Mouse Lineage Research Articles

Mechanism of Action of an Autovaccine Against Canine DLBCL Lymphoma Leading to a Long Overall Survival

An autovaccine against canine DLBCL lymphomas associated to a CHOP protocol has proven to greatly improve the overall survival and the time to progression of a series of dogs against another series only treated by the CHOP protocol (1). This vaccine was obtained by purifying specific proteins and peptides from a node biopsy. In order that the vaccine matches the heterogeneity of the tumor, one vaccine was produced for each dog.The biopsy was homogenised then the cell debris were removed by centrifugation. The proteins in suspension in the supernatant were precipitated by a high salt concentration. Once the proteins were suspended in buffer, the solution was passed through a column of HA (hydroxylapatite)- particles. The particles were then washed by different concentrations of phosphate buffer in order to concentrate heat shock proteins at the surface of the HA-particles and eliminate contaminating proteins. The vaccine was thus made of HA- particles used as carrier and adjuvant and proteins and peptides enriched in heat shock proteins (gp96 and HSP70). The preparation resulted in 1500 µg of proteins and 40 mg of HA for each dose. Vaccination protocol consisted in 4 injections within four weeks and one injection a month for four months. In order to establish the mechanism of action, vaccines were tested for their ability to cross prime the CD8 and to be link the CD91 and TLR2 and 4. We also checked that the HA-particles could activate the inflammasome.Materials and methods, and results:CD8 cross-priming:Murine epithelial cells (4T1) were used to induce a tumor in isogenic mice (Balb/c). A vaccine was prepared from a tumor and was used to stimulate in vitrodendritic cells of the same mouse lineage. These late cells are then incubated with CTLs of a mouse already in contact with the 4T1. It appeared that the CTLs synthesize more IFN-gamma than the negative control indicating a cross priming of the CD8 lymphocytes by the vaccine particles in a dose dependent manner.TLR2 and 4 labelling:The vaccine proteins (from vaccines used for the vaccinated group of DLBCL dogs) were removed from their HA carrier by washing the HA- particles with a 0.5 M KHPO4solution. They were then introduced in a culture of monocytes overexpressing TLR2 or TLR4. TLR2 and 4 were co transfected with reporter gene of SEAP which is fused with NF-κβ and AP-1. It appeared that the HA-powder alone did not activate the TLR receptors while the proteins released from the powder were ligand of both TLR2 and 4.CD91 labelling:Proteins released from the HA carriers were associated to a peroxydase and then introduced in a culture of a monocyte line expressing CD91 (RAW264) with or without anti- CD91. The cells were labelled by the peroxydase /proteins complexes. Furthermore, it appeared that only the cells grown without the antibody were labelled.Inflammasome activation by the HA-particles:HA-powders having different characteristics, surface area, granulometry, sintering temperature, shape, were introduced in culture of THP-1 cells and the IL-1B excretion was measured and compared to positive contols known to activate the inflammasome. It was demonstrated that depending on the concentration, some powders activated the inflammasome more than the positive control.Discussion:It is generally accepted that CD8 cross- priming against cancer antigens is essential for the elimination of cancer cells by the immune system. CD91 labelling by the vaccine proteins suggest that the heat shock proteins play a key-role in the cross-priming. CD91 are specific receptors for HSPs found at the surface of antigen presenting cells. HSPs are essential molecules in the antigen processing for their expression at the APCs surface. It is also known that these proteins are ligand of the TLR2 and TLR4.Conclusions:It is thus possible to purify specific proteins from nodes of dogs with DLBCL lymphoma and associate them to an adjuvant material to obtain a CD8 cross-priming against the tumor cells. These biological results could be related to a better clinical outcome for vaccinated dogs(1) Laura Marconato, Patrick Frayssinet, Nicole Rouquet, Stefano Comazzi, Vito Ferdinando Leone, Paola Laganga, Federica Rossi, Lorenzo Pezzoli, Luca Aresu, Randomized, placebo-controlled, double-blinded chemo-immunotherapy clinical trial in a Pet Dog model of Diffuse Large B-cell Lymphoma: a successful combination. Clin Cancer Res. 2014 Feb 1;20(3):668-77 DisclosuresFrayssinet:Urodelia: Employment. Rouquet:Urodelia: Employment. Marconato:Urodelia: Research Funding. Rossi:Urodelia: Research Funding.

