Murine Strains Research Articles

Analysis of 24-h recovery of transfused stored RBCs in recipient mice of distinct genetic backgrounds.

Human studies have demonstrated substantial donor-to-donor variation in refrigerated RBC storage with respect to several variables, including 24-h post-transfusion RBC recovery. However, the human studies leading to these observations are mostly performed using autologous transfusions of stored RBCs, thereby avoiding issues of infectious disease transmission and alloimmunization. Accordingly, one cannot distinguish whether variability in 24-h RBC recovery is due to alterations in RBC storage, differences in phagocytic activity of the recipient's reticuloendothelial system or both. Similar to humans, genetically distinct inbred mouse strains have substantial differences in RBC storage biology, including 24-h post-transfusion RBC recovery. In this report, we juxtaposed 24-h recoveries in 15 distinct inbred strains of mice, holding the RBC donor constant to isolate transfusion recipient variation as an independent variable. Strains were chosen for differences in baseline reticulocyte count and haemoglobin, which may correlate to RBC life span and turnover. Unlike large differences observed in storage of RBCs obtained from different strains of mice, only subtle strain-to-strain differences were observed regarding 24-h post-transfusion RBC recoveries. These findings indicate that the murine strains examined are not likely to be useful in sorting out mechanisms of clearance of stored RBCs, and suggest that such mechanisms may be generally conserved in the strains of mice analysed.

Improved biomechanical and biological outcomes in the MRL/MpJ murine strain following a full-length patellar tendon injury.

Musculoskeletal injuries greatly affect the U.S. population and current clinical approaches fail to restore long-term native tissue structure and function. Tissue engineering is a strategy advocated to improve tendon healing; however, the field still needs to establish biological benchmarks for assessing the effectiveness of tissue-engineered structures. Investigating superior healing models, such as the MRL/MpJ, offers the opportunity to first characterize successful healing and then apply experimental findings to tissue-engineered therapies. This study seeks to evaluate the MRL/MpJ's healing response following a central patellar tendon injury compared to wildtype. Gene expression and histology were assessed at 3, 7, and 14 days following injury and mechanical properties were measured at 2, 5, and 8 weeks. Native patellar tendon biological and mechanical properties were not different between strains. Following injury, the MRL/MpJ displayed increased mechanical properties between 5 and 8 weeks; however, early tenogenic expression patterns were not different between the strains. Furthermore, expression of the cyclin-dependent kinase inhibitor, p21, was not different between strains, suggesting an alternative mechanism may be driving the healing response. Future studies will investigate collagen structure and alignment of the repair tissue and characterize the complete healing transcriptome to identify mechanisms driving the MRL/MpJ response.

Animal models of lupus and lupus nephritis.

This article reviews the commonly used murine strains for studying lupus and lupus nephritis, including strains that develop lupus spontaneously, congenic strains, induced models of lupus, as well as genetically engineered mouse models of lupus bearing transgenes or knockouts. The review then summarizes the main cellular and molecular pathways that lead to the pathogenesis of this autoimmune disease, including autoantibodies. Finally, it concludes with therapeutic insights gained from using mouse models of lupus. To sum, much of what we have learned about lupus has arisen from studying mouse models of the disease, and the laboratory mouse continues to be one of the best tools for studying human SLE.

CD201 and CD27 identify hematopoietic stem and progenitor cells across multiple murine strains independently of Kit and Sca-1

Identification and isolation of hematopoietic stem cells (HSCs) in mice is most commonly based on the expression of surface molecules Kit and Sca-1 and the absence of markers of mature lineages. However, Sca-1 is absent or weakly expressed in hematopoietic progenitors in many strains, including nonobese diabetic (NOD), BALB/c, C3H, and CBA mice. In addition, both Kit and Sca-1 levels are modulated following bone marrow injury. In these cases, other markers and dye exclusion methods have been employed to identify HSCs, yet there is no antibody-based stain that enables identification of HSCs and early progenitors when Kit and Sca-1 are inadequate. CD201 is a marker that is highly restricted to HSCs and progenitors, and CD27 is expressed at moderate-to-high levels on HSCs. We show here that combining CD201 and CD27 enables highly efficient isolation of long-term HSCs in NOD mice as well as in other strains, including SJL, FVB, AKR, BALB/c, C3H, and CBA. We also find that HSCs appear to maintain expression of CD201 and CD27 after hematopoietic injury when Kit expression is downregulated. These results suggest a widely applicable yet simple alternative for HSC isolation in settings where Kit and Sca-1 expression are insufficient.

