Differentiation Invivo Research Articles

A Novel Inhibitor of Mutant IDH1 Induces Differentiation in Vivo and Prolongs Survival in a Mouse Model of Leukemia

Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in patients with glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, cholangiocellular carcinoma and chondrosarcoma. Mutant IDH produces R-2-hydroxyglutarate (R2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. We recently established an in vivo mouse model and investigated the function of mutant IDH1. By computational drug screening, we identified an inhibitor of mutant IDH1 (HMS-101), which inhibits mutant IDH1 cell proliferation, decreases R2HG levels in vitro, and efficiently blocks colony formation of AML cells from IDH1 mutated patients but not of normal CD34+ bone marrow cells. In the present study we investigated the effect of the inhibitor in our IDH1/HoxA9-induced mouse model of leukemia in vivo.To identify the maximally tolerated dose of HMS-101, we treated normal C57BL/6 mice with variable doses of HMS-101 for 9 days and measured the serum concentration. Mice receiving 0.5 mg and 1mg intraperitoneally once a day tolerated the drug well with mean plasma concentrations of 0.1 to 0.3 µM. To evaluate the effect of HMS-101 in the IDH1 mouse model, we transduced IDH1 R132C in HoxA9-immortalized murine bone marrow cells. Sorted transgene positive cells were then transplanted into lethally irradiated mice. After 5 days of transplantation, mice were treated with HMS-101 intraperitoneally for 5 days/week. The R/S-2HG ratio in serum was reduced 3-fold in HMS-101 treated mice after 8 weeks of treatment compared to control treated mice. HMS-101 or PBS treated mice had similar levels of transduced leukemic cells in peripheral blood at 2 and 6 weeks after transplantation. However, from week 6 to week 15 leukemic cells in peripheral blood decreased from 76% to 58, 63% to 29%, 67% to 7%, and 74% to 38% in 4/6 mice treated with HMS-101. In one mouse the percentage of leukemic cells was constant, and in one mouse it increased from week 6 to week 15 after transplantation. Leukemic cells increased constantly in peripheral blood until death in control treated mice. While the control cohort developed severe leukocytosis, anemia and thrombocytopenia around 8 to 10 weeks post transplantation, mice treated with HMS-101 still had normal WBC, RBC and platelet counts at 15 weeks after transplantation. Moreover, the HMS-101 treated mice had significantly more differentiated Gr1+CD11b+ cells in peripheral blood than control mice at 6 weeks and 15 weeks after transplantation and at death (P=.01). Morphologic evaluation of blood cells at 15 weeks or death from HMS-101 treated mice revealed a high proportion of mature neutrophils that were GFP positive and thus derived from IDH1 transduced cells, whereas control treated mice had monocytic morphology with a high proportion of immature cells. Importantly, HMS-101 treated mice survived significantly longer with a median latency of 87 days (range 80-118), whereas PBS-treated mice died with a median latency of 66 days (range 64-69) after transplantation (P<.001). Of note, HMS-101 was found to be specific for mutant IDH1, as mutant IDH2 cells were not preferentially inhibited over IDH2 wildtype cells in vitro.This data demonstrates that HMS-101 specifically inhibits R2HG-production of mutant IDH1 in vivo, inhibits proliferation, induces differentiation in leukemic cells, and thus prolongs survival of IDH1mutant leukemic mice. Therefore, HMS-101 - a novel inhibitor of mutant IDH1 - shows promising activity in vivo and warrants further development towards clinical use in IDH1 mutated patients. DisclosuresChaturvedi:Hannover Medical School: Patents & Royalties. Preller:Hannover Medical School: Patents & Royalties. Heuser:Hannover Medical School: Patents & Royalties.

Knockdown of nuclear factor erythroid 2-related factor 2 by lentivirus induces differentiation of glioma stem-like cells.

Glioma stem cells (GSCs) are key in the progression and recurrence of glioblastoma. Inducing the differentiation of GSCs is an important therapeutic target for glioblastoma. Nuclear factor erythroid 2-related factor 2 (Nrf2) has been reported to be important in maintaining the stem cell status of GSCs; however, its association with differentiation has not been studied. Herein, we knocked down Nrf2 from GSCs to investigate the role of Nrf2 in the differentiation of GSCs. First, Nrf2 expression was observed at different stages of differentiation; then, Nrf2 was knocked down and the association of Nrf2 with differentiation degree was observed invitro. Finally, GSCs were planted in nude mice to study the association of Nrf2 with differentiation invivo. The expression of Nrf2 decreased with the differentiation process. Following Nrf2 knockdown, the proportion of sphere-like colonies decreased and the dendritic cells in spheres increased; the expression of Nrf2 significantly decreased while the expression of differentiation marker glial fibrillary acidic protein (GFAP) and βIII-tubulin increased both at the protein and the gene level. In the xenografts of nude mice, the differentiation of tumor cells was improved. These results suggest that Nrf2 is a key factor inhibiting the differentiation of GSCs, and knockdown of Nrf2 may promote the differentiation process, providing a therapy target for GSCs.