Recombinant DEK Regulates Hematopoietic Stem and Progenitor Cells in a CXCR2- GαI Protein-Dependent Manner

DEK, a non-histone nuclear phosphoprotein involved in heterochromatin remodeling, is released from cells and can regulate hematopoiesis (Broxmeyer et al., 2012, Stem Cells Dev., 21: 1449; 2013, Stem Cells, 31: 1447). Marrow from DEK-/- mice manifest increased hematopoietic progenitor cell (HPC) numbers and cycling status and decreased long-term and secondary hematopoietic stem cells (HSC) engrafting capability. Moreover, recombinant mouse (rmu) DEK inhibited HPC colony formation in vitro. We now show that rmuDEK is myelosuppressive in vitro in an S-phase specific manner and reversibly decreases numbers and cycling status of CFU-GM, BFU-E, and CFU-GEMM in vivo, with DEK-/- mice being much more sensitive than control mice to this suppression. In addition, rmuDEK treatment of WT mice in vivo reversibly reduces the phenotypic number of ST-HSC, MPP, CMP, GMP, and MEP in the marrow. In vivo administration of rmuDEK to DEK-/- mice greatly enhanced the number of phenotypic LT-HSC and functional HSC in these mice (competitive HSC repopulation in lethally irradiated mice). DEK-/- mice are also more sensitive to 5-FU than control mice resulting in slower hematopoietic cell recovery, possibly due to the enhanced cycling status of HPC in DEK-/- mice. To determine the biological role of extracellular DEK, we utilized a reagent that could inactivate its function. This inactivating agent, but not its control, neutralized the inhibitory effect of rmuDEK. In addition, treating marrow cells in vitro with truncated rmuDEK by pretreating the DEK with the enzyme DPP4 (which DEK has targeted truncation sites for) also blocked the inhibitory effects of DEK suggesting that DEK must be in its full length form in order to perform its functionUpon our discovery that the DEK protein has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, we hypothesized that DEK may manifest at least some of its actions through CXCR2, known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case we first confirmed expression of CXCR2 on the surface of HSC and HPC. To determine whether rmuDEK’s inhibitory function is mediated through the CXCR2 receptor, a neutralizing monoclonal antibody against CXCR2 was utilized. Marrow treated in vitro with rmuDEK, rhIL-8, or rmuMIP-2 inhibited colony formation; however pretreating marrow cells with the neutralizing CXCR2 antibody blocked the inhibitory effect of these proteins. Marrow treated with rmuMIP-1α (a chemokine that does not bind to CXCR2) also inhibited colony formation; however neutralizing CXCR2 antibody had no effect on the ability of MIP-1α to inhibit colony formation. Neutralizing CXCR4, a chemokine receptor that binds SDF-1 but not IL-8, MIP-2 or MIP-1α, had no effect on the inhibition of colony formation. DEK inhibition of CFU-GM colony formation is dependent on Gαi-protein-coupled receptor signaling as determined through the use of pertussis toxin. This is a unique mechanism since IL-8 and MIP-1α had been previously reported by us to be insensitive to the inhibitory effects of pertussis toxin.As extracellular DEK can remodel chromatin in non-hematopoietic cells in vitro (Kappes et al., 2011, Genes Dev., 25: 673; Saha et al., 2013, PNAS, 110: 6847), we next assessed the effects of DEK on the heterchromatin marker H3K9Me3 in the nucleus of purified mouse Lineage negative, Sca-1 positive, c-kit positive (LSK) marrow cells by imaging flow cytometry. RmuDEK enhanced the presence of H3K9Me3 in the nucleus of DEK-/- LSK marrow cells indicating that rmuDEK can be internalized by LSK cells and mediate heterchromatin formation. These results add to emerging evidence that DEK, a unique nuclear protein, plays a role in regulating hematopoiesis through a CXCR2/Gαi protein signaling pathway. Its modulation may be of practical use. DisclosuresNo relevant conflicts of interest to declare.

