stem cells (mBMSCs) in vitro. In the present study, third generation lentiviral (Lv) vectors transducing the IL-1Ra/green fluorescent protein (copGFP) gene were constructed and transfected into mBMSCs to establish the Lv.IL-1Ra.copGFP/mBMSCs, which were evaluated using fluorescence microscopy, flow cytometry, cell viability analysis using a cell counting kit-8 kit, Trypan blue staining and an MTT growth kinetics assay. The expression of IL-1Ra was analyzed using reverse transcription-quantitative polymerase chain reaction and western blotting. The results demonstrated that the Lv.IL-1Ra/copGFP vector was successfully constructed. The mBMSCs exhibited a short proliferation life, however they had good growth kinetics at an early stage and improved viability following efficient transduction of the IL-1Ra gene. IL-1Ra was overexpressed following transfection of mBMSCs. In conclusion, lentiviral vector transduced mBMSCs were able to efficiently express exogenous Il-1Ra under certain conditions and had a marked capacity for proliferation. [ABSTRACT FROM AUTHOR]

Background: Experimental models of pneumonia with penicillin non-susceptible Streptococcus pneumoniae (PNSSP) are hard to reproduce because the majority of strains with clinical relevance (like serotypes 6B, 9 V and 19 F) have low murine virulence. By optimization of culture and inoculum conditions of PNSSP (using porcine mucin), our aim was to develop a suitable, reliable and reproducible pneumonia mouse model for anti-infective pharmacology research. Results: Seven PNSSP strains, including serotypes 6B, 9 V, 14 and 19 F were included. Strain INS-E611 displayed the highest murine virulence and was chosen to validate the lung model. Nose-instilled pneumococci grew between 2.1 and 2.5 log10 CFU/g of lung in 24 hours when an optimized culture of bacterial cells was used, but animals were all alive and recovered of infection after 36 h. In contrast, inoculum supplementation with mucin led to 100% mortality related to a successful lung infection confirmed by histopathology. These findings were reproduced with all seven PNSSP strains in neutropenic mice. Immunocompetent animals cleared all strains spontaneously. Conclusions: This pneumonia model produces a progressive and uniformly fatal lung infection with diverse serotypes of PNSSP independently of their intrinsic murine virulence. [ABSTRACT FROM AUTHOR]

Experience-dependent synaptic plasticity is widely expressed in the mammalian brain and is believed to underlie memory formation. Persistent forms of synaptic plasticity in the hippocampus, such as long-term potentiation (LTP) and long-term depression (LTD) are particularly of interest, as evidence is accumulating that they are expressed as a consequence of, or at the very least in association with, hippocampus-dependent novel learning events. Learning-facilitated plasticity describes the property of hippocampal synapses to express persistent synaptic plasticity when novel spatial learning is combined with afferent stimulation that is subthreshold for induction of changes in synaptic strength. In mice it occurs following novel object recognition and novel object-place recognition. Calmodulin-dependent kinase II (CAMKII) is strongly expressed in synapses and has been shown to be required for hippocampal LTP in vitro and for spatial learning in the water maze. Here, we show that in mice that undergo persistent inhibitory autophosphorylation of αCAMKII, object-place learning is intact. Furthermore, these animals demonstrate a higher threshold for induction of persistent (>24 h) hippocampal LTP in the hippocampal CA1 region during unrestrained behaviour. The transgenic mice also express short-term depression in response to afferent stimulation frequencies that are ineffective in controls. Furthermore, they express stronger LTD in response to novel learning of spatial configurations compared to controls. These findings support that modulation of αCAMKII activity via autophosphorylation at the Thr305/306 site comprises a key mechanism for the maintenance of synaptic plasticity within a dynamic range. They also indicate that a functional differentiation occurs in the way spatial information is encoded: whereas LTP is likely to be critically involved in the encoding of space per se , LTD appears to play a special role in the encoding of the content or features of space. [ABSTRACT FROM AUTHOR]

The aim of this study was to examine the impact of moderate-intensity treadmill exercise on the structure and function of the murine heart and its associated impact on Akt- AMPK- mTOR signaling. A secondary aim was to test whether the exercise phenotype was altered following a cardiotoxic bolus dose of doxorubicin ( DOX). Two-month-old C57Bl/6J female mice remained sedentary ( SED, n = 12) or were progressively trained with treadmill running for 2 months up to 18 m/min; 60 min/day, 5 days/weeks ( EX, n = 11) or EX + DOX (15 mg/kg/dose) ( EX + DOX, n = 6). Following treadmill training, mice underwent graded exercise tolerance testing and echocardiography. Training improved graded exercise tolerance by 68 ± 5% relative to SED, and this effect was not altered with bolus DOX. There were no changes in relative heart size with EX or EX + DOX versus SED. Regional posterior wall thickening was improved in EX and abrogated in EX + DOX. EX had a reduced cardiomyocyte cross-sectional area ( CSA) relative to SED, and CSA was further attenuated with DOX. Following EX, AMPK-associated phosphorylation of ULK1(ser317) tended to be lower relative to SED. Akt-associated phosphorylation of TSC2(thr1462) and mTOR(ser2448) were also decreased relative to SED. We observed an increase in AMPK activity with DOX that was not translated to downstream AMPK phosphorylation sites. We conclude that 2 months of moderate treadmill exercise training improves regional cardiac function and exercise capacity, but does not induce relative physiologic hypertrophy in female mice. Differential responses in Akt- AMPK- mTOR signaling may mediate the observed phenotype. [ABSTRACT FROM AUTHOR]

ABSTRACT Supraspinatus (SS) tendon tears are common musculoskeletal injuries whose surgical repair exhibits the highest incidence of re-tear of any tendon. Development of therapeutics for improving SS tendon healing is impaired by the lack of a model that allows biological perturbations to identify mechanisms that underlie ineffective healing. The objective of this study was to develop a mouse model of supraspinatus insertion site healing by creating a reproducible SS tendon detachment and surgical repair which can be applied to a wide array of inbred mouse strains and genetic mutants. Anatomical and structural analyses confirmed that the rotator cuff of the mouse is similar to that of human, including the presence of a coracoacromial (CA) arch and an insertion site that exhibits a fibrocartilagenous transition zone. The surgical repair was successfully conducted on seven strains of mice that are commonly used in Orthopaedic Research suggesting that the procedure can be applied to most inbred strains and genetic mutants. The quality of the repair was confirmed with histology through 14 days after surgery in two mouse strains that represent the variation in mouse strains evaluated. The developed mouse model will allow us to investigate mechanisms involved in insertion site healing. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:25-32, 2015. [ABSTRACT FROM AUTHOR]

Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors. [ABSTRACT FROM AUTHOR]

