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Cancer-intrinsic immune evasion mechanisms and pleiotropy are a barrier to cancer immunotherapy. This is apparent in certain highly fatal cancers, including high-grade gliomas and glioblastomas (GBM). In this study, we evaluated two murine syngeneic glioma models (GL261 and CT2A) as preclinical models for human GBM using functional genetic screens, single-cell transcriptomics and machine learning approaches. Through CRISPR genome-wide co-culture killing screens with various immune cells (cytotoxic T cells, natural killer cells, and macrophages), we identified three key cancer-intrinsic evasion mechanisms: NFκB signaling, autophagy/endosome machinery, and chromatin remodeling. Additional fitness screens identified dependencies in murine gliomas that partially recapitulated those seen in human GBM (e.g., UFMylation). Our single-cell analyses showed that different glioma models exhibited distinct immune infiltration patterns and recapitulated key immune gene programs observed in human GBM, including hypoxia, interferon, and TNF signaling. Moreover, in vivo orthotopic tumor engraftment was associated with phenotypic shifts and changes in proliferative capacity, with murine tumors recapitulating the intratumoral heterogeneity observed in human GBM, exhibiting propensities for developmental- and mesenchymal-like phenotypes. Notably, we observed common transcription factors and cofactors shared with human GBM, including developmental (Nfia and Tcf4), mesenchymal (Prrx1 and Wwtr1), as well as cycling-associated genes (Bub3, Cenpa, Bard1, Brca1, and Mis18bp1). Perturbation of these genes led to reciprocal phenotypic shifts suggesting intrinsic feedback mechanisms that balance in vivo cellular states. Finally, we used a machine-learning approach to identify two distinct immune evasion gene programs, one of which represents a clinically-relevant phenotype and delineates a subpopulation of stem-like glioma cells that predict response to immune checkpoint inhibition in human patients. This comprehensive characterization helps bridge the gap between murine glioma models and human GBM, providing valuable insights for future therapeutic development. [ABSTRACT FROM AUTHOR]

Background: Enhancing venous return during cardiopulmonary resuscitation (CPR) can lead to better hemodynamics and improved outcome after cardiac arrest (CA). Peripheral Intravenous Analysis (PIVA) provides feedback on venous flow changes and may indicate an increase in venous return and cardiac output during CPR. We hypothesize PIVA can serve as an early indicator of increased venous return, preceding end-tidal CO2 (etCO2) increase, before the return of spontaneous circulation (ROSC) in a rat model of CA and CPR. Results: Eight male Wistar rats were intubated and ventilated, and etCO2 was measured. Vessels were cannulated in the tail vein, femoral vein, femoral artery, and central venous and connected to pressure transducers. Ventilation was discontinued to achieve asphyxial CA. After 8 min, CPR began with ventilation, epinephrine, and automated chest compressions 200 times per minute until mean arterial pressure increased to 120 mmHg. Waveforms were recorded and analyzed. PIVA was calculated using a Fourier transformation of venous waveforms. Data are mean ± SE. Maximum PIVA values occurred in the tail vein 34.7 ± 2.9 s before ROSC, with subsequent PIVA peaks in femoral vein and centrally at 30.9 ± 5.4 and 25.1 ± 5.0 s, respectively. All PIVA peaks preceded etCO2 increase (21.5 ± 3.2 s before ROSC). Conclusion: PIVA consistently detected venous pressure changes prior to changes in etCO2. This suggests that PIVA has the potential to serve as an important indicator of venous return and cardiac output during CPR, and also as a predictor of ROSC. [ABSTRACT FROM AUTHOR]

Muscle fatigue is the diminution of force required for a particular action over time. Fatigue may be particularly pronounced in aging muscles, including those used for swallowing actions. Because risk for swallowing impairment (dysphagia) increases with aging, the contribution of muscle fatigue to age-related dysphagia is an emerging area of interest. The use of animal models, such as mice and rats (murine models) allows experimental paradigms for studying the relationship between muscle fatigue and swallowing function with a high degree of biological precision that is not possible in human studies. The goal of this article is to review basic experimental approaches to the study of murine tongue muscle fatigue related to dysphagia. Traditionally, murine muscle fatigue has been studied in limb muscles through direct muscle stimulation and behavioral exercise paradigms. As such, physiological and bioenergetic markers of muscle fatigue that have been validated in limb muscles may be applicable in studies of cranial muscle fatigue with appropriate modifications to account for differences in muscle architecture, innervation ratio, and skeletal support. Murine exercise paradigms may be used to elicit acute fatigue in tongue muscles, thereby enabling study of putative muscular adaptations. Using these approaches, hypotheses can be developed and tested in mice and rats to allow for future focused studies in human subjects geared toward developing and optimizing treatments for age-related dysphagia. [ABSTRACT FROM AUTHOR]

Studies on vitamin D supplementation have been performed in experimental and clinical investigations considering gestational diabetes and/or vitamin D deficiency in pregnancy. However, the results are controversial and few present the effects and mechanisms of this micronutrient on pregestational diabetes. The objective of this study was to evaluate the effect of vitamin D on the pregnancy of rats with pre-existing diabetes and their fetuses. Pregestational diabetes was induced in Sprague–Dawley rats at birth. The adult diabetic and nondiabetic rats were orally administered with vitamin D (cholecalciferol) throughout the pregnancy. The diabetes status was monitored during pregnancy by an oral glucose tolerance test (OGTT). At the end of the pregnancy, pancreas and blood samples were collected for morphological analyses and lipid peroxidation measurements, respectively. The influence of vitamin D treatment on reproductive outcomes, fetal growth, and development were compared to those of untreated diabetic and nondiabetic pregnant rats. P < 0.05 was considered a significant statistical limit. The diabetic rats given vitamin D had a greater number of insulin-positive cells, contributing to reduced blood glucose levels and thiobarbituric acid reactive substance concentrations (TBARS—an indicator of membrane lipid peroxidation), and increased reduced thiol group levels, contributing to suitable intrauterine conditions for better fetal development, which was confirmed by higher fetal viability rates. Thus, this study shows the effects and mechanisms of vitamin D supplementation on pre-existing diabetes in pregnant rats, confirming its beneficial effects on maternal redox status and glycemic control, and the decline of adverse maternal–fetal repercussions. [ABSTRACT FROM AUTHOR]

Extracranial arteriovenous malformation (AVM) is a congenital vascular anomaly causing disfigurement, bleeding, ulceration, and pain. Most lesions are associated with somatic MAP2K1 activating mutations in endothelial cells (ECs). The purpose of this study was to determine if EC expression of mutant activated MAP2K1 is sufficient to produce vascular malformations in mice. We generated mice with a ROSA26 allele containing a lox-stop-lox gene trap (GT), Map2k1 cDNA with an activating p.K57N missense mutation, an internal ribosomal entry site, and green fluorescent protein cDNA (R26GT−Map2k1−GFP). We expressed mutant MAP2K1 and GFP in ECs of fetal and newborn mice using Tg-Cdh5Cre or Tg-Cdh5CreER alleles. Tg-Cdh5Cre+/−;R26GT−Map2k1−GFP/+ animals that express mutant MAP2K1 in ECs in utero developed diffuse vascular abnormalities and died by embryonic (E) day 16.5. Tg-Cdh5CreER+/−;R26GT−Map2k1−GFP/+ animals in which mutant MAP2K1 expression was induced in ECs by tamoxifen at postnatal (P) day 1 developed vascular malformations in the brain, ear, and intestines by P23. The lesions consisted of abnormal networks of blood vessels containing recombined and non-recombined ECs. In conclusion, expression of MAP2K1 p.K57N is sufficient to cause vascular malformations in mice. This model can be used to study the malformation process and for pre-clinical pharmacologic studies. [ABSTRACT FROM AUTHOR]