Toxoplasma gondii oral infection induces intestinal inflammation and retinochoroiditis in mice genetically selected for immune oral tolerance resistance.

Toxoplasmosis is a worldwide disease with most of the infections originating through the oral route and generates various pathological manifestations, ranging from meningoencephalitis to retinochoroiditis and inflammatory bowel disease. Animal models for these pathologies are scarce and have limitations. We evaluated the outcome of Toxoplasma gondii oral infection with 50 or 100 cysts of the ME-49 strain in two lines of mice with extreme phenotypes of susceptibility (TS) or resistance (TR) to immune oral tolerance. Therefore, the aim of this study was to evaluate the behaviour of TS and TR mice, orally infected by T. gondii, and determine its value as a model for inflammatory diseases study. Mortality during the acute stage of the infection for TR was 50% for both dosages, while 10 and 40% of the TS died after infection with these respective dosages. In the chronic stage, the remaining TS succumbed while TR survived for 90 days. The TS displayed higher parasite load with lower intestinal inflammation and cellular proliferation, notwithstanding myocarditis, pneumonitis and meningoencephalitis. TR presented massive necrosis of villi and crypt, comparable to inflammatory bowel disease, with infiltration of lymphoid cells in the lamina propria of the intestines. Also, TR mice infected with 100 cysts presented intense cellular infiltrate within the photoreceptor layer of the eyes, changes in disposition and morphology of the retina cell layers and retinochoroiditis. During the infection, high levels of IL-6 were detected in the serum of TS mice and TR mice presented high amounts of IFN-γ and TNF-α. Both mice lineages developed different disease outcomes, but it is emphasized that TR and TS mice presented acute and chronic stages of the infection, demonstrating that the two lineages offer an attractive model for studying toxoplasmosis.

Principles of regulatory information conservation between mouse and human.

To broaden our understanding of the evolution of gene regulation mechanisms, we generated occupancy profiles for 34 orthologous transcription factors (TFs) in human–mouse erythroid progenitor, lymphoblast and embryonic stem-cell lines. By combining the genome-wide transcription factor occupancy repertoires, associated epigenetic signals, and co-association patterns, here we deduce several evolutionary principles of gene regulatory features operating since the mouse and human lineages diverged. The genomic distribution profiles, primary binding motifs, chromatin states, and DNA methylation preferences are well conserved for TF-occupied sequences. However, the extent to which orthologous DNA segments are bound by orthologous TFs varies both among TFs and with genomic location: binding at promoters is more highly conserved than binding at distal elements. Notably, occupancy-conserved TF-occupied sequences tend to be pleiotropic; they function in several tissues and also co-associate with many TFs. Single nucleotide variants at sites with potential regulatory functions are enriched in occupancy-conserved TF-occupied sequences.Supplementary informationThe online version of this article (doi:10.1038/nature13985) contains supplementary material, which is available to authorized users.

Wild-type KRAS inhibits oncogenic KRAS-induced T-ALL in mice.

The role of hyperactive RAS signaling is well established in myeloid malignancies but less clear in T-cell malignancies. The Kras2(LSL)Mx1-Cre (KM) mouse model expresses endogenous KRAS(G12D) in hematopoietic cells and is widely used to study mechanisms and treatment of myeloproliferative neoplasms (MPN). The model displays an intriguing shift from MPN to acute T-cell leukemia (T-ALL) after transplantation to wild-type mice, but the mechanisms underlying this lineage shift is unknown. Here, we show that KRAS(G12D) increases proliferation of both myeloid and T-cell progenitors, but whereas myeloid cells differentiate, T-cell differentiation is inhibited at early stages. Secondary mutations in the expanded pool of T-cell progenitors accompany T-ALL development, and our results indicate that the shift from myeloid to T-lymphoid malignancy after transplantation is explained by the increased likelihood for secondary mutations when the tumor lifespan is increased. We demonstrate that tumor lifespan increases after transplantation because primary KM mice die rapidly, not from MPN, but from KRAS(G12D) expression in nonhematopoietic cells, which causes intestinal bleeding and severe anemia. We also identify loss of the wild-type KRAS allele as a secondary mutation in all T-ALL cells and provide evidence that wild-type KRAS acts as a tumor suppressor in the T-cell lineage in mice.