The aim of the present study was to characterize a murine model of acute respiratory distress syndrome (ARDS) abiding by the Berlin definition of human ARDS and guidelines for animal models of ARDS. To this end, C57BL/6NCrl mice were challenged with lipopolysaccharide (LPS; 15 mg/kg, i.p.) followed 18 h later by injection of oleic acid (OA; 0.12 mL/kg, i.v.). Controls received saline injection at both time points. Haemodynamics were monitored continuously. Arterial blood gas analyses were performed just before and every 30 min after OA challenge. Ninety minutes after OA challenge, the chest of mice was scanned using micro-computed tomography (CT). Cytokine concentrations were measured in plasma samples. Lungs were harvested 90 min after OA challenge for histology, immunohistochemistry, lung weight measurements and tissue cytokine detection. A histological lung injury score was determined. Eighteen hours after LPS challenge, mice exhibited a severe systemic inflammatory response syndrome. Oxygenation declined significantly after OA injections ( Pa o2/ Fi o2 283 ± 73 and 256 ± 71 mmHg at 60 and 90 min, respectively; P < 0.001). Bilateral patchy infiltrates were present on the micro-CT scans. Histology revealed parenchymal damage with accumulation of polymorphonuclear neutrophils, intra-alveolar proteinacous debris and few hyaline membranes. The lung wet : dry ratio indicated damage to the alveolar capillary membrane. Cytokine patterns evidenced a severe local and systemic inflammatory state in plasma and lung tissue. In conclusion, the described two-hit model of ARDS shows a pathological picture of ARDS closely mimicking human ARDS according to the Berlin definition and may facilitate interpretation of prospective experimental results. [ABSTRACT FROM AUTHOR]

Background As low bone mineral density is a risk factor for fracture in childhood, optimizing age appropriate bone mass is recommended and might lower the impact of bone loss related to age. Consumption of omega-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic and docosahexaenoic (DHA) acids have been shown to beneficially modulate bone metabolism. The objective of this study was to determine the incidence of fracture in neonates receiving a fish compared with soybean oil-based intravenous lipid emulsion and evaluate the effect of varying dietary omega-3 PUFA consumption on growing bone in young mice. Materials and methods Eligibility criteria for the clinical study included gestational age ≤37 wk and parenteral nutrition-dependence for ≥4 wk. Radiographs were reviewed after lipid initiation to identify radiologic bone fracture. The animal study evaluated female C57/Bl6 mice randomized into one of five groups from age 3-12 wk, at which time femurs were harvested for micro-computed tomography and light microscopy analysis. Results A lower incidence of bone fracture was found in neonates maintained on fish compared with soybean oil. In the animal study, findings suggest the DHA diet provides the best protection against trabecular bone loss as evidenced by increased bone volume fraction, increased trabecular number, and decreased trabecular separation on micro-computed tomography. These protective effects appeared to affect the bone microstructure alone. Conclusions The lower fracture risk observed in fish oil fed neonates in combination with the protective effects of DHA observed in the femurs of young C57/BL6 mice suggest an important role for omega-3 PUFAs on bone growth. [ABSTRACT FROM AUTHOR]

Purpose: Accurate dosimetry is essential when irradiating mice to ensure that functional and molecular endpoints are well understood for the radiation dose delivered. Conventional methods of prescribing dose in mice involve the use of a single dose rate measurement and assume a uniform average dose throughout all organs of the entire mouse. Here, the authors report the individual average organ dose values for the irradiation of a 12, 23, and 33 g mouse on a 320 kVp x-ray irradiator and calculate the resulting error from using conventional dose prescription methods. Methods: Organ doses were simulated in the Geant4 application for tomographic emission toolkit using the MOBY mouse whole-body phantom. Dosimetry was performed for three beams utilizing filters A (1.65 mm Al), B (2.0 mm Al), and C (0.1 mm Cu + 2.5 mm Al), respectively. In addition, simulated x-ray spectra were validated with physical half-value layer measurements. Results: Average doses in soft-tissue organs were found to vary by as much as 23%-32% depending on the filter. Compared to filters A and B, filter C provided the hardest beam and had the lowest variation in soft-tissue average organ doses across all mouse sizes, with a difference of 23% for the median mouse size of 23 g. Conclusions: This work suggests a new dose prescription method in small animal dosimetry: it presents a departure from the conventional approach of assigning a single dose value for irradiation of mice to a more comprehensive approach of characterizing individual organ doses to minimize the error and uncertainty. In human radiation therapy, clinical treatment planning establishes the target dose as well as the dose distribution, however, this has generally not been done in small animal research. These results suggest that organ dose errors will be minimized by calibrating the dose rates for all filters, and using different dose rates for different organs. [ABSTRACT FROM AUTHOR]

Abstract: The in vitro activities of fluconazole (FLC), amphotericin B (AmB) and caspofungin (CSP) were evaluated against three isolates of Candida lusitaniae using time–kill curves. AmB showed in vitro fungicidal activity, whilst FLC and CSP exerted mainly strain-dependent fungistatic activity. The in vivo efficacies of the three drugs were evaluated in a murine model of disseminated infection. The doses administered were FLC 50mg/kg/day, AmB 0.8mg/kg/day and CSP 5mg/kg/day. All three drugs were able to reduce the fungal burden in the kidneys of infected mice, with AmB showing the highest efficacy, followed by CSP. At least in this model, FLC, AmB and CSP are good candidates for treating invasive infections by C. lusitaniae. [Copyright &y& Elsevier]

Highlights: [•] NDV-3 vaccine is protective against murine vulvovaginal candidiasis (VVC). [•] Protection requires both T and B lymphocytes for cell-mediated and humoral immunity. [•] Reduction in vaginal fungal count is accompanied by reduced neutrophil influx. [•] These results warrant further development of NDV-3 against VVC. [ABSTRACT FROM AUTHOR]

Abstract: The conventional PCR technique was used for studying the schistosomicidal effect of Mirazid® in the murine model. Results of the molecular study were compared with the parasitological results (ova and worm count). The used PCR technique was more sensitive than the Kato-Katz thick smears. Mirazid® showed some schistosomicidal effects against murine Schistosoma mansoni. However, it was not efficient enough to cure any of the studied mice. [Copyright &y& Elsevier]