Structural determinants of phenotypic diversity and replication rate of human prions.

The infectious pathogen responsible for prion diseases is the misfolded, aggregated form of the prion protein, PrPSc. In contrast to recent progress in studies of laboratory rodent-adapted prions, current understanding of the molecular basis of human prion diseases and, especially, their vast phenotypic diversity is very limited. Here, we have purified proteinase resistant PrPSc aggregates from two major phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD), determined their conformational stability and replication tempo in vitro, as well as characterized structural organization using recently emerged approaches based on hydrogen/deuterium (H/D) exchange coupled with mass spectrometry. Our data clearly demonstrate that these phenotypically distant prions differ in a major way with regard to their structural organization, both at the level of the polypeptide backbone (as indicated by backbone amide H/D exchange data) as well as the quaternary packing arrangements (as indicated by H/D exchange kinetics for histidine side chains). Furthermore, these data indicate that, in contrast to previous observations on yeast and some murine prion strains, the replication rate of sCJD prions is primarily determined not by conformational stability but by specific structural features that control the growth rate of prion protein aggregates.

Correlates of Renal Atrophy in Murine 2 Kidney 1 Clip Hypertension

Renal artery stenosis (RAS) is a common disorder associated with both cardiovascular and renal morbidity and mortality. Recent studies have shown that stenting of the stenotic kidney fails to improve renal or cardiovascular outcomes, prompting a search for more effective therapies. Strain‐specific variability in renal outcomes is a well recognized problem associated with murine models of chronic injury. The objective of this study was to define strain specific differences in renal outcome and correlation with parameters of injury in a murine model of RAS.We used a 0.25 mm polytetrafluoroethylene cuff to establish RAS in mice (n=292 total). We examined the correlation between age, time following surgery, body weight, blood pressure, heart and kidney weights, ratio of STK/CLK weight, and plasma angiotensin level in 3 murine strains. Percent atrophy in the STK as a function of time following RAS surgery is summarized in the table below: Strain 1 Week 2 Week 4 Week 6 Week 129 21% 31% XX 45% C57BLKS/J 25% 51% 63% 72% C57BL/6J XX XX 39% 41% The right kidney weight and the STK/CLK weight ratio correlated with %atrophy in all strains, with the strongest correlation observed in C57BL/6J mice. In C57BL/6J mice, blood pressure correlated with %atrophy. CLK weight correlated with heart weight, with the strongest correlation observed in 129Sv mice. In BLKS mice, the time following surgery and age correlated with CLK weight and %atrophy in the STK; blood pressure correlated with plasma angiotensin level.We conclude that RAS produces severe chronic renal injury in the STK of all strains studied, with the most severe injury observed in C57BLKS/J mice.The STK/CLK weight ratio correlated most strongly with %atrophy and can be used to predict development of chronic renal damage in all strains. Hypertrophy of the CLK, as measured by CLK weight, strongly correlated with heart weight.

Coincidental loss of DOCK8 function in NLRP10-deficient and C3H/HeJ mice results in defective dendritic cell migration

Dendritic cells (DCs) are the primary leukocytes responsible for priming T cells. To find and activate naïve T cells, DCs must migrate to lymph nodes, yet the cellular programs responsible for this key step remain unclear. DC migration to lymph nodes and the subsequent T-cell response are disrupted in a mouse we recently described lacking the NOD-like receptor NLRP10 (NLR family, pyrin domain containing 10); however, the mechanism by which this pattern recognition receptor governs DC migration remained unknown. Using a proteomic approach, we discovered that DCs from Nlrp10 knockout mice lack the guanine nucleotide exchange factor DOCK8 (dedicator of cytokinesis 8), which regulates cytoskeleton dynamics in multiple leukocyte populations; in humans, loss-of-function mutations in Dock8 result in severe immunodeficiency. Surprisingly, Nlrp10 knockout mice crossed to other backgrounds had normal DOCK8 expression. This suggested that the original Nlrp10 knockout strain harbored an unexpected mutation in Dock8, which was confirmed using whole-exome sequencing. Consistent with our original report, NLRP3 inflammasome activation remained unaltered in NLRP10-deficient DCs even after restoring DOCK8 function; however, these DCs recovered the ability to migrate. Isolated loss of DOCK8 via targeted deletion confirmed its absolute requirement for DC migration. Because mutations in Dock genes have been discovered in other mouse lines, we analyzed the diversity of Dock8 across different murine strains and found that C3H/HeJ mice also harbor a Dock8 mutation that partially impairs DC migration. We conclude that DOCK8 is an important regulator of DC migration during an immune response and is prone to mutations that disrupt its crucial function.