Abstract 3045: Defining an elusive multiple myeloma stem cell population in mice

Abstract Multiple Myeloma (MM) is a debilitating disease of proliferating and malignant plasma cells that is currently incurable. The ability of monoclonal recurrence of disease suggests it might arise from a stem cell-like population capable of self-renewal. The difficulty to isolate the cancer stem-like cell in MM in both mice and men has introduced confusion toward this hypothesis. XBP1s is a key positive regulator for unfolded protein response and a master transcription factor for plasma cell development. It was previously reported that transgenic overexpression of XBP1s in the B cell lineage of mice (XBP1s-Tg), induces a multiple myeloma phenotype resembling human MM. We have now defined a post-germinal center, pre-plasma B cell population that was significantly increased in XBP1s-Tg mice as the hallmarks of MM was demonstrated. This non-memory B cell population resembled a stem/progenitor cell population, with increased expression of aldehyde dehydrogenase (ALDH), Notch1 and c-kit. Importantly, utilizing an in vitro culture for differentiating plasma cells, the increased population began to differentiate into plasma cells, increasing in size and gaining CD138 expression, the hallmark marker for plasma cells. In conclusion, we have defined a transitional pre-plasma cell population that could potentially be a stem/progenitor population of multiple myeloma. These results provide a novel insight into stem cell biology with regard to multiple myeloma and the use of murine models of myeloma to study the disease. Citation Format: Joshua Kellner, Yunpeng Hua, Bei Liu, Zihai Li. Defining an elusive multiple myeloma stem cell population in mice. [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 3045. doi:10.1158/1538-7445.AM2014-3045

Levels of circulating anti-muscarinic and anti-adrenergic antibodies and their effect on cardiac arrhythmias and dysautonomia in murine models of Chagas disease.

SUMMARY Antibodies (Ab) recognizing G-protein coupled receptors, such as β 1 and β 2 adrenergic (anti-β 1-AR and anti-β 2-AR, respectively) and muscarinic cholinergic receptors (anti-M2-CR) may contribute to cardiac damage, however their role in chronic chagasic cardiomyopathy is still controversial. We describe that Trypanosoma cruzi-infected C3H/He mice show increased P and QRS wave duration, and PR and QTc intervals, while the most significant ECG alterations in C57BL/6 are prolonged P wave and PR interval. Echocardiogram analyses show right ventricle dilation in infected animals of both mouse lineages. Analyses of heart rate variability (HRV) in chronically infected C3H/He mice show no alteration of the evaluated parameters, while C57BL/6 infected mice display significantly lower values of HRV components, suggesting autonomic dysfunction. The time-course analysis of anti-β 1-AR, anti-β 2-AR and anti-M2-CR Ab titres in C3H/He infected mice indicate that anti-β 1-AR Ab are detected only in the chronic phase, while anti-β 2-AR and anti-M2-CR are observed in the acute phase, diminish at 60dpi and increase again in the chronic phase. Chronically infected C57BL/6 mice presented a significant increase in only anti-M2-CR Ab titres. Furthermore, anti-β 1-AR, anti-β 2-AR and anti-M2-CR, exhibit significantly higher prevalence in chronically T. cruzi-infected C3H/He mice when compared with C57BL/6. These observations suggest that T. cruzi infection leads to host-specific cardiac electric alterations.

MicroRNA-125 family members exert a similar role in the regulation of murine hematopoiesis

MicroRNAs (miRNAs) are crucial for proper functioning of hematopoietic stem and progenitor cells (HSPCs). Members of the miRNA-125 family (consisting of miR-125a, miR-125b1, and miR-125b2) are known to confer a proliferative advantage on cells upon overexpression, to decrease the rate of apoptosis by targeting proapoptotic genes, and to promote differentiation toward the myeloid lineage in mice. However, many distinct biological effects of the three miR-125 species have been reported as well. In the current study, we set out to assess whether the three miRNA-125s that carry identical seed sequences could be functionally different. Our data show that overexpression of each of the three miR-125 family members preserves HSPCs in a primitive state invitro, results in a competitive advantage upon serial transplantation, and promotes skewing toward the myeloid lineage. All miR-125 family members decreased the pool of phenotypically defined Lin(-)Sca(+)Kit(+)CD48(-)CD150(+) long-term hematopoietic stem cells, simultaneously increasing the self-renewal activity upon secondary transplantation. The downregulation of miR-125s in hematopoietic stem cells abolishes these effects and impairs long-term contribution to blood cell production. The introduction of a point mutation within the miRNA-125 seed sequence abolishes all abovementioned effects and leads to the restoration of normal hematopoiesis. Our results show that all miR-125 family members are similar in function, they likely operate in a seed-sequence-dependent manner, and they induce a highly comparable hematopoietic phenotype.