The mouse has been the dominant model organism in studies on the development, genetics and evolution of the mammalian skull and associated soft-tissue for decades. There is the potential to take advantage of this well studied model and the range of mutant, knockin and knockout organisms with diverse craniofacial phenotypes to investigate the functional significance of variation and the role of mechanical forces on the development of the integrated craniofacial skeleton and musculature by using computational mechanical modelling methods (e.g. finite element and multibody dynamic modelling). Currently, there are no detailed published data of the mouse masticatory musculature available. Here, using a combination of micro-dissection and non-invasive segmentation of iodine-enhanced micro-computed tomography, we document the anatomy, architecture and proportions of the mouse masticatory muscles. We report on the superficial masseter (muscle, tendon and pars reflecta), deep masseter, zygomaticomandibularis (anterior, posterior, infraorbital and tendinous parts), temporalis (lateral and medial parts), external and internal pterygoid muscles. Additionally, we report a lateral expansion of the attachment of the temporalis onto the zygomatic arch, which may play a role in stabilising this bone during downwards loading. The data presented in this paper now provide a detailed reference for phenotypic comparison in mouse models and allow the mouse to be used as a model organism in biomechanical and functional modelling and simulation studies of the craniofacial skeleton and particularly the masticatory system. [ABSTRACT FROM AUTHOR]

Abstract: Background: Heterotopic ossification (HO) frequently causes complications following orthopaedic and trauma surgery and may drastically reduce the postoperative outcome due to pain and joint contracture. Current therapeutic options include NSAID''s and local radiation. However, both options of prevention show disadvantages such as delayed fracture healing and impaired ossification as well as other side effects.9 Our goal was to investigate a novel approach in the prevention of heterotopic ossification by pharmacologically interfering with the molecular signalling pathways involved in this process. Hypoxia leads to numerous effects on a cellular level, one of which is the activation of the transcriptional complex hypoxia-inducible factor (HIF).19 Among several other actions, the HIF1-α signalling pathway in turn regulates angiogenesis through induction of the expression of vascular endothelial growth factor (VEGF).21 We hypothesised that by pharmacologically interfering with the HIF-1α signalling pathway, the amount of HO formation may be reduced. Echinomycin is a known inhibitor of HIF-1-alpha and was used in our study with the aim to prevent HO from forming. Methods: We examined the effect of Echinomycin on HO formation in a murine model where an Achilles tenotomy was performed. This has previously been shown to reliably produce islets of heterotopic ossification within the soft tissue of mouse hind limbs at 10 weeks after surgery. The control group underwent Achilles tenotomy only, whereas the Echinomycin group additionally received Echinomycin subcutaneously. After trial completion, the limbs were harvested and Micro-CT was performed. Heterotopic bone volume was then identified in 3d images and quantified. Results: We found a highly significant reduction in the bone volume following subcutaneous administration of Echinomycin compared to the control group. Conclusion: Although a substantial reduction could be achieved, it was not possible to completely prevent heterotopic ossification from forming. Further studies have yet to be conducted to optimise the results by altering the dosage and duration of administration as well as investigate the mechanism by which Echinomycin led to the reduction of HO formation. [Copyright &y& Elsevier]

Abstract: Chlamydia trachomatis, although commonly asymptomatic in women, can result in chronic sequelae, such as pelvic inflammatory disease, ectopic pregnancy and infertility. However, a clear relationship has not been determined between specific serovars and the ability to lead to upper genital tract infection or infertility. Thus, in order to investigate differences in pathogenicity, C3H/HeN mice were infected in the ovarian bursa with the C. trachomatis strains D (UW-3/Cx), F (N.I.1), F (IC-Cal-3) and E (Bour). Differences both in the amount of vaginal shedding as well as subsequent fertility rates were observed between D (UW-3/Cx) and F (N.I.1) compared to F (IC-Cal-3) and E (Bour). Approximately 50% of the mice infected with the D (UW-3/Cx) and F (N.I.1) strains had vaginal shedding for up to 3–4 weeks after infection and fertility rates of less than 25%. Furthermore, mice inoculated with D (UW-3/Cx) and F (N.I.1) showed infertility even in the absence of medroxy progesterone acetate (MPA) treatment. In contrast, both MPA and non-MPA treated mice infected with F (IC-Cal-3) or E (Bour) did not show vaginal shedding and had fertility rates between 45 and 88%. Mutations in the CT135 open reading frame have been associated with virulence. However, no nucleotide differences were found among the four isolates for CT135. This murine model of infection with C. trachomatis may help with the understanding of disease pathology in humans and ultimately vaccine development. [Copyright &y& Elsevier]

Abstract: A comparison of the clinicopathology of European bat lyssavirus (EBLV) types-1 and -2 and of rabies virus was undertaken. Following inoculation of mice at a peripheral site with these viruses, clinical signs of rabies and distribution of virus antigen in the mouse brain were examined. The appearance of clinical signs of disease varied both within and across the different virus species, with variation in incubation periods and weight loss throughout disease progression. The distribution of viral antigen throughout the regions of the brain examined was similar for each of the isolates during the different stages of disease progression, suggesting that antigen distribution was not associated with clinical presentation. However, specific regions of the brain including the cerebellum, caudal medulla, hypothalamus and thalamus, showed notable differences in the proportion of virus antigen positive cells present in comparison to other brain regions suggesting that these areas are important in disease development irrespective of virus species. [Copyright &y& Elsevier]

Highlights: [•] Semaphorin 4A protein and mRNA levels are downregulated as CD4−CD8− cells mature. [•] Semaphorin 4A protein is localized to the thymic medulla. [•] Semaphorin 4A mRNA and protein are present on CD4−CD8−CD44+CD25− (DN1) thymocytes. [•] SEMA4A protein in DN1 cells is restricted to the CD117− subset of DN1 cells. [•] Sema4A modulation in thymocytes suggests a complex regulation of migratory cues. [ABSTRACT FROM AUTHOR]

We previously showed that interstitial fluid pressure (IFP) may be an alternate regulator of angiogenesis in solid tumors. Given the accepted link between hypoxia-induced factor and angiogenesis this study investigated the effect of IFP on hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) in human osteosarcoma xenografts in SCID mice and in different hypoxic environments. Tumors were grown either at heterotopic (flank) or orthotopic (medullary canal of the proximal tibia) sites in the host animal. Microfluidic probes determined pH, O2-saturation, IFP, and peripheral blood flow perfusion continuously. We assessed tumor growth in the orthotopic site ( n = 15) by softex radiographs weekly, 3D microCT, histological evaluation, and for molecular responses. An increased cytoplasmic immunohistostaining of cells for HIF-1α ( p = 0.03) and VEGF-A ( p = 0.004) on the outer periphery was noted compared to the tumor center, with VEGFR2 uniformly stained throughout. This paralleled a raised state of interstitial hypertension ( p = 0.007) in the tumor center relative to the peripheral surface but was inconsistent with a state of hypoxia ( p = 0.03) in the tumor center. In vitro culture of human osteosarcoma cell lines (HOS, U2OS) and a human osteoblast control at 0- and 20-mmHg of hydrostatic pressure revealed suppression of HIF-1α ( p = 0.02) and VEGF-A ( p = 0.02) gene expression when IFP was raised, while the effect on VEGFR1 was equivocal. This study proposes an alternative regulatory angiogenic pathway via the influence of IFP on cancer cell function. The identification of a mechanistic cellular link to the physical parameter becomes an important tool to evaluate cancer cell growth within solid tumors. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:2038-2045, 2012 [ABSTRACT FROM AUTHOR]