Spinocerebellar ataxias (SCAs) represent a large group of hereditary degenerative diseases of the nervous system, in particular the cerebellum, and other systems that manifest with a variety of progressive motor, cognitive, and behavioral deficits with the leading symptom of cerebellar ataxia. SCAs often lead to severe impairments of the patient's functioning, quality of life, and life expectancy. For SCAs, there are no proven effective pharmacotherapies that improve the symptoms or substantially delay disease progress, i.e., disease-modifying therapies. To study SCA pathogenesis and potential therapies, animal models have been widely used and are an essential part of pre-clinical research. They mainly include mice, but also other vertebrates and invertebrates. Each animal model has its strengths and weaknesses arising from model animal species, type of genetic manipulation, and similarity to human diseases. The types of murine and non-murine models of SCAs, their contribution to the investigation of SCA pathogenesis, pathological phenotype, and therapeutic approaches including their advantages and disadvantages are reviewed in this paper. There is a consensus among the panel of experts that (1) animal models represent valuable tools to improve our understanding of SCAs and discover and assess novel therapies for this group of neurological disorders characterized by diverse mechanisms and differential degenerative progressions, (2) thorough phenotypic assessment of individual animal models is required for studies addressing therapeutic approaches, (3) comparative studies are needed to bring pre-clinical research closer to clinical trials, and (4) mouse models complement cellular and invertebrate models which remain limited in terms of clinical translation for complex neurological disorders such as SCAs. [ABSTRACT FROM AUTHOR]

Talaromycosis (penicilliosis) caused by Talaromyces marneffei is one of the most important opportunistic infection diseases in tropical countries of South and Southeast Asia. Most infections occurred in individuals with human immunodeficiency virus (HIV) and the primarily reason for the increase in the number of the cases is HIV pandemic. The pathogenesis of T. marneffei infection is unclear. There is still no ideal animal model for studying talaromycosis. In this study, we developed a stable, safe and maneuverable murine model that mimics human T. marneffei disseminated infection using T. marneffei yeast intraperitoneal injected to BALB/c nude mice. We successfully observed symptoms similar to those seen in clinical patients in this murine model, including skin lesions, hepatosplenomegaly, pulmonary infection and mesenteric lesions. We further studied the pathological changes of various tissues and organs in the infected animals to help better understand the severity of the infection. This model may provide a good tool for studying disseminated infection induced by T. marneffei. [ABSTRACT FROM AUTHOR]

Jasonia glutinosa (L.) DC., known as rock tea (RT), is traditionally used in Spain as a digestive due to its beneficial properties in bowel disorders. The pharmacological nature of these properties has not been established yet. The aim of this work was to evaluate the therapeutic utility of RT in experimental colitis and to identify chemical constituents with anti-inflammatory and/or anti-oxidative properties. RT extract was prepared with ethanol in a Soxhlet apparatus and analysed by HPLC–DAD. Superoxide radical scavenging properties, xanthine oxidase and lipoxygenase (5-LOX) inhibitory activity, and capability to lower nitric oxide (NO) and tumor necrosis factor α (TNF-α) levels were measured in cell-free and cell-based assays. In the 2.5%-dextran-sodium sulphate (DSS) injury-repair model of ulcerative colitis (UC), mice were daily treated with sulfasalazine (SSZ, as reference drug, 100 mg/kg bw), RT (5, 25 and 50 mg/kg bw, p.o.), or vehicle over 20 days. Colitis was scored daily. Colon samples were examined macroscopically and histopathologically. Protein levels of myeloperoxidase (MPO), interleukins 6, and 10 (IL-6, IL-10), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2) were studied as markers of oxidative stress and inflammatory activity. The integrity of the apical epithelial layer was assessed by immunofluorescence staining of zonula ocludens-1 (ZO-1). Finally, intestinal contractility was also evaluated by isometric myography. Fifteen phenolic compounds and three pigments were identified and quantified, of which caffeoylquinic acids, and the flavonoid, quercetin-3-O-galactoside, were the most abundant. RT extract significantly scavenged superoxide radicals, inhibited 5-LOX activity, and lowered NO and TNF-α levels. DSS-treated mice receiving RT scored clinically lower than controls during the first 3 days of DSS treatment and during the recovery period. SSZ was less effective than RT. Anatomical and histological examination of colon samples revealed that RT significantly prevented colon shortening, increased colon thickness, and lowered the macroscopic damage score. RT also significantly prevented the increase of MPO activity, IL-6 levels, iNOS and COX-2 expression, the loss of ZO-1 apical expression, and normalized contractility disturbances. In conclusion, daily administration of RT showed therapeutic properties in the DSS-model of UC. The benefits of RT can likely be attributed to its anti-inflammatory and antioxidant phenolic and flavonoid constituents. [ABSTRACT FROM AUTHOR]

Diphtheria toxin (DT) efficiently inhibits protein synthesis in human cells, resulting in severe disease diphtheria. The sensitivity towards DT varies between mammalian species. Mice and rats are resistant to DT. However, the reason underlying this insensitivity is controversially discussed and not well understood. Therefore, we investigated the steps of DT uptake, i.e. receptor binding and internalization into mouse J774A.1 macrophages and primary rat fibroblasts. We exploited the non-toxic DT-mutant cross-reacting material 197 (CRM197) and three additional receptor binding-deficient mutants (250 nM each) to investigate binding to cell surface and internalization into murine cells via flow cytometry and stimulated emission depletion (STED) super-resolution optical microscopy. Dual-color STED imaging unveiled CRM197 interacting with the murine precursor of the heparin-binding epidermal growth factor-like growth factor (HB-EGF). Moreover, we identified CRM197's transmembrane domain as an additional HB-EGF binding site, which is also involved in the receptor-mediated internalization into murine cells. However, we do not find evidence for translocation of the catalytically active subunit (DTA) into the cytosol when 250 nM DT were applied. In conclusion, we provide evidence that the resistance of murine cells to DT is caused by an insufficiency of DTA to escape from endosomes and reach the cytosol. Possibly, a higher affinity interaction of DT and the HB-EGF is required for translocation, which highlights the role of the receptor in the endosomes during the translocation step. We extend the current knowledge about cellular uptake of the medically relevant DT and CRM197. [ABSTRACT FROM AUTHOR]

Purpose of Review: This article aimed to review animal models of antifungals and identifies human literature to assess if the extrapolation of results is reliable. Recent Findings: Animal studies have helped identify area under the concentration curve to minimum inhibitory concentration ratio targets for new drugs and formulations such as isavuconazole and delayed-release posaconazole that have translated to successful outcomes in humans. Models have also been influential in the identification of possible combination therapies for the treatment of aspergillosis, such as voriconazole and echinocandins. However, challenges are endured with animal models when it comes to replicating the pharmacokinetics of humans which has been exemplified with the newest itraconazole formulation. Additionally, animal models have displayed a survival benefit with the use of iron chelators and amphotericin for mucormycosis which was not demonstrated in humans. Summary: Animal models have been a staple in the development and optimization of antifungal agents. They afford the ability to investigate uncommon diseases, such as invasive fungal infections, that would otherwise take years and many resources to complete. Although there are many benefits of animal models, there are also shortcomings. This is why the reliability of extrapolating data from animal models to humans is often scrutinized. [ABSTRACT FROM AUTHOR]