Murine cytomegalovirus strains co-replicate at multiple tissue sites and establish co-persistence in salivary glands in the absence of Ly49H-mediated competition.

Infection with multiple genetically distinct strains of pathogen is common and can lead to positive (complementation) or negative (competitive) within-host interactions. These interactions can alter aspects of the disease process and help shape pathogen evolution. Infection of the host with multiple strains of cytomegalovirus (CMV) occurs frequently in humans and mice. Profound, NK-cell-mediated (apparent) competition has been identified in C57BL/6 mice, and prevented the replication and shedding of certain co-infecting CMV strains. However, the frequency of such strong competition has not been established. Other within-host interactions such as complementation or alternative forms of competition remain possible. Moreover, high rates of recombination in both human CMV and murine CMV (MCMV) suggest prolonged periods of viral co-replication, rather than strong competitive suppression. An established model was employed to investigate the different possible outcomes of multi-strain infection in other mouse strains. In this study, co-replication of up to four strains of MCMV in the spleen, liver and salivary glands was observed in both MCMV-susceptible and MCMV-resistant mice. In the absence of apparent competition, no other forms of competition were unmasked. In addition, no evidence of complementation between viral strains was observed. Importantly, co-replication of MCMV strains was apparent for up to 90 days in the salivary glands. These data indicated that competition was not the default outcome of multi-strain CMV infection. Prolonged, essentially neutral, co-replication may be the norm, allowing for multi-strain transmission and prolonged opportunities for recombination.

Vaccination with the Live Attenuated <i>Francisella</i> <i>novicida</i> Mutant <i>FTN0109</i> Protects against Pulmonary Tularemia

Francisella tularensis is considered a potential bioterrorism agent due to its low infectious dose, high mortality rate, and ability to be spread via the aerosol route. We characterized the F. tularensis subspecies novicida mutant strain FTN0109 as a potential vaccine candidate against tularemia. This strain, which lacks an outer membrane lipoprotein, is attenuated in vitro and in vivo, as it exhibits reduced replication within murine J774 macrophages and has a pulmonary LD50 in BALB/c and C57BL/6 mice of >105 CFU (compared to WT parental strain U112, LD50 FTN0109 also conferred complete protection in BALB/c mice against subsequent pulmonary challenge with 10 LD50 (60,000 CFU) of the murine virulent Francisella strain LVS. We also have demonstrated partial protection (50%) against the highly human virulent subspecies tularensis strain SCHU S4 (25 LD50, 12,500 CFU) following intratracheal vaccination in the Fischer 344 rat, a second rodent model for tularemia. Overall, our results suggest that FTN0109 serves as a potential putative vaccine candidate against pulmonary tularemia.

Epigenetic modification of Rta (ORF50) promoter is not responsible for distinct reactivation patterns of murine gammaherpesviruses.

Gammaherpesviruses-encoded replication and transcription activator (Rta) (ORF50) plays an essential role in the initiation of viral lytic gene expression and reactivation from latency. The Rta expression is influenced by many viral and cellular factors, including epigenetic modifications, mainly DNA methylation and histone modifications. Murine gammaherpesvirus 68 (MHV-68), belonging to the species Murid herpesvirus (MuHV-4), is widely used as amodel to study human gammaherpesvirus infections in vitro as well as in vivo. Recent studies of the MHV-68 Rta promoter revealed the effect of DNA demethylation and histone acetylation, induced by the inhibitor of histone deacetylase trichostatin A(TSA), on the MHV-68 reactivation from latency. Two other strains of MuHV-4, MHV-72 and MHV-4556, possess several unique properties, which distinguish them from strain MHV-68. Recently discovered reduced capacity of MHV-72 and MHV-4556 to reactivate from latency may be related to different methylation/demethylation patterns of the promoter regulatory region of the Rta. Here, we investigated the epigenetic regulation of the Rta promoter of three murine gammaherpesvirus strains, MHV-68, MHV-72 and MHV-4556, during latency and reactivation in vivo. However, we did not find any differences between Rta of MHV-68, MHV-72 and MHV-4556 and its epigenetic regulation during lytic infection, latency and de novo infection after ex vivo and in vivo reactivation induced by TSA. We confirmed that the treatment with TSA successfully induced demethylation of the Rta promoter regions of all three studied strains. Moreover, we have shown that the primary sequence of Rta and its promoter is identical for all three strains.