Recipient total bone marrow irradiation has no long-term effect on clonal behavior of transplanted murine hematopoietic stem cells

Hematopoietic stem cell (HSC) transplantation is commonly used for treatment of hematopoietic and non-hematopoietic diseases and to investigate HSC function in an experimental setting. Preparation for transplantations often includes myeloablative treatment by total body irradiation (TBI). TBI damages both the host HSCs and their niche, results in high donor cell engraftment levels and is suggested to induce chemokines that attract HSCs to the bone marrow. Studies over the last decade demonstrated that upon transplantation into irradiated recipients, individual murine HSCs differ dramatically in their repopulation potential, myeloid/lymphoid cell output and cycling rate. It is currently unclear whether recipient irradiation contributes to the observed heterogeneity. To address this question, we transplanted different doses of freshly isolated highly purified HSCs into irradiated or untreated mice. As recipients we used C57BL/6W41/W41 mice that bear a c-kit mutation that negatively affects HSC numbers and function. We detected a pronounced delay in reconstitution kinetics of the B cell lineage in non-irradiated mice while the donor chimerism levels in T lymphocytes and short-lived granulocytes developed more quickly. To test how many stem cells engraft in the two transplant scenarios, we utilized a retroviral DNA barcoding method to uniquely label HSCs prior to their transplantation into irradiated or untreated mice and followed barcode contributions in blood for 15 months. The same delay in B-cell reconstitution was observed. However, barcode analysis in peripheral blood demonstrated similar clonal kinetics and heterogeneity of HSC clone sizes, cycling behavior and developmental potential in both irradiated and non-irradiated mice. Upon sacrifice, we were not able to detect differences in the number of contributing HSC clones in the bone marrow, demonstrating that the HSC homing was not affected by irradiation. In summary, our data demonstrate that clonal fluctuations and heterogeneity in the HSC pool are stem cell intrinsic and independent of the effects of irradiation on the niche.

Identification of hematopoietic-specific regulatory elements from the CD45 gene and use for lentiviral tracking of transplanted cells

The development of a hematopoietic reporter is crucial for determining the fate of lineages derived from cell-based therapies. A marking system will enable safer embryonic stem and induced pluripotent stem cell-based derivation of blood lineages and facilitate the development of efficient cellular reprogramming strategies based on direct fibroblast conversion. Here we report that the protein tyrosine phosphatase CD45 is an ideal candidate gene on which to base a hematopoietic reporter. CD45 regulatory elements were discovered by analyzing transcription factor chromatin occupancy (ChIP-seq) and promoter nuclease sensitivity (DNase-seq) to identify minimally sufficient sequences required for expression. After cloning the CD45 regulatory elements into an attenuated lentiviral backbone, we found that two transcriptional initiation regions were essential for high-level expression. Expressing CD45 promoters containing these regions and tethered to green fluorescent protein (GFP) in a primary B-cell differentiation assay and a transplantation model resulted in high levels of GFP in lymphoid, myeloid, and nucleated erythroid cells in mouse and human blood cell lineages. Moreover, GFP levels remained high 5 months after secondary transplantation, indicating persistence of the reporter. No CD45-driven GFP expression is observed after fibroblast or embryonic stem cell transduction. The GFP reporter is seen only after embryonic stem cells differentiate into hematopoietic cell progenitors and lineages, suggesting that this hematopoietic reporter system could be useful in validating potential autologous blood cell therapies.