Although widely used as a preclinical model for studying cardiovascular diseases, there is a scarcity of in vivo hemodynamic measurements of the naïve murine system in multiple arterial and venous locations, from head-to-toe, and across sex and age. The purpose of this study is to quantify cardiovascular hemodynamics in mice at different locations along the vascular tree while evaluating the effects of sex and age. Male and female, adult and aged mice were anesthetized and underwent magnetic resonance imaging. Data were acquired from four co-localized vessel pairs (carotid/jugular, suprarenal and infrarenal aorta/inferior vena cava (IVC), femoral artery/vein) at normothermia (core temperature 37 ± 0.2 °C). Influences of age and sex on average velocity differ by location in arteries. Average arterial velocities, when plotted as a function of distance from the heart, decrease nearly linearly from the suprarenal aorta to the femoral artery (adult and aged males: − 0.33 ± 0.13, R2 = 0.87; − 0.43 ± 0.10, R2 = 0.95; adult and aged females: − 0.23 ± 0.07, R2 = 0.91; − 0.23 ± 0.02, R2 = 0.99). Average velocity of aged males and average volumetric flow of aged males and females tended to be larger compared to adult comparators. With cardiovascular disease as the leading cause of death and with the implications of cardiovascular hemodynamics as important biomarkers for health and disease, this work provides a foundation for sex and age comparisons in pathophysiology by collecting and analyzing hemodynamic data for the healthy murine arterial and venous system from head-to-toe, across sex and age. [ABSTRACT FROM AUTHOR]

Lysozyme is an important component of the innate immune system and has roles in peptidoglycan cleavage of gram-positive organisms. Myeloid cells highly express the isoform, lysozyme M, and its promoter has been used to direct Cre recombinase expression to target deletion of floxed genes in myeloid cells. However, generation of the LysMCre mouse effectively disrupts the LysM gene, and mice homozygous for the Cre allele lack the LysM gene product. To test the contribution of LysM in sterile acute lung injury, we generated LysMCre mice homozygous for the Cre allele (+/+) or wild-type allele (−/−). These mice were challenged with LPS delivered via oropharygneal aspiration. Mice were monitored and weighed daily, and BAL cell counts, differential, protein, and cytokine levels were assessed at days 2 and 4. LysMCre+/+ and LysMCre−/− had similar weight loss and recovery, and similar inflammatory responses to LPS at days 2 and 4. These findings indicate that loss of LysM and expression of Cre recombinase are non-contributory in sterile acute lung injury. [ABSTRACT FROM AUTHOR]

Endometriosis is a chronic inflammatory disease. Dysfunctional regulation of chemokines and chemokine receptors is a crucial aspect of endometriosis pathogenesis. Chemokine G-protein-coupled receptors (GPCRs) are important drug targets that regulate inflammation and immunity. Recently, CXCR7, a C-X-C motif containing GPCR, has been identified as a receptor for chemokine ligand CXCL12, one of the best characterized chemokines for cell trafficking, angiogenesis, and cell proliferation in cancer and inflammation. Here, we investigated the expression and localization of CXCR7 in human endometriosis and a murine model of the disease. Normal endometrial epithelium and stroma showed undetectable or very low expression of CXCR7, without any significant changes across phases of the menstrual cycle in humans. CXCR7 is significantly upregulated in endometriosis, showing higher staining in glands and in associated vessels. The mouse model recapitulated the human findings. In conclusion, overexpression of CXCR7 in different cellular populations of endometriosis microenvironment may play a role in the pathogenesis and represent a novel target for treatment. [ABSTRACT FROM AUTHOR]

Increasing prevalence of Cryptosporidium raises the importance to explore different aspects of its infection. In the absence of reproducible in vitro culturing, animal model is the only experimental method to study Cryptosporidium. Our study evaluated Cryptosporidium infection using coproscopy, copro-antigen and copro-DNA for early detection of murine cryptosporidiosis. Hundred and forty albino mice (neonates and adult) were divided into two groups, control group received sterile PBS solution, and infected groups were inoculated with molecularly characterized Cryptosporidium parvum oocysts and further subdivided into three subgroups for infectious dose response detection. Mice fecal samples were collected every 4 h on the first day and then daily and examined for fecal oocysts, copro-antigen and copro-DNA. Four mice from each subgroup were killed at 12, 24 and 48 h post-infection (P-I), and their intestines were examined for cryptosporidial mucosal DNA. Cryptosporidium copro-antigen and copro-DNA were detected 4 and 8 h P-I in infected neonatal and adult mice, respectively, and intestinal mucosal DNA was detected after 12 h in both. Microscopy was able to detect oocysts 48 h P-I. Inoculated C. parvum oocysts were recovered in feces of infected mice without genotypic changes. Neonate mice showed higher susceptibility for cryptosporidial infection than adults without statistical differences for the given infectious doses. Both copro-immunoassay and copro-nPCR assays can early detect Cryptosporidium infection; however, nPCR was able to identify Cryptosporidium species, making nPCR a reliable biomarker for early detection in murine model. [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]

Background: Asthma is a chronic airway inflammatory disease that has a high prevalence nowadays, and seeking the means of relieving asthmatic symptoms is now an issue with increased importance. While mesenchymal stem cells have been demonstrated to display immunomodulatory effects, the effect of fetus-type mesenchymal stem cells (MSCs) on asthmatic symptoms in vivo have not been reported to date. Methods: Female BALB/c mice at 8 weeks of age were sensitized by ovalbumin, and MSCs derived from Wharton's jelly of human umbilical cord mesenchymal stem cells (hUCMSCs) were injected into the asthmatic mice. Airway hyper-responsiveness, lung eosinophil infiltration, cytokine level in splenocyte cultures and serum immunoglobulin level were measured. Enzyme-linked immunosorbent assay was used to determine cytokine and immunoglobulin levels. Results: This current study demonstrated that hUCMSCs attenuated both lung lymphocyte and eosinophil infiltration, and significantly decreased the concentration of Th2 cytokines interleukin-5 in splenocyte cultures. Conclusions: Human umbilical cord mesenchymal stem cells have the advantage of being easily harvested non-invasively and are capable of rapid proliferation, therefore an ideal material for stem cell-based immune therapies. The current study showed that fetal-type MSCs were able to suppress asthmatic symptoms efficiently, and its immunomodulatory effect resulted primarily from suppressing the Th2 pathway in the animal model. This study suggested that hUCMSCs could be an ideal candidate for cell-based therapies of asthma. [ABSTRACT FROM AUTHOR]

Presbyphagia affects approximately 40 % of otherwise healthy people over 60 years of age. Hence, it is a condition of primary aging rather than a consequence of primary disease. This distinction warrants systematic investigations to understand the causal mechanisms of aging versus disease specifically on the structure and function of the swallowing mechanism. Toward this goal, we have been studying healthy aging C57BL/6 mice (also called B6), the most popular laboratory rodent for biomedical research. The goal of this study was to validate this strain as a model of presbyphagia for translational research purposes. We tested two age groups of B6 mice: young (4-7 months; n = 16) and old (18-21 months; n = 11). Mice underwent a freely behaving videofluoroscopic swallow study (VFSS) protocol developed in our lab. VFSS videos (recorded at 30 frames per second) were analyzed frame-by-frame to quantify 15 swallow metrics. Six of the 15 swallow metrics were significantly different between young and old mice. Compared to young mice, old mice had significantly longer pharyngeal and esophageal transit times ( p = 0.038 and p = 0.022, respectively), swallowed larger boluses ( p = 0.032), and had a significantly higher percentage of ineffective primary esophageal swallows ( p = 0.0405). In addition, lick rate was significantly slower for old mice, measured using tongue cycle rate ( p = 0.0034) and jaw cycle rate ( p = 0.0020). This study provides novel evidence that otherwise healthy aging B6 mice indeed develop age-related changes in swallow function resembling presbyphagia in humans. Specifically, aging B6 mice have a generally slow swallow that spans all stages of swallowing: oral, pharyngeal, and esophageal. The next step is to build upon this foundational work by exploring the responsible mechanisms of presbyphagia in B6 mice. [ABSTRACT FROM AUTHOR]