Experimental Approaches to Tissue Injury and Repair in Advanced Age.

Cutaneous wound healing is a complex physiological process. This process can be altered by multiple physiological and pathological factors. Multiple pathophysiological disturbances act to impair resolution of cutaneous wound injury, including obesity, diabetes, peripheral vascular disease, and advanced age. As our longevity increases without a concomitant increase in healthy living years, it is plausible to assume that problematic wound closure will continue to consume a large portion of our health care resources. Furthermore, advanced age is associated with numerous alterations in the innate and adaptive immune responses that complicate outcomes following cutaneous injury, trauma, or infection. Thus, models that examine the impact of advanced age on cutaneous wound repair will be of great benefit to the development of potential therapeutics that target age-related aberrancies in tissue repair. Herein, we detail two animal models of tissue injury, excisional wound injury and burn injury, that can be used to evaluate wound healing in the context of advanced age. We also describe modifications of these methods to examine wound infection following either excisional or burn injury. Lastly, we discuss methods of subsequent tissue analysis following injury. Models described below can be further adapted to genetically engineered murine strains to study the effects of aging and other co-morbidities on wound healing.

Cordycepin an Adenosine Analogue Executes Anti Rotaviral Effect by Stimulating Induction of Type I Interferon

Abstract Objectives: Rotavirus is the single most common etiological pathogen of severe diarrhea in infants causing death of over half a million infants a year. In spite of such a huge disease burden, still no effective antivirals are available against rotavirus. Cordycepin (3’-deoxyadenosine), an adenosine analogue, has been reported to modulate cell proliferation, platelet aggregation and provide protection against HIV infection. Herein, attempts were made to analyze efficacy of cordycepin for protection against rotavirus and underlying mechanism. Methods Cordycepin’s anti-rotaviral activity was elucidated against simian rotavirus strain SA11 (H96), rhesus strain RRV, human strain Wa (in vitro) and murine strain EW (in BALB/c). Plaque assay, qPCR for viral transcripts and immunoblotting of viral proteins were done to examine viral abundance. Effect of cordycepin on Interferon pathway was analyzed by immunoblotting, co-immunoprecipitation and molecular docking. Results Cordycepin was found to reduce propagation of different rotavirus strains such as SA11, RRV, Wa at 64μM with minimal cytotoxicity in vitro and EW in BALB/c mice. Protection was obtained upto 24 hours, after virus infection at both low and high multiplicity of infection. Sensitivity of rotavirus infection to Interferons has been documented. In cordycepin treated cells increased induction of Interferon and downstream antiviral proteins were observed. Cordycepin treatment leads to RIGI-MAVS interaction, IRF3 activation, and Interferon induction. By using different software probable docking sites and orientation of cordycepin and RIG-I were predicted. Cordycepin was found to have relatively higher binding probability for ATP binding domain than other domains. In Vero cells,deficient in Interferon signalling, cordycepin failed to protect against rotavirus. This suggests that though cordycepin may have other effects but anti-rotaviral effects were due to boosting of cellular innate immune responses mediated by Interferon. Conclusions Overall this study highlights that cordycepin exerts its anti-rotaviral activity by modulating immune signalling through enhanced Interferon production.

Interleukin-15 modulates adipose tissue by altering mitochondrial mass and activity.

Interleukin-15 (IL-15) is an immunomodulatory cytokine that affects body mass regulation independent of lymphocytes; however, the underlying mechanism(s) involved remains unknown. In an effort to investigate these mechanisms, we performed metabolic cage studies, assessed intestinal bacterial diversity and macronutrient absorption, and examined adipose mitochondrial activity in cultured adipocytes and in lean IL-15 transgenic (IL-15tg), overweight IL-15 deficient (IL-15−/−), and control C57Bl/6 (B6) mice. Here we show that differences in body weight are not the result of differential activity level, food intake, or respiratory exchange ratio. Although intestinal microbiota differences between obese and lean individuals are known to impact macronutrient absorption, differing gut bacteria profiles in these murine strains does not translate to differences in body weight in colonized germ free animals and macronutrient absorption. Due to its contribution to body weight variation, we examined mitochondrial factors and found that IL-15 treatment in cultured adipocytes resulted in increased mitochondrial membrane potential and decreased lipid deposition. Lastly, IL-15tg mice have significantly elevated mitochondrial activity and mass in adipose tissue compared to B6 and IL-15−/− mice. Altogether, these results suggest that IL-15 is involved in adipose tissue regulation and linked to altered mitochondrial function.