Myor/ABF-1 Mrna Expression Marks Follicular Helper T Cells but Is Dispensable for Tfh Cell Differentiation and Function In Vivo

Follicular T helper cells (Tfh) are crucial for effective antibody responses and long term T cell-dependent humoral immunity. Although many studies are devoted to this novel T helper cell population, the molecular mechanisms governing Tfh cell differentiation have yet to be characterized. MyoR/ABF-1 is a basic helix-loop-helix transcription factor that plays a role in the differentiation of the skeletal muscle and Hodgkin lymphoma. Here we show that MyoR mRNA is progressively induced during the course of Tfh-like cell differentiation in vitro and is expressed in Tfh responding to Alum-precipitated antigens in vivo. This expression pattern suggests that MyoR could play a role in the differentiation and/or function of Tfh cells. We tested this hypothesis using MyoR-deficient mice and found this deficiency had no impact on Tfh differentiation. Hence, MyoR-deficient mice developed optimal T-dependent humoral responses to Alum-precipitated antigens. In conclusion, MyoR is a transcription factor selectively up-regulated in CD4 T cells during Tfh cell differentiation in vitro and upon response to alum-protein vaccines in vivo, but the functional significance of this up-regulation remains uncertain.

The Isolation, Differentiation, and Survival In Vivo of Multipotent Cells from the Postnatal Rat filum terminale

Neural stem cells (NSCs) are undifferentiated cells in the central nervous system (CNS) that are capable of self-renewal and can be induced to differentiate into neurons and glia. Current sources of mammalian NSCs are confined to regions of the CNS that are critical to normal function and surgically difficult to access, which limits their therapeutic potential in human disease. We have found that the filum terminale (FT), a previously unexplored, expendable, and easily accessible tissue at the caudal end of the spinal cord, is a source of multipotent cells in postnatal rats and humans. In this study, we used a rat model to isolate and characterize the potential of these cells. Neurospheres derived from the rat FT are amenable to in vitro expansion in the presence of a combination of growth factors. These proliferating, FT-derived cells formed neurospheres that could be induced to differentiate into neural progenitor cells, neurons, astrocytes, and oligodendrocytes by exposure to serum and/or adhesive substrates. Through directed differentiation using sonic hedgehog and retinoic acid in combination with various neurotrophic factors, FT-derived neurospheres generated motor neurons that were capable of forming neuromuscular junctions in vitro. In addition, FT-derived progenitors that were injected into chick embryos survived and could differentiate into both neurons and glia in vivo.

Extensive Methylation of Promoter Sequences Silences Lentiviral Transgene Expression During Stem Cell Differentiation In Vivo

Lentiviral vectors (LV) are widely used to stably transfer genes into target cells investigating or treating gene functions. In addition, gene transfer into early murine embryos may be improved to efficiently generate transgenic mice. We applied lentiviral gene transfer to generate a mouse model transgenic for SET binding protein-1 (Setbp1) and enhanced green fluorescent protein (eGFP). Neither transgenic founders nor their vector-positive offspring transcribed or expressed the transgenes. Bisulfite sequencing of the internal spleen focus-forming virus (SFFV) promoter demonstrated extensive methylation of all analyzed CpGs in the transgenic mice. To analyze the impact of Setbp1 on epigenetic silencing, embryonic stem cells (ESC) were differentiated into cardiomyocytes (CM) in vitro. In contrast to human promoters in LV, virally derived promoter sequences were strongly methylated during differentiation, independent of the transgene. Moreover, the commonly used SFFV promoter (SFFVp) was highly methylated with remarkable strength and frequency during hematopoietic differentiation in vivo in LV but less in γ-retroviral (γ-RV) backbones. In summary, we conclude that LV using an internal SFFVp are not suitable to generate transgenic mice or perform constitutive expression studies in differentiating cells. Choosing the appropriate promoter is also crucial to allow stable transgene expression in clinical gene therapy.