Toxoplasma gondii 70 kDa Heat Shock Protein: Systemic Detection Is Associated with the Death of the Parasites by the Immune Response and Its Increased Expression in the Brain Is Associated with Parasite Replication

The heat shock protein of Toxoplasma gondii (TgHSP70) is a parasite virulence factor that is expressed during T. gondii stage conversion. To verify the effect of dexamethasone (DXM)-induced infection reactivation in the TgHSP70-specific humoral immune response and the presence of the protein in the mouse brain, we produced recombinant TgHSP70 and anti-TgHSP70 IgY antibodies to detect the protein, the specific antibody and levels of immune complexes (ICs) systemically, as well as the protein in the brain of resistant (BALB/c) and susceptible (C57BL/6) mice. It was observed higher TgHSP70-specific antibody titers in serum samples of BALB/c compared with C57BL/6 mice. However, the susceptible mice presented the highest levels of TgHSP70 systemically and no detection of specific ICs. The DXM treatment induced increased parasitism and lower inflammatory changes in the brain of C57BL/6, but did not interfere with the cerebral parasitism in BALB/c mice. Additionally, DXM treatment decreased the serological TgHSP70 concentration in both mouse lineages. C57BL/6 mice presented high expression of TgHSP70 in the brain with the progression of infection and under DXM treatment. Taken together, these data indicate that the TgHSP70 release into the bloodstream depends on the death of the parasites mediated by the host immune response, whereas the increased TgHSP70 expression in the brain depends on the multiplication rate of the parasite.

Comparison of dynamic expressions of Tim-3 and PD-1 in the brains between toxoplasmic encephalitis-resistant BALB/c and -susceptible C57BL/6 mice

T cells and IFN-γ are essential for controlling the reactivation of toxoplasmic encephalitis (TE), regardless of whether mice are susceptible or resistant to TE. It has been demonstrated that CD8(+) T cells exhausted in chronic Toxoplasma gondii infection result in TE reactivation in C57BL/6 mice. However, this phenomenon had not been reported in genetically TE-resistant BALB/c mice. To explore the immune mechanism of TE in different backgrounds of mice, the dynamic expressions of Tim-3, programmed cell death 1 (PD-1), and their ligands (galectin-9, PD-L1, PD-L2) in brain tissues were compared between TE-resistant BALB/c and -susceptible C57BL/6 mice infected with Prugniaud (Pru, a type II strain) of T. gondii in this study. Compared with infected BALB/c mice, there were remarkable pathological changes with significantly higher histological scores in the brains of C57BL/6 mice at 14, 35, 50, and 70 days postinfection (p.i., P < 0.01); significantly increased mRNA expressions of Tim-3 at 35 (P < 0.05) and 70 (P < 0.01) days p.i.; and significantly increased PD-1 at all the times p.i. (P < 0.01) in the brains of infected C57BL/6 mice. Furthermore, there were significantly increased mRNA expressions of PD-L1 in the brain of C57BL/6 mice than that in BALB/c mice at all the times p.i. (P < 0.01). Although the mRNA expressions of galectin-9 (ligand of Tim-3) were increased in the brains of both lineages of mice at all the times p.i., it showed no differences between the two lineages of mice. Our data suggest that the differences of Tim-3 and PD-1/PD-L1 expressions may contribute to the different immune responses between TE-resistant BALB/c and -susceptible C57BL/6 mice infected with Pru strain of T. gondii.

An antisense promoter in mouse L1 retrotransposon open reading frame-1 initiates expression of diverse fusion transcripts and limits retrotransposition

Between 6 and 30% of human and mouse transcripts are initiated from transposable elements. However, the promoters driving such transcriptional activity are mostly unknown. We experimentally characterized an antisense (AS) promoter in mouse L1 retrotransposons for the first time, oriented antiparallel to the coding strand of L1 open reading frame-1. We found that AS transcription is mediated by RNA polymerase II. Rapid amplification of cDNA ends cloning mapped transcription start sites adjacent to the AS promoter. We identified >100 novel fusion transcripts, of which many were conserved across divergent mouse lineages, suggesting conservation of potential functions. To evaluate whether AS L1 transcription could regulate L1 retrotransposition, we replaced portions of native open reading frame-1 in donor elements by synonymously recoded sequences. The resulting L1 elements lacked AS promoter activity and retrotransposed more frequently than endogenous L1s. Overexpression of AS L1 transcripts also reduced L1 retrotransposition. This suppression of retrotransposition was largely independent of Dicer. Our experiments shed new light on how AS fusion transcripts are initiated from endogenous L1 elements across the mouse genome. Such AS transcription can contribute substantially both to natural transcriptional variation and to endogenous regulation of L1 retrotransposition.