Functional and Genetic Heterogeneity of Distinct Leukemic Stem Cell Populations in CD34- Human Acute Myeloid Leukemia

Lack of progress in curing AML is likely, in part, to be due to the genetic and functional heterogeneity of AML. ~13 Tier 1 mutations occur per patient sample arrayed in 2-5 clones (CGARN, N Engl J Med, 2013). Not all AML cell populations may be equally chemosensitive; for example, leukemia-propagating leukemic stem cells (LSC) are more chemoresistant. (Ishikawa et al., Nat Biotechnol, 2007). Additionally, the impact of genetic heterogeneity on LSC function is unclear.In ~ 70% of primary human AML with >2% CD34+ cells, LSCs exist within both CD34+CD38- and CD34+CD38+ compartments (Taussig et al., Blood, 2008), and have progenitor-like transcriptional programmes (Goardon et al., Cancer Cell, 2011). ~30% of AML with <2% CD34+ cells (CD34- AML) are genetically distinct, enriched for mutations in NPM1 and co-associated mutations (FLT3, IDH1/2, TET2, and DNMT3A). Here, LSC activity has been detected in both CD34+ and CD34- compartments (Taussig et al., Blood, 2010, Martelli et al., Blood, 2010, Sarry et al., J Clin Invest, 2011). Questions remain about CD34- AML LSC populations: (i) What is the relationship between CD34- and CD34+ LSCs? (ii) What are the nearest counterpart normal haemopoietic cells to LSCs at a global transcriptional level? (iii) What is the impact of genetic heterogeneity on LSC function? Do all clones have equal LSC potential?Of a sequential cohort of 49 CD34- samples, 55% of 38 samples karyotyped had normal karyotype. 29/49 (59%) had mutated NPM1. Co-occurring mutations were FLT3 (54%), IDH1/2 (54%) and DNMT3A (26%). 11/28 samples with sufficient cells engrafted AML confirmed on mutation analysis. In 8/11 samples (7 were NPM1-mutated) sufficient available cells were available for detailed studies. LSC populations in serial transplant assays were present in both minor CD34+ and CD34-; CD34- populations were CD117+ and CD244+ or CD244-. Limit dilution analysis showed similar LSC frequencies in CD34+ and CD34- fractions from the same patient. Unexpectedly, there was no hierarchy with respect to CD34 expression and CD34 expression did not mark functionally distinct LSC populations. RNA-sequencing of 5 CD34+ and 14 CD34- LSC populations from 8 patient samples showed only 42 differentially protein coding genes out of 15539 expressed genes.We used ANOVA analysis to identify 300 top-ranking differentially expressed genes between normal stem/progenitor and myeloid and erythroid precursor populations. Using this signature, principal component analysis showed CD34- AML LSCs (CD34+ and CD34-) are closest to CD34-CD117+CD244+ populations that are promyelocytes have no D14 progenitor function in CFC assays and express late granulocytic macrophage (GM) genes. CD244 separates normal CD34-CD117+ populations into GM (CD244+) from erythroid (CD244-) precursors so allowing greater precision in mapping CD34-AML LSC to normal GM counterparts. CD34-AML LCS were not only enriched for a GM precursor signature, but also for a transcriptional signature seen in normal HSC. CD34- AML LSC expressed 63/100 transcription factor (TF) genes expressed in HSC including HOX and HOX co-factors. Interestingly, LSCs in CD34- AML had a distinct RNA-Seq profile from CD34+ progenitor-like LSCs populations.To explore genetic and functional heterogeneity of LSC populations, bulk and single cell genotyping of LSC populations revealed: (i) branching subclonal structures; with up to 5 genetically distinct LSC clones/patient; (ii) intermediate genotypes where the order of mutation acquisition was identified; (iii) some but not all patient LSC clones could be propagated in mice suggesting that current immunodeficient murine strains do not accurately model human AML. Thus, studies of LSC function have to combine both studies in mice and of primary human samples.In summary, within CD34- AML there are multiple, non-hierarchically arranged LSC populations with transcriptional programmes most closely related to normal CD34- GM precursors. Unlike these normal mature cells, LSCs also express HSC transcriptional signatures. Functional and genetic analysis of single cells and populations from patient LSC, non-LSC compartments and of xenografts reveals clonal structure, order of acquisition of mutations, how subclones are distributed in different immunophenotypic populations, some with different functional properties and, differences in subclonal representation between patients and xenografts. DisclosuresNo relevant conflicts of interest to declare.