Vascular smooth muscle cells (VSMCs) play important roles in cardiovascular disorders and biology. Outlined in this paper is a step-by-step procedure for isolating aortic VSMCs from adult C57BL6J male mice by enzymatic digestion of the aorta using collagenase. The plating, culturing, and subculturing of the isolated cells are discussed in detail along with techniques to characterize VSMC phenotype by gene expression and immunofluorescence. Traction force microscopy was used to characterize contractility of single subcultured VSMCs at baseline. [ABSTRACT FROM AUTHOR]

Diet restriction (DR), the implementation of a reduced calorie diet, without starvation, increases lifespan in a number of model organisms including mammals. How DR extends lifespan remains unclear. Genetic studies have shown that life extension is not a universal response to DR; instead, the effects of DR are strain dependent. We previously mapped a quantitative trait locus (QTL) specifying differential response to DR to chromosome 15 in the inbred long-sleep (ILS) × inbred short-sleep (ISS) recombinant inbred panel of mice. This QTL named Fedr2 (fuel efficiency response to DR)-2 modifies body weight (BW) and hair growth (HG) in response to DR. The QTL has been previously mapped using the ISS.Lore5LA (5LA) congenic strain. Here, we have used the reciprocal congenic strain ILS.Lore5SA (5SA) to further investigate Fedr2. The 5LA congenic showed increased ability to maintain BW and HG under DR. For the reciprocal congenic, 5SA, we expected the reciprocal response that the 5SA congenic would have a lesser ability to maintain BW and HG under DR. This expectation was mostly met. The Fedr2S allele conferred lower BW maintenance under DR in both females and males. For females, the difference in BW was significant for the entirety of DR, and for males, the difference became significant at week 17 of DR. For HG, the Fedr2S allele conferred decreased HG ability under DR in males, but not for females. These results highlight the genetic basis for variation in DR response and support the previous observations that Fedr2 mediates BW and HG during DR. [ABSTRACT FROM AUTHOR]

In vitro differentiation of spermatogonial stem cells (SSCs) promotes the understanding of the mechanism of spermatogenesis. The purpose of this study was to isolate spermatogonial stem cell-like cells from murine testicular tissue, which then were induced into haploid germ cells by retinoic acid (RA). The spermatogonial stem cell-like cells were purified and enriched by a two-step plating method based on different adherence velocities of SSCs and somatic cells. Cell colonies were present after culture in M1-medium for 3 days. Through alkaline phosphatase, RT-PCR and indirect immunofluorescence cell analysis, cell colonies were shown to be SSCs. Subsequently, cell colonies of SSCs were cultured in M2-medium containing RA for 2 days. Then the cell colonies of SSCs were again cultured in M1-medium for 6-8 days, RT-PCR and indirect immunofluorescence cell analysis were chosen to detect haploid male germ cells. It could be demonstrated that 10 mol l of RA effectively induced the SSCs into haploid male germ cells in vitro. [ABSTRACT FROM AUTHOR]

Molecular subtypes of glioblastoma (GBM) with distinct alterations have been identified. There is need for reproducible, versatile preclinical models that resemble specific GBM phenotypes to facilitate preclinical testing of novel therapies. We present a cell line-based murine proneural GBM model and characterize its response to radiation therapy. Proneural gliomas were generated by injecting PDGF-IRES-Cre retrovirus into the subcortical white matter of adult mice that harbor floxed tumor suppressors (Pten and p53) and stop-floxed reporters. Primary cell cultures were generated from the retrovirus induced tumors and maintained in vitro for multiple passages. RNA sequencing-based expression profiling of the resulting cell lines was performed. The tumorigenic potential of the cells was assessed by intracranial injection into adult naïve mice from different strains. Tumor growth was assessed by bioluminescence imaging (BLI). BLI for tumor cells and brain slices were obtained and compared to in vivo BLI. Response to whole-brain radiation was assessed in glioma-bearing animals. Intracranial injection of PdgfPtenp53luciferase glioma cells led to formation of GBM-like tumors with 100 % efficiency ( n = 48) and tumorigenesis was retained for more than 3 generations. The cell lines specifically resembled proneural GBM based on expression profiling by RNA-Seq. PdgfPtenp53luciferase cell number correlated with BLI signal. Serial BLI measured tumor growth and correlated with size and location by ex vivo imaging. Moreover, BLI predicted tumor-related mortality with a 93 % risk of death within 5 days following a BLI signal between 1 × 10 and 5 × 10 photons/s cm. BLI signal had transient but significant response following radiotherapy, which corresponded to a modest survival benefit for radiated mice ( p < 0.05). Intracranial injection of PdgfPtenp53luciferase cells constitutes a novel and highly reproducible model, recapitulating key features of human proneural GBM, and can be used to evaluate tumor-growth and response to therapy. [ABSTRACT FROM AUTHOR]

Endothelial cells control vascular tone, permeability and leukocyte transmigration and are modulated by pro-inflammatory mediators. Schistosomiasis is an intravascular disease associated with inflammation, therefore altering endothelial cells' phenotype. Purinergic P2X7 receptors (P2X7R) play an important role in inflammation; however, the impact of the disease upon endothelial P2X7R function or expression has not been explored. Using ethidium bromide uptake to investigate P2X7R function, we observed that the effects of ATP (3 mM) and the P2X7R agonist 3′- O-(4-benzoyl)-ATP (BzATP) were smaller in mesenteric endothelial cells from the Schistosoma mansoni-infected group than in the control group. In the control group, BzATP induced endothelial nitric oxide production, which was blocked by the P2X7R antagonists KN-62 and A740003. However, in the infected group, we observed a reduced effect of BzATP and no effect of both P2X7R antagonists, suggesting a downregulation of endothelial P2X7R in schistosomiasis. We observed similar results in both infected and P2X7R groups, which were also comparable to data obtained with KN-62- or A740004-treated control cells. Data from Western blot and immunocytochemistry assays confirmed the reduced expression of P2X7R in the infected group. In conclusion, our data show a downregulation of P2X7R in schistosomiasis infection, which likely limits the infection-related endothelial damage. [ABSTRACT FROM AUTHOR]

Emerging evidence suggests sex and apolipoprotein E ( APOE) genotype separately modify outcomes after intracerebral hemorrhage (ICH). We test the hypothesis that an interaction exists between sex and APOE polymorphism in modifying outcomes after ICH and is altered by administration of exogenous apoE-mimetic peptide. To define the effects of sex and APOE polymorphism in ICH, we created collagenase-induced ICH in male and female APOETR mice (targeted replacement mice homozygous for APOE3 or APOE4 alleles; n = 12/group) and assessed performance on Rotarod (RR) and Morris water maze (MWM). To evaluate hematoma formation, we used hematoxylin and eosin staining at 24 h after injury ( n = 8/group). Using separate cohorts ( n = 12/group), apoE-mimetic peptide (COG1410 at 2 mg/kg) was administered after ICH, and mice were assessed by RR and MWM. Female mice outperformed male mice via RR and MWM by over 190% improvement through 7 days (RR) and 32 days (MWM) of testing after ICH ( p < 0.01). Female APOE3TR mice demonstrated improved function compared with all other groups ( p < 0.05) without any difference in hematoma volume at 24 h after injury in any group. Administration of a therapeutic apoE-mimetic peptide improved RR latencies through 7 days after ICH in male and female APOE4TR mice and MWM latencies over days 28-32 after ICH in male APOE4TR mice ( p < 0.05). Sex and APOE polymorphism influence functional outcomes in our murine model of ICH. Moreover, administration of exogenous apoE-mimetic peptide after injury differentially modifies the interaction between sex and APOE polymorphism. [ABSTRACT FROM AUTHOR]