Objective. Interleukin (IL)-33, which mediates the Th2 allergic pathway, may play a key role in allergic airway inflammation. This study was conducted to evaluate the therapeutic potential of anti-IL-33 antibody for treatment of allergic inflammation of the lower airway in a murine model. Methods. Twenty-four BALB/c mice were used in this study. Saline was used for sensitization and challenge of mice in Group A (control group, n = 6). Mice in Group B (ovalbumin (OVA) group, n = 6) received intraperitoneal (ip) and intranasal OVA challenge. In Group C (control IgG group, n = 6), mice received ip injection with control IgG prior to OVA challenge. Mice in Group D (anti-IL-33 group, n = 6) received an ip injection of anti-IL-33 prior to challenge. Measurements of serum total and OVA-specific IgE and the number of eosinophils, neutrophils, and lymphocytes in bronchoalveolar lavage (BAL) fluid were performed. We performed histopathologic examination to evaluate the degree of eosinophilic infiltration in lung tissue. Airway hyperreactivity was measured according to change of enhanced pause (Penh). Results. A significant decrease in serum total and OVA-specific IgE and the number of eosinophils and neutrophils in BAL fluid was observed in Group D, compared with Group B or Group C ( p < .05). In Group D, treatment with anti-IL-33 resulted in a significant decrease in eosinophilic infiltration in lung tissue, compared with Group B and Group C ( p < .05). Degree of airway hyperreactivity, measured by Penh, showed a significant decrease in the anti-IL-33 treatment group, compared with the OVA group or the control IgG treatment group ( p < .01, at 50 mg/mL of methacholine). Conclusions: Anti-IL-33 has therapeutic potential for treatment of allergic inflammation of the lower airway. [ABSTRACT FROM AUTHOR]

The heavily O-glycosylated mucin MUC2 constitutes the major protein in the mucosal layer that acts as a physical barrier protecting the epithelial layer in the colon. In this study, Muc2 was purified from mucosal scrapings from the colon of wild-type (WT) mice, core 3 transferase knockout (C3Gnt−/−) mice and intestinal epithelial cell-specific core 1 knockout (IEC C1Galt1−/−) mice. The Muc2 O-glycans were released by reductive β-elimination and analyzed with liquid chromatography-mass spectrometry in the negative-ion mode. Muc2 from the distal colon of WT and C3Gnt−/− knockout mice carried a mixture of core 1- or core 2-type glycans, whereas Muc2 from IEC C1Galt1−/− mice carried highly sialylated core 3- and core 4-type glycans. A large portion of NeuAc in all mouse models was positioned on disialylated N-acetyllactosamine units, an epitope not reported on human colonic MUC2. Mass spectra and proton NMR spectroscopy revealed an abundant NeuAc linked to internally positioned N-acetylglucosamine on colonic murine Muc2, which also differs markedly from human MUC2. Our results highlight that murine colonic Muc2 O-glycosylation is substantially different from human MUC2, which could be one explanation for the different commensal microbiota of these two species. [ABSTRACT FROM AUTHOR]

Objectives. The aim of this study is to define the kinetics of the pulmonary inflammatory response in cockroach allergen (CRA) sensitized and challenged outbred mice. Methods. Asthma-like pulmonary inflammation was induced with three pulmonary exposures to CRA, without the use of adjuvants. Mice were sacrificed at multiple time points and asthma-like pulmonary inflammation quantified. Results. Several pulmonary parameters showed a pronounced biphasic inflammatory response with an early stage (1.5 hours post challenge) and late stage (24 hours). The initial phase was characterized by the production of multiple inflammatory mediators, including CXC chemokines, and the recruitment of neutrophils to the lung. The number of pulmonary eosinophils decreased in the early phase but quickly rebounded. Both the early and late phases had increases in TNF production in addition to airways hyperreactivity. The model also demonstrated early production of mucin with clearance by 12 hours followed by new accumulation of mucin in the pulmonary epithelial cells. Eotaxins within the lung peaked at about 12 hours and the numbers of eosinophils in the lung remained constant throughout the 48 hours of the study. Conclusions. The pulmonary inflammatory parameters in response to a clinically relevant allergen define a biphasic response. These data may be used to investigate the pathogenesis of the disease and develop targeted therapies for the distinct phases. [ABSTRACT FROM AUTHOR]

Background: Biomarkers facilitate early detection of disease and measurement of therapeutic efficacy, both at clinical and experimental levels. Recent advances in analytics and disease models allow comprehensive screening for biomarkers in complex diseases, such as asthma, that was previously not feasible. Objective: Using murine and nonhuman primate (NHP) models of asthma, identify biomarkers associated with early and chronic stages of asthma and responses to steroid treatment. Methods: The total protein content from thymic stromal lymphopoietin transgenic (TSLP Tg) mouse BAL fluid was ascertained by shotgun proteomics analysis. A subset of these potential markers was further analyzed in BAL fluid, BAL cell mRNA, and lung tissue mRNA during the stages of asthma and following corticosteroid treatment. Validation was conducted in murine and NHP models of allergic asthma. Results: Over 40 proteins were increased in the BAL fluid of TSLP Tg mice that were also detected by qRT-PCR in lung tissue and BAL cells, as well as in OVA-sensitive mice and house dust mite-sensitive NHP. Previously undescribed as asthma biomarkers, KLK1, Reg3γ, ITLN2, and LTF were modulated in asthmatic mice, and Clca3, Chi3l4 (YM2), and Ear11 were the first lung biomarkers to increase during disease and the last biomarkers to decline in response to therapy. In contrast, GP-39, LCN2, sICAM-1, YM1, Epx, Mmp12, and Klk1 were good indicators of early therapeutic intervention. In NHP, AMCase, sICAM-1, CLCA1, and GP-39 were reduced upon treatment with corticosteroids. Conclusions and clinical relevance: These results significantly advance our understanding of the biomarkers present in various tissue compartments in animal models of asthma, including those induced early during asthma and modulated with therapeutic intervention, and show that BAL cells (or their surrogate, induced sputum cells) are a viable choice for biomarker examination. [ABSTRACT FROM AUTHOR]