Abstract B14: Gain of function contribution of mutant AKT-E17K to lung and mammary tumorigenesis in mouse models

Abstract The hotspot Akt1-E17K mutation that results in a glutamic acid to lysine substitution in the seventeenth amino acid position in the pleckstrin homology domain of Akt1 occurs in approximately 0.6-2% of human lung cancers and in 6-8% of human breast cancer. To determine whether this Akt1 variant is a bona-fide activating mutation and to investigate the mechanistic basis of the Akt1 gain of function dependent mechanisms in lung and breast tumorigenesis we generated a transgenic Cre-inducible murine strain in the ROSA26 (R26) locus (R26-Akt1-E17K mice). To analyse the effects of mutant Akt1 in lung epithelium, we made use of two different systems to activate the transgene into pulmonary cells: intranasal instillation of an Adenovirus carrying the Cre recombinase (Ad-Cre) and the mating of R26-Akt1-E17K mice with Thyroid transcription factor-1 (TTF-1)-Cre mice that allow expression of the transgenes in lung epithelium. We report that in murine lung epithelium, mutant Akt1-E17K induces bronchial and/or bronchiolar hyperplastic lesions that at low frequency progress to frank carcinoma and accelerates tumor formation induced by chemical carcinogens. To assess the potential role of Akt1 as an oncogene in mammary epithelium, we examined the consequences of the mutant Akt1-E17K in the mammary gland of mice by mating of R26-Akt1-E17K mice with Mouse mammary tumor virus (MMTV)-Cre mice. We provide strong evidences that the expression of mutant Akt1-E17K in the mammary gland, driven by the MMTV promoter, was sufficient to produce a tumoral response in mice starting from 5-6 months of age. Transgenic mice developed invasive and in situ carcinoma in 45% of the cases and, in agreement with these results, mice that express activated Akt1 in the mammary gland showed poor survival Citation Format: Donatella Malanga, Stefania Belmonte, Stefania Belmonte, Carmela De Marco, Orlando Paciello, Alfonso Baldi, Giuseppe Viglietto. Gain of function contribution of mutant AKT-E17K to lung and mammary tumorigenesis in mouse models. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B14.

Lupus-prone mice fail to raise antigen-specific T cell responses to intracellular infection.

Systemic lupus erythematosus (SLE) is characterized by multiple cellular abnormalities culminating in the production of autoantibodies and immune complexes, resulting in tissue inflammation and organ damage. Besides active disease, the main cause of morbidity and mortality in SLE patients is infections, including those from opportunistic pathogens. To understand the failure of the immune system to fend off infections in systemic autoimmunity, we infected the lupus-prone murine strains B6.lpr and BXSB with the intracellular parasite Toxoplasma gondii and survival was monitored. Furthermore, mice were sacrificed days post infection and parasite burden and cellular immune responses such as cytokine production and cell activation were assessed. Mice from both strains succumbed to infection acutely and we observed greater susceptibility to infection in older mice. Increased parasite burden and a defective antigen-specific IFN-gamma response were observed in the lupus-prone mice. Furthermore, T cell:dendritic cell co-cultures established the presence of an intrinsic T cell defect responsible for the decreased antigen-specific response. An antigen-specific defect in IFN- gamma production prevents lupus-prone mice from clearing infection effectively. This study reveals the first cellular insight into the origin of increased susceptibility to infections in SLE disease and may guide therapeutic approaches.

An interleukin-1 beta-encoding retrovirus exhibits enhanced replication in vivo.