The genetic and physiological similarities between mice and humans have focused considerable attention on rodents as potential models of human health and disease. Together with the wealth of resources, knowledge, and technologies surrounding the mouse as a model system, these similarities have propelled this species to the forefront of biomedical research. The advent of genomic manipulation has quickly led to the creation and use of genetically engineered mice as powerful tools for cutting edge studies of human disease research including the discovery, refinement, and utility of many currently available therapeutic regimes. In particular, the creation of genetically modified mice as models of human disease has remarkably changed our ability to understand the molecular mechanisms and cellular pathways underlying disease states. Moreover, the mouse models resulting from gene transfer technologies have been important components correlating an individual's gene expression profile to the development of disease pathologies. The objective of this review is to provide physician-scientists with an expansive historical and logistical overview of the creation of mouse models of human disease through gene transfer technologies. Our expectation is that this will facilitate on-going disease research studies and may initiate new areas of translational research leading to enhanced patient care. [ABSTRACT FROM AUTHOR]

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]

Understanding mechanotransduction pathways leading to thrombosis will require progressive steps, including determination of the mechanical behavior of the platelet membrane in response to applied loads. The platelet membrane deformation capacity, as quantified by membrane progression into a borosilicate glass micropipette of defined internal diameter, was probed in murine platelets using a controlled range of negative pressure (0-7 cm HO). Based on our observations that the platelet portion outside the micropipette was mostly spherical and that the platelet volume did not change upon aspiration, a novel continuum mechanics-based model of the platelet micropipette aspiration experiment was created, and a new hyperelastic isotropic material model including membrane residual tension was proposed for the platelet membrane. Murine platelet membranes maintained an average linear deformation behavior: L/ R = 146,100 p × R + 19.923, where L is the platelet length aspirated in the micropipette (m), R is micropipette radius (m) and p is the aspiration pressure (Pa). The theoretical model was used to generate material constants for the murine platelet membrane that allowed for an accurate simulation of the micropipette aspiration experiments. From published results, another set of material constants was established for the human platelet membrane. Limited cases of platelet lysis upon aspiration were analyzed using the theoretical model to determine preliminary membrane tension strength values. [ABSTRACT FROM AUTHOR]

It has recently been demonstrated that not only mature B lymphocytes, but also non-lymphoid cells, including cancer cells and neurons, express IgG. In the eye, an important immune privileged site, the presence of IgG has been ascribed to IgG entering the eye through breaches of the blood-ocular barrier. Here we demonstrate that the eye itself can produce IgG intrinsically. Applying immunohistochemistry, in situ hybridization, and RT-PCR, several intraocular structures were found to express proteins and mRNA transcripts of IgG heavy chains, light chains, V(D)J rearrangements, and enzymes required for V(D)J recombination. IgG receptors were also detected in the intraocular epithelium and endothelium. The extensive distribution of IgG and its receptors in intraocular structures indicates that locally produced IgG could play a significant role in maintaining the ocular microenvironment and protection of the eyes, and it might also be involved in the pathogenesis of age-related macular degeneration and some inflammatory diseases. [ABSTRACT FROM AUTHOR]

Both the incidence of invasive fungal infections and the number of antifungal agents available to clinicians have expanded significantly over the past two decades. Successes with pharmacokinetic and pharmacodynamic evaluations of other antimicrobial agents in animal models and their clinical correlations with patient outcomes have led to an increased number of studies evaluating both old and new antifungal agents. Recently, animal models have successfully defined target pharmacodynamic indices for many antifungal agents and fungal infections, but validation of these targets in human studies is frequently lacking. This article evaluates the potential pros and cons of extrapolating to humans the animal data on antifungal pharmacodynamics. [ABSTRACT FROM AUTHOR]

Human multipotent mesenchymal stromal cells (MSCs) exhibit multilineage differentiation potential, support hematopoiesis, and inhibit proliferation and effector function of various immune cells. On the basis of these properties, MSC are currently under clinical investigation in a range of therapeutic applications including tissue repair and immune-mediated disorders such as graft-versus-host-disease refractory to pharmacological immunosuppression. Although initial clinical results appear promising, there are significant concerns that application of MSC might inadvertently suppress antimicrobial immunity with an increased risk of infection. We demonstrate here that on stimulation with inflammatory cytokines human MSC exhibit broad-spectrum antimicrobial effector function directed against a range of clinically relevant bacteria, protozoal parasites and viruses. Moreover, we identify the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) as the underlying molecular mechanism. We furthermore delineate significant differences between human and murine MSC in that murine MSC fail to express IDO and inhibit bacterial growth. Conversely, only murine but not human MSC express inducible nitric oxide synthase on cytokine stimulation thus challenging the validity of murine in vivo models for the preclinical evaluation of human MSC. Collectively, our data identify human MSC as a cellular immunosuppressant that concurrently exhibits potent antimicrobial effector function thus encouraging their further evaluation in clinical trials. [ABSTRACT FROM AUTHOR]

Objective: To investigate liver damage and abscess formation in murine, secondary peritonitis. Subjects: Male C57BL/6 mice. Treatment: Intraperitoneal injection with 10 CFU Klebsiella pneumoniae and treatment with gentamicin 5 mg/kg/day (BID), subcutaneously. Methods: Animals were killed at 12, 24, 48 and 72 h after infection. Bacterial burden was determined in the blood and the liver. Liver abscess formation was assessed macroscopically and microscopically. Plasma levels of alkaline phosphatase (ALP) and alanine transaminase (ALT) were measured. Polymorphonuclear leukocyte (PMN) accumulation was assessed via tissue myeloperoxidase (MPO) concentrations. Liver interleukin-10 (IL-10) levels were determined by ELISA. Results: K. pneumoniae was detectable in the blood and the liver at 12 h after infection. Liver abscess formation was visible earliest at 24 h after infection and most pronounced within the right liver lobes. ALP and ALT levels peaked at 12 and 24 h after infection, respectively. MPO was elevated in the right and left liver lobes at 12 h but only in the right lobes at 48 h after infection, compared to tissue levels in naïve mice. Liver IL-10 concentrations were not significantly increased. Conclusion: Peritonitis led to liver injury and abscess formation but did not significantly affect tissue concentrations of anti-inflammatory IL-10. [ABSTRACT FROM AUTHOR]