Abstract: Objective: To identify more effective and less toxic drugs to treat animal toxoplasmosis. Methods: Efficacy of seven kinds of sulfonamides against Toxoplasma gondii (T. gondii) in an acute murine model was evaluated. The mice used throughout the study were randomly assigned to many groups (10 mice each), which either remained uninfected or were infected intraperitoneally with tachyzoites of T. gondii (strains RH and CN). All groups were then treated with different sulfonamides and the optimal treatment protocol was determined candidates. Sulfadiazine-sodium (SD) was used for comparison. Results: The optimal therapy involved gavaging mice twice per day with 250 mg/kg bw of sulfachloropyrazine-sodium (SPZ) for five days. Using this protocol, the average survival time and the time-point of 50% fatalities were prolonged significantly compared with SD treatment. Treatment with SPZ protected 40% of mice from death, and the heart and kidney tissue of these animals was parasite-free, as determined by nested-PCR. SPZ showed excellent therapeutic effects in the treatment of T. gondii in an acute murine model and is therefore a promising drug candidate for the treatment and prevention of T. gondii in animals. Conclusions: It can be concluded that the effective drug sulfachloropyrazine may be the new therapeutic options against animal toxoplasmosis. [Copyright &y& Elsevier]

Abstract: Surface contaminants, such as bacterial debris and manufacturing residues, may remain on orthopedic implants after sterilization procedures and affect osseointegration. The goals of this study were to develop a murine model of osseointegration in order to determine whether removing surface contaminants enhances osseointegration. To develop the murine model, titanium alloy implants were implanted into a unicortical pilot hole in the mid-diaphysis of the femur and osseointegration was measured over a five week time course. Histology, backscatter scanning electron microscopy and X-ray energy dispersive spectroscopy showed areas of bone in intimate physical contact with the implant, confirming osseointegration. Histomorphometric quantification of bone-to-implant contact and peri-implant bone and biomechanical pullout quantification of ultimate force, stiffness and work to failure increased significantly over time, also demonstrating successful osseointegration. We also found that a rigorous cleaning procedure significantly enhances bone-to-implant contact and biomechanical pullout measures by two-fold compared with implants that were autoclaved, as recommended by the manufacturer. The most likely interpretation of these results is that surface contaminants inhibit osseointegration. The results of this study justify the need for the development of better detection and removal techniques for contaminants on orthopedic implants and other medical devices. [Copyright &y& Elsevier]

We examined if lipopolysaccharide (LPS) treatment of mice affected cardiolipin (CL) synthesis. Mice were injected i.p. with LPS, the liver harvested, and CL synthase (CLS) enzyme activity and its mRNA expression examined. Treatment of mice with LPS resulted in a 55% decrease ( p < 0.01) in mRNA expression of murine CLS compared to controls, but CLS enzyme activity was unaltered. The pool size of liver CL and other phospholipids were unaltered by LPS treatment. A similar effect was observed in murine epidermal fat pad and in vitro in RAW mouse macrophages and in human HepG2 cells. LPS treatment of HepG2 cells transiently expressing a histidine-tagged human cardiolipin synthase-1 (hCLS1) reduced hCLS1 mRNA and newly synthesized CLS activity indicating that LPS inhibits production of newly synthesized hCLS1 via reduction in hCLS1 mRNA. The results clearly indicate that CLS mRNA levels cannot be correlated with CLS enzyme activity nor CL content in the LPS model of inflammation. [ABSTRACT FROM AUTHOR]

Abstract: Pluripotent embryonic stem cells (ESCs) were first isolated nearly three decades ago from mice, yet efficient ESC isolation has been limited to rodents and primates to date. We report a novel and robust technique for isolating ESCs from mammalian pre-implantation embryos by altering the epigenotype of embryonic explants and using pressed zona pellucida-free blastocysts. We first examined this technique for murine ESC derivation. Compared with controls, murine ESCs were efficiently derived when explants were exposed to 1μM 5-azacytidine, an epigenetic modifier that causes DNA demethylation (56.1% vs 31.6%; P < 0.01). Mouse ESCs stained positively for alkaline phosphatase, expressed markers of pluripotency including Oct4, Rex1 and SSEA1 and formed teratomas when injected into Severe Combined Immuno-Deficient (SCID) mice. The approach was subsequently used for bovine ESC derivation. In bovine a higher concentration of 5-azacytidine (5 μM) was required to elicit a response. This technique resulted in up to 18 times more efficient isolation of pluripotent cells than traditional methods (71.4% vs 4.0%; P < 0.001). These putative bovine ESCs expressed OCT4, REX1 mRNA and SSEA-1 and SSEA-4 proteins; and were able to form embryoid bodies in vitro and teratomas when injected in Severe Combined Immuno Deficient (SCID) mice. This is the first report on derivation of ESCs with both in vitro and in vivo differentiation potential in a livestock species. [Copyright &y& Elsevier]

Atrial fibrillation (AF), the most common cardiac arrhythmia seen in general practice, can be promoted by conduction slowing. Cardiac impulse conduction depends on gap junction channels, which are composed of connexins (Cxs). While atrial Cx40 and Cx43 are equally expressed, AF studies have primarily focused on Cx40 reductions. The G60S Cx43 mutant (Cx43G60S/+) mouse model of Oculodentodigital dysplasia has a 60% reduction in Cx43 in the atria. Cx43G60S/+ mice were compared with Cx40-deficient (Cx40-/-) mice to determine the role of Cxs in atrial tachycardia/fibrillation (AT/F). Intracardiac electrophysiological studies were done in 6-mo-old male C57BL/6 Cx43G60S/+ mutant, littermate (Cx43+/+), Cx40-/-, and C57BL/6 wild-type (WT) mice. AT/F induction used an extra stimulus during sinus rhythm, programmed electrical stimulation, or burst pacing (1-ms pulses, 50-Hz, 400-ms train) in the absence and presence of carbachol (CCh). Atrial effective refractory periods did not differ between strains. Cx43G60S/+ mice were more susceptible to induction of sustained AT/F (duration >2 min, 9 of 12; maximum >35 min) compared with Cx43+/+ mice (3 of 11; χ2 = 5.24; P = 0.02). CCh enhanced sustained AT/F susceptibility in WT (from 1 of 12 without, to 7 of 10 with CCh; χ2 = 8.98; P < 0.01) but not in Cx40-/- mice (1 of 13 without vs. 2 of 9 with CCh; χ2 = 0.95; P = NS). The pattern of epicardial recordings during AT/F in Cx43G60S/+ mice was left preceding right, with left atrial fractionated activation patterns consistent with clinical observations of AF. In conclusions, while Cx43G60S/+ mice had severe AT/F, Cx40-/- mice were resistant to CCh-induced AT/F. [ABSTRACT FROM AUTHOR]