Bacillus subtilis Spores as Vaccine Adjuvants: Further Insights into the Mechanisms of Action

Bacillus subtilis spores have received growing attention regarding potential biotechnological applications, including the use as probiotics and in vaccine formulations. B. subtilis spores have also been shown to behave as particulate vaccine adjuvants, promoting the increase of antibody responses after co-administration with antigens either admixed or adsorbed on the spore surface. In this study, we further evaluated the immune modulatory properties of B. subtilis spores using a recombinant HIV gag p24 protein as a model antigen. The adjuvant effects of B. subtilis spores were not affected by the genetic background of the mouse lineage and did not induce significant inflammatory or deleterious effects after parenteral administration. Our results demonstrated that co-administration, but not adsorption to the spore surface, enhanced the immunogenicity of that target antigen after subcutaneous administration to BALB/c and C57BL/6 mice. Spores promoted activation of antigen presenting cells as demonstrated by the upregulation of MHC and CD40 molecules and enhanced secretion of pro-inflammatory cytokines by murine dendritic cells. In addition, in vivo studies indicated a direct role of the innate immunity on the immunomodulatory properties of B. subtilis spores, as demonstrated by the lack of adjuvant effects on MyD88 and TLR2 knockout mouse strains.

Fibroblast Growth Factor-2 Isoform (Low Molecular Weight/18 kDa) Overexpression in Preosteoblast Cells Promotes Bone Regeneration in Critical Size Calvarial Defects in Male Mice

Repair of bone defects remains a significant clinical problem. Bone morphogenetic protein 2 (BMP2) is US Food and Drug Administration-approved for fracture healing but is expensive and has associated morbidity. Studies have shown that targeted overexpression of the 18-kDa low-molecular-weight fibroblast growth factor 2 isoform (LMW) by the osteoblastic lineage of transgenic mice increased bone mass. This study tested the hypotheses that overexpression of LMW would directly enhance healing of a critical size calvarial bone defect in mice and that this overexpression would have a synergistic effect with low-dose administration of BMP2 on critical size calvarial bone defect healing. Bilateral calvarial defects were created in LMW transgenic male mice and control/vector transgenic (Vector) male mice and scaffold with or without BMP2 was placed into the defects. New bone formation was assessed by VIVA-computed tomography of live animals over a 27-week period. Radiographic and computed tomography analysis revealed that at all time points, healing of the defect was enhanced in LMW mice compared with that in Vector mice. Although the very low concentration of BMP2 did not heal the defect in Vector mice, it resulted in complete healing of the defect in LMW mice. Histomorphometric and gene analysis revealed that targeted overexpression of LMW in osteoblast precursors resulted in enhanced calvarial defect healing due to increased osteoblast activity and increased canonical Wnt signaling.

Differential protein network analysis of the immune cell lineage.

Recently, the Immunological Genome Project (ImmGen) completed the first phase of the goal to understand the molecular circuitry underlying the immune cell lineage in mice. That milestone resulted in the creation of the most comprehensive collection of gene expression profiles in the immune cell lineage in any model organism of human disease. There is now a requisite to examine this resource using bioinformatics integration with other molecular information, with the aim of gaining deeper insights into the underlying processes that characterize this immune cell lineage. We present here a bioinformatics approach to study differential protein interaction mechanisms across the entire immune cell lineage, achieved using affinity propagation applied to a protein interaction network similarity matrix. We demonstrate that the integration of protein interaction networks with the most comprehensive database of gene expression profiles of the immune cells can be used to generate hypotheses into the underlying mechanisms governing the differentiation and the differential functional activity across the immune cell lineage. This approach may not only serve as a hypothesis engine to derive understanding of differentiation and mechanisms across the immune cell lineage, but also help identify possible immune lineage specific and common lineage mechanism in the cells protein networks.