Diffuse malignant mesothelioma is an aggressive tumor which displays a median survival of 11.2 months and a 5-year survival of less than 5% emphasizing the need for more effective treatments. This study uses an orthotopic model of malignant mesothelioma established in syngeneic, immunocompetent C57Bl/6 mice which produce malignant ascites and solid tumors that accurately replicate the histopathology of the human disease. Host stromal and immune cell accumulation within malignant ascites and solid tumors was determined using immunofluorescent labeling with confocal microscopy and fluorescence-activated cell sorting. An expression profile of cytokines and chemokines was produced using quantitative real-time PCR arrays. Tumor spheroids and solid tumors show progressive growth and infiltration with host stromal and immune cells including macrophages, endothelial cells, CD4 and CD8 lymphocytes, and a novel cell type, myeloid derived suppressor cells (MDSCs). The kinetics of host cell accumulation and inflammatory mediator expression within the tumor ascites divides tumor progression into two distinct phases. The first phase is characterized by progressive macrophage and T lymphocyte recruitment, with a cytokine profile consistent with regulatory T lymphocytes differentiation and suppression of T cell function. The second phase is characterized by decreased expression of macrophage chemotactic and T-cell regulating factors, an increase in MDSCs, and increased expression of several cytokines which stimulate differentiation of MDSCs. This cellular and expression profile suggests a mechanism by which host immune cells promote diffuse malignant mesothelioma progression. [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]

Purpose: Systemic toxicity coupled with long treatment regimes of approved topical chemotherapeutic agents such as imiquimod and 5-fluorouracil (5-FU) are limiting. There is now more focus on the potential use of topical terpene agents as skin cancer treatments. Here, we show for the first time that topical Melaleuca alternifolia (tea tree) oil (TTO), abundant in terpenes, has in vivo antitumour activity. Method: Topical TTO formulations applied to immunocompetent tumour-bearing mice were assessed for antitumour efficacy by monitoring tumour growth and by histological analysis following treatment. Results: Four, daily, topical treatments of 10% TTO/DMSO regressed subcutaneous AE17 mesotheliomas in mice for a period of 10 days and significantly retarded the growth of subcutaneous B16-F10 melanomas. The antitumour effect of topical 10% TTO/DMSO was accompanied by skin irritation similar to other topical chemotherapeutic agents, but unlike other approved topical agents, quickly and completely resolved. Furthermore, we show that topical 10% TTO/DMSO caused an influx of neutrophils and other immune effector cells in the treated area, with no evidence of systemic toxicity. Conclusion: TTO combined with an effective carrier significantly inhibited the growth of aggressive, subcutaneous, chemo-resistant tumours in immunocompetent mice. Taken together, these findings highlight the potential of topical TTO as an alternative topical antitumour treatment. [ABSTRACT FROM AUTHOR]

Mammographic density (MD) is the area of breast tissue that appears radiologically white on mammography. Although high MD is a strong risk factor for breast cancer, independent of BRCA1/2 mutation status, the molecular basis of high MD and its associated breast cancer risk is poorly understood. MD studies will benefit from an animal model, where hormonal, gene and drug perturbations on MD can be measured in a preclinical context. High and low MD tissues were selectively sampled by stereotactic biopsy from operative specimens of high-risk women undergoing prophylactic mastectomy. The high and low MD tissues were transferred into separate vascularised biochambers in the groins of SCID mice. Chamber material was harvested after 6 weeks for histological analyses and immunohistochemistry for cytokeratins, vimentin and a human-specific mitochondrial antigen. Within-individual analysis was performed in replicate mice, eliminating confounding by age, body mass index and process-related factors, and comparisons were made to the parental human tissue. Maintenance of differential MD post-propagation was assessed radiographically. Immunohistochemical staining confirmed the preservation of human glandular and stromal components in the murine biochambers, with maintenance of radiographic MD differential. Propagated high MD regions had higher stromal ( p = 0.0002) and lower adipose ( p = 0.0006) composition, reflecting the findings in the original human breast tissue, although glands appeared small and non-complex in both high and low MD groups. No significant differences were observed in glandular area ( p = 0.4) or count ( p = 0.4) between high and low MD biochamber tissues. Human mammary glandular and stromal tissues were viably maintained in murine biochambers, with preservation of differential radiographic density and histological features. Our study provides a murine model for future studies into the biomolecular basis of MD as a risk factor for breast cancer. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells contribute to the graft-versus-leukemia effect after allogeneic stem cell transplantation. However, the efficacy of NK cell-mediated tumor cell lysis is limited due to target cell resistance, and target cell-induced apoptosis (TiA) was proposed to contribute to differences in susceptibility to NK cells. Here we analyzed the effects of target cells on the apoptosis of cytokine-activated NK cells in vitro. We found no association of target cell susceptibility and TiA of NK cells in an array of human and murine target-effector cell combinations. Incubation of NK cells with caspase inhibitors blocked TiA incompletely, indicating that TiA is partly based on caspase-independent mechanisms. Modulating NK cell susceptibility against TiA by caspase inhibition did not influence cytotoxic efficacy. Furthermore, we found cytotoxic potential of NK cells to be markedly decreased following first target cell contact. Exhaustion of NK cell activity by first target cell contact was, however, not mediated by TiA. In addition, we found no relevant TiA by lymphoma cell lines against activated murine NK cells. We conclude that TiA represents only a minor factor of target cell resistance against NK cell-mediated cytolysis. [ABSTRACT FROM AUTHOR]

We investigated the activity of caspofungin against a Candida tropicalis clinical isolate showing paradoxical growth in vitro. BALB/c mice immunosuppressed by cyclophosphamide were infected intraperitoneally using 107 CFU/mouse. Caspofungin was administered intraperitoneally once daily for 5 days or as a single dose using the following doses: 0.12, 0.25, 1, 2, 3, 5, and 15 mg/kg. The single dose of caspofungin was effective only at 5 and 15 mg/kg concentrations (100% survival). Five-day caspofungin treatment led to 100% survival at doses of 1 mg/kg or higher. Caspofungin treatment significantly decreased the number of viable yeasts in the peritoneal lavage samples as well as in the infected abscesses at doses 1, 3, 5, and 15 mg/kg caspofungin as compared to the untreated control ( P<0.001 in all cases), and even to the group treated with 0.12 mg/kg caspofungin ( P<0.05 in all cases). At 2 mg/kg caspofungin dose, sterilization of the internal organs was reproducibly incomplete, suggesting that the role of paradoxical growth in the late clinical failure cannot be excluded. [ABSTRACT FROM AUTHOR]

Brain iron homeostasis is maintained by a balance of both iron uptake and release, and accumulating evidence has revealed that brain iron concentrations increase with aging. Hepcidin, an iron regulatory hormone produced by hepatocytes in response to inflammatory stimuli, iron, and hypoxia, has been shown to be the long-sought hormone responsible for the regulation of body iron balance and recycling in mammals. In this study, we report that hepcidin is widely expressed in the murine brain. In cerebral cortex, hippocampus and striatum, hepcidin mRNA levels increased with aging. Injection of hepcidin into the lateral cerebral ventricle resulted in decreased Fpn1 protein levels in cerebral cortex, hippocampus, and striatum. Additionally, treatment of primary cultured neurons with hepcidin caused decreased neuronal iron release and Fpn1 protein levels. Together, our data provide further evidence that hepcidin may be involved in the regulation of brain iron metabolism. [ABSTRACT FROM AUTHOR]

This study characterized bone structure, composition, and mechanical properties in growing male mice. The development of the collagen network during maturation was monitored, and the effect of voluntary physical exercise was investigated. We hypothesized that increased bone loading from exercise would increase the amount and improve the properties of the collagen network during growth and maturation. Half of the mice (total n = 168) had access to running wheels, while half were kept sedentary. Weight and running activity were recorded, and groups of mice were killed at 1, 2, 4, and 6 months of age. The collagen network was assessed by biochemical evaluation of collagen content and cross-links and by tensile testing of decalcified bone. Mineralized femur was analyzed with pQCT and three-point-bending and femoral neck–strength tests. After 6 months, the exercising mice had 10% lower body weight than the sedentary group. There was no difference in the amount of collagen or collagen cross-links, while tensile testing had higher breaking force and stiffness of the collagen network in runners after 4 months but not after 6 months. The bone mineral density and cross-sectional area were higher in the running group after 6 months. Runners also showed higher breaking force and stiffness of the diaphysis and the femoral neck at 2 and 6 months. The significant modulation of mechanical properties of the collagen network without any change in collagen content indicates that physical exercise improves properties of the collagen network in maturing bone. The improvement after exercise of the properties of mineralized bone appears to be more pronounced and long-lasting compared to the early improved properties of the collagen network. [ABSTRACT FROM AUTHOR]