Background: Embryonic stem cells are cells derived from early-stage embryos that are characterized by pluripotency and self-renewal capacity. The in vitro cultured murine embryonic stem cells can indefinitely propagate in an undifferentiated state in the presence of leukemia inhibitory factor (LIF). However, when stimulated, these cells can differentiate into cell lines derived from all three embryonic germ layers. The trichostatin A (TSA) is an epigenetic modifier agent and several studies have used the TSA to stimulate cellular differentiation. However, most of these studies only assessed one TSA concentration. Therefore, this study aimed to evaluate the effects of different TSA concentrations on histone hyperacetylation during in vitro cell differentiation of murine pluripotent embryonic stem cells, cultured with or without LIF, in the quest of to standardize their application on early cultures of embryonic stem cells. Materials, Methods & Results: Undifferentiated murine embryonic stem cells were plated in the presence of different TSA concentrations (0 nM, 15 nm, 50 nM and 100 nM) in the presence or absence of LIF. Thus, the treatments were evaluated in undifferentiated embryonic stem cells cultured in the presence of LIF (Control group: 0 nM LIF+; Group 15 nM LIF+; Group 50 nM LIF+ and Group 100 nM LIF+), and in embryonic stem cells cultured in the absence of LIF (Control group: 0 nM LIF-; Group 15 nM LIF-; Group 50 nM LIF- and Group 100 nM LIF-). Treatment with TSA was performed for 24 h. After that the medium was replaced with fresh medium without TSA. Samples were collected at 0, 12, 24, 36 and 48 h after the beginning of the experiment. Three replicates were performed in each experimental group. The relative amount of Histone H3 lysine 9 acetylation was analyzed in all groups, as well as the cell proliferation in the embryonic stem cells cultured in the presence of LIF. In the control group (0 nM), the absence of LIF resulted in higher levels (P <0.05) of H3lys9ac compared to the cultures supplemented with LIF. In the embryonic stem cells cultured in the presence of LIF, the 50 nM and 100 nM treatments resulted in higher levels (P <0.05) of H3lys9ac when compared with 0 nM and 15 nM treatments. Evaluating the Hoechst area in the 0 nM group, it was observed that the number of cells increased (P <0.05) according to the time of culture. Treatment with 15 nM also reflected a similar distribution, but the Hoechst area in 15 nM group was lower (P <0.05) at 24 and 48h when compared to the observed in the control group. In the 100 nM treatment, was observed that the area of Hoechst was lower (P <0.05) to that obtained in the control group at 12, 24 and 48h. In addition, it was observed that treatment with TSA induces greater cellular differentiation when compared to control groups in stem cells cultured in the presence of LIF as well as in the absence of LIF. Discussion: In the present study it was observed that TSA treatment increased the levels of histone acetylation in murine embryonic stem cells at a 50 nM concentration, making it possible to reduce the concentration recommended in the literature (100 nM). In addition, it was concluded that the lower TSA concentrations utilized (15 nm and 50 nM) was less harmful to cellular proliferation than the 100 nM TSA concentration. [ABSTRACT FROM AUTHOR]

Persistent pressure overload can cause cardiac hypertrophy and progressive heart failure (HF). The authors developed a pressure-overload HF model of juvenile mice to study the cardiac response to pressure overload that may be applicable to clinical processes in children. Severe thoracic aortic banding (sTAB) was performed using a 28-gauge needle for 40 juvenile (age, 3 weeks) and 47 adult (age, 6 weeks) C57BL/6 male mice. To monitor the structural and functional changes, M-mode echocardiography was performed for conscious mice that had undergone sTAB and sham operation. Cardiac hypertrophy, dilation, and HF occurred in both juvenile and adult mice after sTAB. Compared with adults, juvenile HF is characterized by greater impairment of ventricular contractility and less hypertrophy. In addition, juvenile mice had significantly higher rates of survival than adult mice during the early postoperative weeks. Consistent with clinical HF seen in children, juvenile banded mice demonstrated a lower growth rate than either adult banded mice or juvenile control mice that had sham operations. The authors first developed a juvenile murine model of pressure-overload HF. Learning the unique characteristics of pressure-overload HF in juveniles should aid in understanding age-specific pathologic changes for HF development in children. [ABSTRACT FROM AUTHOR]

Abstract: The potential role of CXC chemokines bearing the glu-leu-arg (ELR) motif in bone repair was studied using a cranial defect (CD) model in mice lacking the CXC receptor (mCXCR−/− knockout mice), which is homologous to knockout of the human CXC receptor 2 (CXCR2) gene. During the inflammatory stage of bone repair, ELR CXC chemokines are released by inflammatory cells and serve as chemotactic and angiogenic factors. mCXCR−/− mice were smaller in weight and length from base of tail to nose tip, compared to WT littermates. DEXA analysis indicated that bone mineral density (BMD), bone mineral content (BMC), total area (TA), bone area (BA), and total tissue mass (TTM) were decreased in the mCXCR−/− mice at 6, 12, and 18weeks of age. Trabecular bone characteristics in mCXCR−/− (% bone, connectivity, number, and thickness) were reduced, and trabecular spacing was increased as evidenced by μCT. There was no difference in bone formation or resorption indices measured by bone histomorphometry. Trabecular BMD was not altered. Cortical bone volume, BMD, and thickness were reduced; whereas, bone marrow volume was increased in mCXCR−/−. Decreased polar moment of inertia (J) in the tibias/femurs suggested that the mCXCR−/− long bones are weaker. This was confirmed by three-point bending testing of the femurs. CDs created in 6-week-old male mCXCR−/− and WT littermates were not completely healed at 12weeks; WT animals, however, had significantly more bone in-growth than mCXCR−/−. New bone sites were identified using polarized light and assessed for numbers of osteocyte (OCy) lacunae and blood vessels (BlV) around the original CD. In new bone, the number of BlV in WT was >2× that seen in mCXCR−/−. Bone histomorphometry parameters in the cranial defect did not show any difference in bone formation or resorption markers. In summary, studies showed that mCXCR−/− mice have (1) reduced weight and size; (2) decreased BMD and BMC; (3) decreased amounts of trabecular and cortical long bone; (4) decreased femur bone strength; and (5) significantly reduced intramembranous bone formation and number of BlV in new calvarial bone during bone repair. [Copyright &y& Elsevier]

Abstract: ZIPK (zipper-interacting protein kinase) is a Ca2+-independent protein kinase that promotes myosin phosphorylation in both smooth muscle and non-muscle cells. A recent report attempted to clarify a debate over the subcellular localization of ZIPK in non-muscle cells (Shoval et. al. (2007) Plos Genetics. 3: 1884–1883). A species-specific loss of a key phosphorylation site (T299) in murine (mouse and rat) ZIPK seems to direct it to the nucleus, while the presence of the T299 site in human ZIPK correlates with cytoplasmic localization. T299 is immediately adjacent to a putative nuclear localization sequence (NLS) and may mask its function when phosphorylated, therefore explaining the species-specific dichotomy of intracellular localization. However, despite the murine ZIPK (mZIPK) lacking the T299 residue that is critical for controlling human ZIPK (hZIPK) subcellular localization, mutational analysis showed that this NLS control locus is nonfunctional in the murine context. A constitutively active Rho promoted the cytoplasmic retention of a human ZIPK mutant that would otherwise localize to the nucleus. Endogenous hZIPK showed sensitivity to the nuclear export inhibitor leptomycin B, suggesting a continuous shuttling between cytoplasm and nucleus that is dependent upon T299 dephosphorylation. Thus, the C-terminal domain of human and murine ZIPK demonstrated quite divergent nuclear import and export functionality. We conclude that in the case of ZIPK, studies between the species may not be directly comparable to each other given the gross differences in intracellular localization and movement. [ABSTRACT FROM AUTHOR]