Interleukin-1 beta (IL-1β) is an inflammatory cytokine that is secreted in response to inflammasome activation by innate microbe-sensing pathways. Although some retroviruses can trigger IL-1β secretion through the DNA-sensing molecule IFI16, the effect of IL-1β on the course of infection is unknown. To test whether IL-1β secretion affects retroviral replication in vivo, I constructed a novel murine leukemia virus strain (FMLV-IL-1β) that encodes the mature form of IL-1β. This virus replicated with kinetics similar to that of wild-type virus in tissue culture but caused a dramatically more aggressive infection of both C57BL/6 and BALB/c mice. By 7 days postinfection (PI), mice infected with FMLV-IL-1β exhibited splenomegaly and viral loads 300-fold higher than those in mice infected with wild-type FMLV. Furthermore, the enlarged spleens of FMLV-IL-1β-infected mice correlated with a large expansion of Gr-1(+) CD11b(+) myeloid-derived suppressor cells, as well as elevated levels of immune activation. Although FMLV-IL-1β infection was controlled by C57BL/6 mice by 14 days p.i., FMLV-IL-1β was able to establish a significant persistent infection and immune activation in BALB/c mice. These results demonstrate that IL-1β secretion is a powerful positive regulator of retroviral infection and that FMLV-IL-1β represents a new model of proinflammatory retroviral infection. Interleukin-1 beta (IL-1β) is an inflammatory cytokine released in response to activation of innate pathogen-sensing pathways during microbial infection. To examine the potential impact of IL-1β on retroviral replication in vivo, I constructed a novel mouse retrovirus strain (FMLV-IL-1β) that encodes IL-1β and promotes abundant IL-1β secretion from infected cells. This virus replicates with normal kinetics in cultured cells but displays a dramatically enhanced ability to replicate in mice and caused persistent infection and immune activation in the BALB/c strain of mice. These results establish IL-1β as a positive regulator of retroviral replication and suggest that targeting this pathway may have therapeutic benefits in infections with proinflammatory retroviruses. This virus can also be used to further study the impact of inflammatory pathways on retroviral infection.

Abstract 1011: Metformin inhibits angiogenesis, local and metastatic growth of triple-negative breast cancer by targeting two classes of adipose tissue progenitors

Abstract We have recently reported that human white adipose tissue (WAT) contains two classes of CD45-CD34+ progenitors with cooperative roles in triple negative breast cancer progression. In orthotopic murine models we found that 1) purified human WAT CD34+CD13+ mesenchymal adipose stromal cell progenitors (ASCs) were not able to migrate but promoted local tumor growth in the mammary fat pad and 2) purified human WAT CD34+CD31+ endothelial progenitors (EPCs) were able to migrate towards lymph nodes and blood and promoted breast cancer cell EMT, migration, invasion and metastatic growth (Orecchioni et al, 2013). Here we report that in several in vitro and in vivo models the biguanide Metformin, a drug commonly used in type 2 diabetes management, is able to inhibit angiogenesis, local and metastatic growth of triple negative breast cancer by targeting WAT progenitors. First, we and others (Dallaglio et al, in press) have found in collaborative studies that Metformin inhibits the formation of capillary-like networks by HUVEC cells. This effect was partially AMPK-dependent. Metformin inhibited VEGF-dependent activation of ERK1/2, angiogenesis in matrigel pellets in vivo, and decreased microvessel density in tumor-free mice. The effect of Metformin on WAT progenitors was then studied in vivo in several immune-competent and immune-deficient murine strains with and without triple negative breast cancer. In immune-competent mice, low-dose Metformin (2mg/mL drinking water) induced apoptosis and increased hypoxia of CD34+CD31+ WAT EPCs. As a result of low-dose Metformin administration, WAT and cancer vessels were quantitatively reduced, had a significant impairment of the pericyte/endothelial cell ratio and displayed a branching and dysplastic phenotype. This effect was also observed in immune-competent mice on high fat diet receiving Metformin. WAT CD34+ ASC were targeted by higher doses of Metformin, which were associated with a reduced pericyte coverage of new blood vessels. As Metformin is frequently administered along with aspirin and beta-blockers in diabetic and obese patients, we studied in vivo the association of these three drugs in murine models of triple negative breast cancer. In several immune-deficient murine models, the beta-blocker Atenolol significantly synergized with Metformin in inhibiting both local and metastatic breast cancer growth. In immune-competent murine strains, the anti-metastatic effect of Metformin and Atenolol was further enhanced by the addition of aspirin. These effects were observed in several murine models generated by both p53 mutated or p53 wild type triple negative breast cancer cells. We are currently investigating the molecular mechanisms used by Metformin to inhibit WAT progenitors and the most efficient schedules of administration of Metformin, Atenolol and Aspirin to target local and metastatic breast cancer growth. Citation Format: Stefania Orecchioni, Giuliana Gregato, Francesca Reggiani, Patrizia Mancuso, Angelica Calleri, Giovanna Talarico, Valentina Labanca, Francesco Bertolini. Metformin inhibits angiogenesis, local and metastatic growth of triple-negative breast cancer by targeting two classes of adipose tissue progenitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1011. doi:10.1158/1538-7445.AM2014-1011