The Peutz-Jeghers kinase LKB1 suppresses polyp growth from intestinal cells of a proglucagon-expressing lineage in mice.

Liver kinase B1 (LKB1; also known as STK11) is a serine/threonine kinase and tumour suppressor that is mutated in Peutz-Jeghers syndrome (PJS), a premalignant syndrome associated with the development of gastrointestinal polyps. Proglucagon-expressing enteroendocrine cells are involved in the control of glucose homeostasis and the regulation of appetite through the secretion of gut hormones such as glucagon-like peptide-1 (GLP-1) and peptide tyrosine tyrosine (PYY). To determine the role of LKB1 in these cells, we bred mice bearing floxed alleles of Lkb1 against animals carrying Cre recombinase under proglucagon promoter control. These mice (GluLKB1KO) were viable and displayed near-normal growth rates and glucose homeostasis. However, they developed large polyps at the gastro-duodenal junction, and displayed premature mortality (death from 120 days of age). Histological analysis of the polyps demonstrated that they had a PJS-like appearance with an arborising smooth-muscle core. Circulating GLP-1 levels were normal in GluLKB1KO mice and the polyps expressed low levels of the peptide, similar to levels in the neighbouring duodenum. Lineage tracing using a Rosa26tdRFP transgene revealed, unexpectedly, that enterocytes within the polyps were derived from non-proglucagon-expressing precursors, whereas connective tissue was largely derived from proglucagon-expressing precursors. Developmental studies in wild-type mice suggested that a subpopulation of proglucagon-expressing cells undergo epithelial-mesenchymal transition (EMT) to become smooth-muscle-like cells. Thus, it is likely that polyps in the GluLKB1KO mice developed from a unique population of smooth-muscle-like cells derived from a proglucagon-expressing precursor. The loss of LKB1 within this subpopulation seems to be sufficient to drive tumorigenesis.

Early IFN-gamma production after YF 17D vaccine virus immunization in mice and its association with adaptive immune responses.

Yellow Fever vaccine is one of the most efficacious human vaccines ever made. The vaccine (YF 17D) virus induces polyvalent immune responses, with a mixed TH1/TH2 CD4+ cell profile, which results in robust T CD8+ responses and high titers of neutralizing antibody. In recent years, it has been suggested that early events after yellow fever vaccination are crucial to the development of adequate acquired immunity. We have previously shown that primary immunization of humans and monkeys with YF 17D virus vaccine resulted in the early synthesis of IFN-γ. Herein we have demonstrated, for the first time that early IFN-γ production after yellow fever vaccination is a feature also of murine infection and is much more pronounced in the C57BL/6 strain compared to the BALB/c strain. Likewise, in C57BL/6 strain, we have observed the highest CD8+ T cells responses as well as higher titers of neutralizing antibodies and total anti-YF IgG. Regardless of this intense IFN-γ response in mice, it was not possible to see higher titers of IgG2a in relation to IgG1 in both mice lineages. However, IgG2a titers were positively correlated to neutralizing antibodies levels, pointing to an important role of IFN-γ in eliciting high quality responses against YF 17D, therefore influencing the immunogenicity of this vaccine.

Interfollicular epidermal stem cells self-renew via autocrine Wnt signaling.

The skin is a classical example of a tissue maintained by stem cells. However, the identity of the stem cells that maintain the interfollicular epidermis and the source of the signals that control their activity remain unclear. Using mouse lineage tracing and quantitative clonal analyses, we showed that the Wnt target gene Axin2 marks interfollicular epidermal stem cells. These Axin2-expressing cells constitute the majority of the basal epidermal layer, compete neutrally, and require Wnt/β-catenin signaling to proliferate. The same cells contribute robustly to wound healing, with no requirement for a quiescent stem cell subpopulation. By means of double-labeling RNA in situ hybridization in mice, we showed that the Axin2-expressing cells themselves produce Wnt signals as well as long-range secreted Wnt inhibitors, suggesting an autocrine mechanism of stem cell self-renewal.

Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages

It is now recognized that extensive expression heterogeneities among cells precede the emergence of lineages in the early mammalian embryo. To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterised the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.