Abstract: Background: Recent studies suggest that large (>1cm) diameter circular cutaneous wounds induce follicular neogenesis in mice. However, the effects of non-circular wounds or smaller circular wounds on the hair follicle cycle remain poorly understood. Objective: We investigated whether non-circular wounds or smaller (≤1cm) circular wounds could induce early entry of hair follicle stem cells into anagen. Methods: We created different shaped and sized full-thickness incisional wounds on the dorsal skin of 6-wk old mice (BALB/c, C57BL/6, and CD1), and then assessed for hair growth, wound contraction rates, and growth factor and/or immunomodulatory cytokine involvement. Results: By day 16 and through day 26 post-wounding, we observed hair growth in the skin around the wound, but not in distant unwounded skin of BALB/c and C57BL/6 mice. In contrast, CD1 mice showed hair growth in both the wounded and unwounded skin. In all mice, the hair growth pattern was independent of wound type. The area of hair growth induced by a 2cm linear wound was roughly 2-fold that induced by a 1cm linear wound and 4-fold that of a 0.5cm linear wound. Increased γ-glutamyl transpeptidase activity was observed in hair follicles growing in the wounded area as well as an up to 8-fold upregulation of Wnt- and Shh-dependent signaling, consistent with anagen growth. Conclusion: Our data strongly support that small cutaneous wounds induce telogen to anagen transition of murine hair follicle stem cells. [ABSTRACT FROM AUTHOR]

Following burn injury, the host is susceptible to bacterial infections normally cleared by healthy patients. We hypothesized that during the systemic immune response that follows scald injury, the host's altered immune status increases infection susceptibility. Using a murine model of scald injury under inhaled anesthesia followed by intraperitoneal infection, we observed increased neutrophil numbers and function at postburn day (PBD) 1 compared with sham-burned and PBD4 mice. Further, increased mortality, bacteremia, and serum IL-6 were observed in PBD1 mice after Pseudomonas aeruginosa (PA) infection compared with sham-burned and PBD4 mice infected with PA. To examine these disparate responses, we investigated neutrophils isolated at 5 and 24 h following PA infection from PBDI and sham-burned mice. Five hours after infection, there was no significant difference in number of recruited neutrophils; however, neutrophils from injured mice had decreased activation, active-p38, and oxidative burst compared with sham-burned mice. In direct contrast, 24 h after infection, we observed increased numbers, active-p38, and oxidative burst of neutrophils from PBD1 mice. Finally, we demonstrated that in neutrophils isolated from PBD1 mice, the observed increase in oxidative burst was p138 dependent. Altogether, neutrophil activation and function from thermally injured mice are initially delayed and later exacerbated by a p38-dependent mechanism. This mechanism is likely key to the observed increase in bacterial load and mortality of PBD1 mice infected with PA. [ABSTRACT FROM AUTHOR]

The goal of this study was to examine the ability of a commercially available feed additive (OmniGen-AF) to reduce mammary infections caused by a single strain of mastitic pathogens (Streptococcus uberis, Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae) and to examine the effects of the additive on markers of mammary immunity. Four experiments were completed using a murine model of bovine mastitis. Infection progression was examined using Sybr-green- and TaqMan-based quantitative PCR assays of 16S ribosomal DNA. Infection of the mammary gland with all pathogens caused rapid (24 to 48 h) appearance of pathogen DNA in mammary tissue. Provision of the feed additive for 2 weeks before infection significantly (P < 0.05) reduced the extent of pathogen DNA accumulation in models of S. uberis, E. coli and S. aureus infection. The additive was ineffective in reducing mammary infections caused by K. pneumoniae. We examined mechanisms of action of the additive through assessment of mammary concentrations of mammary myeloperoxidase (MPO), major histocompatibility complex 2 class II (MHC) and macrophage inflammatory protein-1α (MIP) messenger RNA (mRNA) concentrations and by examining serum complement C3 concentration. Infection of the mammary gland increased concentrations of MPO and MHC mRNAs (P < 0.05). Ability of the pathogen to elicit changes in mammary MPO and MHC gene expression was enhanced by the provision of the additive for 2 weeks before infection. These data imply that the additive increased the mammary inflammatory response and increased antigen presentation during a mammary infection. Value of the additive in preventing mastitis in cattle awaits additional studies using a bovine model and further evaluation of additional strains of the pathogens used in this study. [ABSTRACT FROM AUTHOR]

Abstract: IL-27 is a heterodimeric cytokine composed of p28 and Epstein Barr virus induced gene 3 (Ebi3) protein subunits. In the present study, we questioned whether murine gammaherpesvirus 68 (HV-68) could induce expression of Ebi3, p28, and IL-27 in this mouse model of an EBV-like infection. Cultured macrophages and dendritic cells exposed to HV-68 upregulated p28 mRNA expression and increased secretion of the p28 and IL-27 (p28+Ebi3) proteins. B220+ and CD11b+ cells also upregulated p28 mRNA expression following in vivo infection with this virus. Surprisingly, no significant increases in p28 or IL-27 protein production were observed in vivo during the acute or mononucleosis phases of the disease. The possibility that HV-68-induced upregulation of p28 mRNA expression primed cells for IL-27 secretion was suggested by the ability of a TLR4 agonist to augment cytokine production. When cultured macrophages and dendritic cells were exposed to virus plus a suboptimal dose of LPS, increased levels of p28 protein expression were observed. More importantly, when latently infected mice were challenged with a sublethal dose of LPS, augmented p28 and IL-27 protein production occurred. Using a model of sepsis, mice latently infected with HV-68 had exaggerated p28 protein production when compared to mice that were singularly infected or subjected to cecal ligation and puncture. Taken together, these studies define expression of HV-68 induced IL-27, and suggest that mice latently infected with this gammaherpesvirus will have exaggerated responses when confronted with other stimuli capable of inducing this member of the IL-12 family of cytokines. [Copyright &y& Elsevier]

The authors discuss a study on the receptor for advanced glycation end-products (RAGE), by van Zoelen and colleagues, published within the issue. The study showed that RAGE-deleted mice and wild-type mice treated with anti-RAGE antibodies have increased bacterial dissemination. The authors reveal that evidence of the antibacterial properties of RAGE ligands is limited. They add that the study is relevant because it notes the need for further research on RAGE.