Protein p0071, which originally was introduced as a member of the p120-subfamily of armadillo proteins, common to desmosomes and adhaerens junctions (AJs) and to several other cell structures (centrosomes, midbodies), has been localized by using a series of novel mono- and polyclonal antibodies generated against various domains of the molecule. By protein analysis and immunolocalization techniques, protein p0071 has been localized as a plaque protein in AJs of diverse epithelia and certain vascular endothelia, in the composite junctions ( areal compositae) of the intercalated disks of cardiomyocytes, and in the punctate or more extended AJs of the vast majority of cell culture types examined, including mitotic states. Using these antibodies, we have also shown that this AJ protein occurs only rarely or is even absent in tissues such as skeletal and smooth muscles, in a series of mesenchymal tissue cells, and in specific desmosome-rich cells such as those of the upper layers of the epidermis and certain other stratified epithelia and Hassall corpuscles of the thymus. We have also demonstrated that p0071 is absent from desmosomes. The occurrence of two major subtypes of lymphatic endothelial cells, one with AJs containing p0071 and one without detectable p0071, is emphasized. Possible structural and functional roles of p0071 are discussed in light of these new findings regarding its localization, and the addition of p0071 to the armamentarium of cytodiagnostic cell-type markers is recommended. [ABSTRACT FROM AUTHOR]

To explore whether the graft-versus-leukemia (GVL) effects could be enhanced and acute graft-versus-host disease (aGVHD) could be relieved by syngeneic bone marrow mixed with G-CSF-primed H-2 haploidentical marrow grafting. Female L615 (H-2k) mice were recipient mice and male (615 × BALB/c) F1 (6BF1) (H-2k × H-2d) mice were donors respectively. Donor mice were injected subcutaneously with G-CSF daily at 0.01 μg/g for 6 days, and splenocytes were harvested on day 7. A total of 615 mice were loaded with L615 tumor cells and received 8.5 Gy (60Co γ-ray) irradiation three days later, followed by a mixed bone marrow transplantation (MBMT). The allo-grafts consisted of a mixture of syngenetic plus G-CSF-mobilized (control diluents) H-2 haploidetical marrow cells. GVL effects were monitored by survival time and survival rate of recipient mice. GVHD was assessed by clinical signs of weight loss, ruffled fur, diarrhea and histological changes of skin, liver and small intestines. Allogeneic chimerism was detected using cytogenetic analysis. Enzyme-linked immunosorbent assay (ELISA) method was used to detect cytokines (IL-2, IL-4 and IFN-γ). Fluorescence-activated cell sorting (FACS) analysis was used to detect T-cell phenotype. (1) The mice merely received L615 leukemia cells were all died of leukemia. The L615 mice of 3:1 and 4:1 MBMT groups survived longer than those post syngeneic BMT ( P < 0.01). (2) The survival time of mice in the G-CSF-treated MBMT groups was longer than that of non-primed MBMT groups ( P < 0.05). Administration of G-CSF-treated 6BF1 mice could markedly increase the survival rate of 3:1 and 4:1 MBMT mice ( P < 0.01) with little or a little GVHD. (3) As the post-transplanted time prolonged, the rates of allogeneic chimerism were decreased. The chimerism rates decreased to zero when the mice died of leukemia relapse. (4) L3T4+ cells and relative ratio in both subsets were significantly reduced in G-CSF-treated donor mice. After G-CSF-treated donors, splenocytes from recipients at day 14 post-MBMT showed an increased production of IL-4 and a decreased production of IL-2 and IFN-γ. Syngeneic bone marrow mixed with H-2 haploidentical marrow grafts had a potential way to increase GVL effects, and this GVL effects could be enhanced with little or a little GVHD by G-CSF pretreatment of donors. Improved survival in recipients of G-CSF-mobilized donors is associated with increased IL-4 production and decreased IL-2 and IFN-γ production. [ABSTRACT FROM AUTHOR]

Tumor protein D52 (TPD52) is involved in transformation and metastasis and has been shown to be over-expressed in tumor cells compared to normal cells and tissues. Murine TPD52 (mD52) shares 86% protein identity with the human TPD52 orthologue (hD52). To study TPD52 protein as a target for active vaccination recombinant, mD52 was administered as a protein-based vaccine. Naïve mice were immunized with either mD52 protein and CpG/ODN as a molecular adjuvant or CpG/ODN alone. Two weeks following the final immunization, mice were challenged s.c. with syngeneic tumor cells that over-express mD52. Two distinct murine tumor cell lines were used for challenge in this model, mKSA and 3T3.mD52. Half of the mice immunized with mD52 and CpG/ODN rejected or delayed onset of mKSA s.c. tumor cell growth, and 40% of mice challenged with 3T3.mD52 rejected s.c. tumor growth, as well as the formation of spontaneous lethal lung metastases. Mice immunized with mD52 and CpG/ODN generated detectable mD52-specific IgG antibody responses indicating that mD52 protein vaccination induced an adaptive immune response. In addition, mice that rejected tumor challenge generated tumor-specific cytotoxic T lymphocytes’ responses. Importantly, microscopic and gross evaluation of organs from mD52 immunized mice revealed no evidence of autoimmunity as assessed by absence of T cell infiltration and absence of microscopic pathology. Together, these data demonstrate that mD52 vaccination induces an immune response that is capable of rejecting tumors that over-express mD52 without the induction of harmful autoimmunity. [ABSTRACT FROM AUTHOR]

Many hematological diseases require long-term transfusion support, which causes production of donor-reactive antibodies in sensitized recipients. Sensitized patients are at an increased risk for graft rejection when they undergo allogeneic hematopoietic stem cell transplantation (allo-HSCT). Here, we established a highly sensitized murine model to investigate the mechanism of donor graft rejection. After BALB/c mice were repeatedly transfused with allogeneic spleen cells from C57BL/6 mice, there was a significant increase in complement-dependent cytotoxicity in the serum of sensitized mice. For transplantation, 1 × 107 bone marrow cells (BMCs) from C57BL/6 mice were injected into lethally irradiated recipient BALB/c mice. Sensitized mice died between 12 and 15 days post-transplantation, while non-sensitized mice remained alive after 28 days. The hematopoietic recovery rate declined over time in sensitized recipients. The homing trace assay showed a rapid disappearance of donor BMCs in the spleen and bone marrow of sensitized recipients. In addition, the recipient cells and antibodies in the sensitized serum were capable of inducing high level of cell- and complement-mediated cytotoxicity to the donor graft. Our finding may explain the impaired hematopoietic stem cell homing and poor hematopoietic engraftment observed in highly sensitized allo-HSCT patients.Bone Marrow Transplantation (2007) 40, 691–698; doi:10.1038/sj.bmt.1705791; published online 30 July 2007 [ABSTRACT FROM AUTHOR]