The impact of coexisting genetic mutations on murine optic glioma biology.

Children with the neurofibromatosis type 1 (NF1) tumor predisposition syndrome are prone to the development of optic pathway gliomas resulting from biallelic inactivation of the NF1 gene. Recent studies have revealed the presence of other molecular alterations in a small portion of these NF1-associated brain tumors. The purpose of this study was to leverage Nf1 genetically engineered mouse strains to define the functional significance of these changes to optic glioma biology. Nf1+/- mice were intercrossed with Nf1(flox/flox) mice, which were then crossed with Nf1(flox/flox); GFAP-Cre mice, to generate Nf1(flox/mut); GFAP-Cre (FMC) mice. These mice were additionally mated with conditional KIAA1549:BRAF knock-in or Pten(flox/wt) mice to generate Nf1(flox/mut); f-BRAF; GFAP-Cre (FMBC) mice or Nf1(flox/mut); Pten(flox/wt); GFAP-Cre (FMPC) mice, respectively. The resulting optic gliomas were analyzed for changes in tumor volume, proliferation, and retinal ganglion cell loss. While KIAA1549:BRAF conferred no additional biological properties on Nf1 optic glioma, FMPC mice had larger optic gliomas with greater proliferative indices and microglial infiltration. In addition, all 3 Nf1 murine optic glioma strains exhibited reduced retinal ganglion cell survival and numbers; however, FMPC mice had greater retinal nerve fiber layer thinning near the optic head relative to FMC and FMBC mice. Collectively, these experiments demonstrate genetic cooperativity between Nf1 loss and Pten heterozygosity relevant to optic glioma biology and further underscore the value of employing genetically engineered mouse strains to define the contribution of discovered molecular alterations to brain tumor pathogenesis.

C5aR, TNF-α, and FGL2 contribute to coagulation and complement activation in virus-induced fulminant hepatitis

Viral fulminant hepatitis (FH) is a disease with a high mortality rate. Activation of the complement system correlates with the development of FH. However, the key factors mediating complement activation in FH remain elusive. Liver tissues were isolated from FH patients infected by hepatitis B virus (HBV) and from mice infected with murine hepatitis virus strain 3 (MHV-3). Wild type mice were treated with or without antagonists of C5aR or TNF-α, and mice deficient for C5aR (C5aR(-/-)), Fgl2 (Fgl2(-/-)), and Tnfα (Tnfα(-/-)) mice were not treated with the antagonists. C5b-9, C5aR, FGL2, CD31, CD11b, fibrin, TNF-α, and complement C3 cleavage products were detected by immunohistochemistry, immunofluorescence, or ELISA. Sorted liver sinusoidal endothelial cells (LSECs) or myeloid-derived (CD11b(+)) cells were stimulated with C5a, TNF-α or MHV-3 in vitro. The mRNA expressions levels of Fgl2 and Tnfα were determined by qRT-PCR analyses. We observed that complement activation, coagulation and pro-inflammatory cytokine production were upregulated in the HBV(+) patients with FH. Similar observations were made in the murine FH models. Complement activation and coagulation were significantly reduced in MHV-3 infected mice in the absence of C5aR, Tnfα or Fgl2. The MHV-3 infected C5aR(-/-) mice exhibited reduced numbers of infiltrated inflammatory CD11b(+) cells and a reduced expression of TNF-α and FGL2. Moreover, C5a administration stimulated TNF-α production by CD11b(+) cells, which in turn promoted the expression of FGL2 in CD31(+) LSEC-like cells in vitro. Administration of antagonists against C5aR or TNF-α ameliorated MHV-3-induced FH. Our results demonstrate that C5aR, TNF-α, and FGL2 form an integral network that contributes to coagulation and complement activation, and suggest that those are potential therapeutic targets in viral FH intervention.