Interleukin-(IL)-4 and IL-13 signal through heterodimeric receptors containing a common IL-4 receptor-α (IL-4Rα) subunit, which is important for protection against helminth infections, including schistosomiasis. Previous studies demonstrated important roles for IL-4Rα-responsive hematopoietic cells, including T cells and macrophages in schistosomiasis. In this study, we examined the role of IL-4Rα responsiveness by nonhematopoietic smooth muscle cells during experimental acute murine schistosomiasis. Comparative Schistosoma mansoni infection studies with smooth muscle cell-specific IL-4Rαdeficient (SM-MHCcreIL-4Rα-/flox) mice, heterozygous control (IL-4Rα-/flox) mice, and global IL-4Rα-deficient (IL-4Ra-/-) mice were conducted. S. mansoni-infected SM-MHCcreIL-4Rα-/flox mice showed increased weight loss and earlier mortalities compared with IL-4Rα-/flox mice, despite comparable TH2/type 2 immune responses. In contrast to highly susceptible IL-4Rα-deficient mice, increased susceptibility in SM-MHCcreIL-4Rα-/flox mice was not accompanied by intestinal tissue damage and subsequent sepsis. However, both susceptible mutant mouse strains failed to efficiently expel eggs, demonstrated by egg reduction in the feces compared with control mice. Reduced egg expulsion was accompanied by impaired IL-4/IL-13-mediated hypercontractile intestinal responses, which was present in the more resistant control mice. Together, we conclude that IL-4Rα responsiveness by smooth muscle cells and subsequent 1L-4- and IL-13-mediated hypercontractility are required for host protection during acute schistosomiasis to efficiently expel S. mansoni eggs and to prevent premature mortality. [ABSTRACT FROM AUTHOR]

Abstract: Specific bacterial lipopolysaccharides (LPS), IFN-γ, and unmethylated cytosine or guanosine-phosphorothioate containing DNAs (CpG) activate host immunity, influencing infectious responses. Macrophages detect, inactivate and destroy infectious particles, and synthetic CpG sequences invoke similar responses of the innate immune system. Previously, murine macrophage J774 cells treated with CpG induced the expression of nitric oxide synthase 2 (NOS2) and cyclo-oxygenase 2 (COX2) mRNA and protein. In this study murine J774 macrophages were exposed to vehicle, interferon γ+lipopolysaccharide (IFN-g/LPS), non-CpG (SAK1), or two-CpG sequence-containing DNA (SAK2) for 0–18h and gene expression changes measured. A large number of immunostimulatory and inflammatory changes were observed. SAK2 was a stronger activator of TNFα- and chemokine expression-related changes than LPS/IFN-g. Up regulation included tumor necrosis factor receptor superfamily genes (TNFRSF’s), IL-1 receptor signaling via stress-activated protein kinase (SAPK), NF-κB activation, hemopoietic maturation factors and sonic hedgehog/wingless integration site (SHH/Wnt) pathway genes. Genes of the TGF-β pathway were down regulated. In contrast, LPS/IFN-g-treated cells showed increased levels for TGF-β signaling genes, which may be linked to the observed up regulation of numerous collagens and down regulation of Wnt pathway genes. SAK1 produced distinct changes from LPS/IFN-g or SAK2. Therefore, J774 macrophages recognize LPS/IFN-g, non-CpG DNA or two-CpG DNA-containing sequences as immunologically distinct. [Copyright &y& Elsevier]

Background: The exact immunopathogenesis of contact hypersensitivity (CH) to mercury remains unclear. Objective: The aim of the present study was to assess the role of CD4+ T cells in mercury-induced CH in mice. Methods: Splenic CD4+ T cells obtained from nonsensitized and mercury-sensitized Balb/c mice were adoptively transferred to groups I and II of the syngeneic recipients, respectively. All recipients were ear-challenged with mercury. The next experiments were to transfer nonsensitized CD4+ T cells to group A of the recipients, whereas mercury-sensitized CD4+ T cells were transferred to groups B and C. Groups A and B were ear-challenged with mercury, whereas group C was ear-challenged with chromium. The ear swelling 24 hours and 48 hours after challenge was used to assess CH. Results: The results of the present study showed that mercury-sensitized but not nonsensitized CD4+ T cells could transfer CH in mice. Furthermore, mercury-sensitized CD4+ T cells could transfer the induction of CH only in the recipients that were challenged with mercury, but not those that were challenged with chromium. Conclusion: The results of the present study suggest that mercury-induced CH may be mediated by mercury-specific CD4+ T cells. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common arrhythmia seen in general practice. Muscarinic ACh receptors (M2R, M3R) are involved in vagally induced AF. M2R and M3R activate the heterotrimeric G proteins, Gi and Gq, respectively, by promoting GTP binding, and these in turn activate distinct K+ channels. Signaling is terminated by GTP hydrolysis, a process accelerated by regulator of G protein signaling (RGS) proteins. RGS2 is selective for Gq and thus may regulate atrial M3R signaling. We hypothesized that knockout of RGS2 (RGS2-/-) would render the atria more susceptible to electrically induced AF. One-month-old male RGS2-/- and C57BL/6 wild-type (WT) mice were instrumented for intracardiac electrophysiology. Atrial effective refractory periods (AERPs) were also determined in the absence and presence of carbachol, atropine, and/or the selective M3R antagonist darifenacin. Susceptibility to electrically induced AF used burst pacing and programmed electrical stimulation with one extrastimulus. Real-time RT-PCR measured atrial and ventricular content of RGS2, RGS4, M2R, M3R, and M4R mRNA. AERP was lower in RGS2-/- compared with `NT mice in both the high right atrium (HRA) (30 ± 1 vs. 34 ± 1 ms, P < 0.05) and mid right atrium (MRA) (21 ± 1 vs. 24 ± 1 ms, P < 0.05). Darifenacin eliminated this difference (HRA: 37 ± 2 vs. 39 ± 2 ms, and MRA: 30 ± 2 vs. 30 ± 1, P > 0.4). RGS2 were more susceptible than WT mice to atrial tachycardia/fibrillation (AT/F) induction (11/22 vs. 1/25, respectively, P < 0.05). Muscarinic receptor expression did not differ between strains, whereas M2R expression was 70-fold higher than M3R (P < 0.01). These results suggest that RGS2 is an important cholinergic regulator in the atrium and that RGS2-/- mice have enhanced susceptibility to AT/F via enhanced M3 muscarinic receptor activity. [ABSTRACT FROM AUTHOR]