In several mammalian species, the retina is capable of synthesizing melatonin and contains an autonomous circadian clock that relies on interlocking transcriptional/translational feedback loops involving several clock genes, such as Per1 and Cry2. Our previous investigations have shown remarkable differences in retinae of melatonin-deficient (C57BL) and melatonin-proficient (C3H) mice with regard to the protein levels of PER1, CRY2, and phosphorylated (p) cyclic AMP response element binding protein (CREB). To elucidate the melatonin receptor type possibly responsible for these differences, we have performed immunocytochemical analyses for PER1, CRY2, and pCREB in retinae of melatonin-proficient wild type (WT) mice and mice with targeted deletions of the MT1 receptor (MelaaBB) or the MT1 and MT2 receptors (Melaabb) at four different time points. Immunoreactions for PER1, CRY2 and pCREB were localized to the nuclei of cells in the inner nuclear layer (INL) and ganglion cell layer (GC) of all strains. Surprisingly, in MelaaBB and Melaabb the day/night rhythm of pCREB, PER1, and CRY2 levels was not abolished, but the maxima and minima of PER1 were 180° out of phase as compared to the WT. These data suggest that MT1 and MT2 melatonin receptors are not necessary to maintain rhythmic changes in clock-gene protein levels in the murine retina, but, as shown for PER1, appear to be involved in internal synchronization. [ABSTRACT FROM AUTHOR]

Targeted expression of interleukin-10 (IL-10) has been proposed as a means to suppress acute and chronic inflammation. We explored the capacity of targeted adenoviral expression of human or viral IL-10 to improve outcome in a zymosan-induced model of acute lung injury and multisystem organ failure. Intratracheal administration of adenovirus expressing either human or viral IL-10 prior to zymosan administration significantly improved survival at a dose of 107 particles (P<0.01), whereas the same recombinant vectors were ineffective at 108 particles and increased mortality at 109 particles. Improved survival after administration of 107 particles of adenovirus expressing viral or human IL-10 was associated with local tissue expression of IL-10 (100–300 pg/g wet wt). In contrast, mortality after administration of 109 particles was associated with markedly elevated IL-10 expression, both in the lung (10 000–70 000 pg/g wet wt) and systemically (1000–3000 pg/ml plasma), with evidence of an exaggerated systemic inflammatory response (plasma IL-6 and TNFα). Targeted gene expression of IL-10 can be used to treat acute inflammatory processes, but increased doses resulting in its systemic release are not associated with improvements in outcome, and may actually exacerbate acute inflammatory processes.Gene Therapy (2006) 13, 276–282. doi:10.1038/sj.gt.3302600; published online 27 October 2005 [ABSTRACT FROM AUTHOR]

Background: Experimental models of colorectal tumor require either laparotomy for induction or anastomosis following resection. The long murine cecum avoids the need for an anastomosis, making the cecum the preferred site for induction. This study aimed to evaluate total colonoscopy with submucosal injection of cecal wall (TCWI) in rats in terms of failure rate (FR), complication rate (CR), and reproducibility (R).Methods: A bolus of bowel prep was given. Anesthesia was injected intraperitoneally. A video fiberscope (5.9 mm outer diameter, 180/90 degrees up/down bending, 100/100 degrees right/left bending, 103 cm working length, 120 degrees view field, and 2.0 mm channel) allowed for irrigation and suction. Saline 1 ml was injected in the cecal wall through a 4-mm-long, 23-gauge needle placed on a 3-mm wire, resulting in a blister. FR was a failure to reach and inject the cecum. Rats were allowed to recover. CR was measured at necropsy. R was assessed by comparing TCWI time, FR, and CR for three investigators. Sample size of 120 (type I error, 0.05; power, 80%) was based on a pilot study. Data are presented as median (range).Results: A total of two of 122 rats (1.6%) died after prep or anesthesia. Bowel prep resulted in 99.1% evacuation of solid feces. A total of 120 male Sprague-Dawley retired breeders weighing 592 g (range, 349-780) underwent TCWI. Scope depth was 28 cm (range, 20-36). Irrigating fluid was 290 ml (range, 100-600). TCWI time was 7 min (range, 4-28). FR was 4%. In three failed cases, the scope reached the ascending colon. CR was 2%. There were two perforations in the ascending colon. All three operators had similar TCWI time (p = 0.673), FR (p > 0.1), and CR (p > 0.1). A total of 98.3% of rats survived to planned sacrifice. At 48-h necropsy, the injection site was macroscopically identified in 118 rats.Conclusions: A safe and reproducible TCWI rat model has been achieved, which may provide a valuable tool in the future for studies of solid colorectal tumors. [ABSTRACT FROM AUTHOR]

Abdominal sepsis due to secondary fecal peritonitis following anastomosis insufficiency is a rare but life threatening complication of colorectal surgery. The induction of IFN-γ by IL-12 is believed to play a key role in sepsis as it promotes antibacterial effector mechanisms such as oxidative burst or nitric oxide induction. The impact of gene deficiency for IL-12 (IL-12p40 KO), oxidative burst (p47phox KO), or NO induction (iNOS KO) on the outcome of fecal peritonitis was characterized using the murine Colon Ascendens Stent Peritonitis model (CASP). In the IL-12p40 KO model, 3 and 12 h after surgery, serum cytokine levels of IL-1β, TNF, IL-18, and IL-10 were analyzed. Expression of IL-1β, IL-10, IP-10, and MIP-1α was measured in lung and liver by RNAse Protection Assay. IL-12p40 and iNOS-deficient mice exhibited a significantly higher susceptibility to CASP as compared to the controls, whereas no significant difference was observed in p47phox KO mice. Absence of IL-12 resulted in delayed expression of proinflammatory cytokines and chemokines in both the liver and the lung, and was associated with significant reduction of IL-1β levels in the serum 12 h after CASP. IL-12 and iNOS possess protective functions in fecal murine peritonitis. Surprisingly, no significant contribution of oxidative burst to the immune response was observed. Overall, these findings suggest that IL-12 deficiency causes a profound delay of the immune response after polymicrobial challenge resulting in significantly increased susceptibility in the CASP model. [ABSTRACT FROM AUTHOR]

Line-1, a weakly immunogenic lung tumor cell line derived from the BALB/c mouse, metastasizes spontaneously to the lungs of mice following subcutaneous administration. The parameters that influence metastasis as well as the progression of metastatic lung disease following surgical resection of primary subcutaneous tumors were characterized. Histological analysis of the lungs obtained from mice bearing different size subcutaneous tumors demonstrated that >90% of the mice developed micrometastatic disease in the lungs when the tumor exceeded 650 mm3 in size. Surgical resection of subcutaneous tumors resulted in the cure of primary disease in 95% of the mice. Macroscopic tumor nodules were grossly visible in the lungs of 75% of the mice 5 weeks after surgery. Serum amyloid A level correlated with primary tumor burden and was diagnostic for the presence of metastatic disease. The efficiency of metastasis, post-surgical primary tumor recurrence and long-term survival were significantly different between BALB/c mice obtained from different suppliers. The Line-1-BALB/c surgical metastasis model provides a clinically relevant tool for the evaluation of anti-cancer therapies, especially those that are designed to target long-term suppression of minimal residual disease following surgical intervention. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are important for their ability to recognize and lyse tumor cells and virus infected cells. NK cells express triggering receptors that are specific for non-MHC ligands. This article describes the 35S release cytotoxic assay, which measures the ability of NK cells derived from spleen cells taken from polyIC-treated mice to lyse B-cell leukemia (BCL1) cells. BCL1 cells express ligands for NKp46 on the cell surface membrane and they are sensitive to allogeneic but not syngeneic IL-2 activated natural killer cells. [ABSTRACT FROM AUTHOR]