Formation of embryoid bodies using dielectrophoresis

Embryoid body (EB) formation forms an important step in embryonic stem cell differentiation invivo. In murine embryonic stem cell (mESC) cultures EB formation is inhibited by the inclusion of leukaemic inhibitory factor (LIF) in the medium. Assembly of mESCs into aggregates by positive dielectrophoresis (DEP) in high field regions between interdigitated oppositely castellated electrodes was found to initiate EB formation. Embryoid body formation in aggregates formed with DEP occurred at a more rapid rate-in fact faster compared to conventional methods-in medium without LIF. However, EB formation also occurred in medium in which LIF was present when the cells were aggregated with DEP. The optimum characteristic size for the electrodes for EB formation with DEP was found to be 75-100 microns; aggregates smaller than this tended to merge, whilst aggregates larger than this tended to split to form multiple EBs. Experiments with ESCs in which green fluorescent protein (GFP) production was targeted to the mesodermal gene brachyury indicated that differentiation within embryoid bodies of this size may preferentially occur along the mesoderm lineage. As hematopoietic lineages during normal development derive from mesoderm, the finding points to a possible application of DEP formed EBs in the production of blood-based products from ESCs.

In vivoevaluation of calcium polyphosphate for bone regeneration

Current problems associated with bone allografts include risk of disease transmission, limited availability, and cost. Synthetic scaffolds have been proposed as substitute graft materials to address these issues. Calcium polyphosphate is a novel synthetic scaffold material that has shown good mechanical properties and biocompatibility. Here, we evaluated calcium polyphosphate in terms of its ability to support cell proliferation and differentiation invivo. Calcium polyphosphate, morsellized cancellous bone, and hydroxyapatite/tricalcium phosphate particles were seeded with marrow stromal cells and implanted subcutaneously in the back of NOD/Scid mice. At 7, 14, and 28 days the samples were harvested and the proliferation characteristics and gene expression were analyzed. All tested graft materials had similar proliferation characteristics and gene expression. The subcutaneous environment had a stronger impact on the proliferation and differentiation of the cells than the scaffold material itself. However, it was shown that calcium polyphosphate is superior to hydroxyapatite/tricalcium phosphate and bone in its ability to support cell survival invivo. The study confirmed that calcium polyphosphate has potential for replacing morsellized cancellous bone as a graft material for bone regeneration.

A20 Tumor Suppressor Deletion and BCL6 Oncogene Activation Cooperate In Deregulating B Cell Differentiation In Vivo

AbstractAbstract 148Diffuse large B-cell lymphoma (DLBCL), the most common type of non-Hodgkin lymphoma, is a heterogeneous disease comprising multiple biologically and clinically distinct subgroups, including germinal center B cell-like (GCB) and activated B cell-like (ABC) DLBCL. Numerous genetic alterations segregate with ABC-DLBCL, namely translocations of the BCL6 proto-oncogene, BLIMP1 inactivation and constitutive NF-κB activation. We recently reported that A20, a negative regulator of NF-κB signaling, is biallelically inactivated by mutations and deletions in one-third of ABC-DLBCL (Compagno et al, Nature, 2009), indicating a tumor suppressor role in this disease. Notably, A20 inactivation is commonly associated with chromosomal translocations deregulating BCL6 (n=11/20 DLBCL cases). Furthermore, the two genes are linked in the same pathway, where NF-κB induced activation of IRF4 leads to BCL6 down-regulation (Saito et al. Cancer Cell 2007) and consequent release of the BCL6 target BLIMP1, a master regulator of plasma cell differentiation. These observations suggest that A20 inactivation and BCL6 translocations cooperate in DLBCL pathogenesis. In order to examine the individual and combined contribution of these two lesions in vivo, we have generated an A20 conditional knockout allele in which a loxP-flanked exon 3 of the A20 gene can be deleted upon Cre-mediated recombination. The resulting mice were crossed with both a Cγ1-Cre deletor strain, which expresses the Cre recombinase in germinal centre (GC) B cells, and the lymphoma-prone Iμ HABCL6 mouse model (Cattoretti et al., Cancer Cell, 2005), which mimics a BCL6 translocation to the immunoglobulin heavy chain locus. When analyzed at 3 months of age, GC B-cell conditional heterozygous (A20Cγ1HET) and homozygous (A20Cγ1KO) A20 knockout mice showed a significant increase in the B220dimCD138+ plasma cell population (0.6% and 0.5%, respectively, versus 0.3% for wild-type littermates) and a corresponding 2-fold increase in IgG1 serum immunoglobulin levels after immunization with sheep red blood cells. Furthermore, A20 knockout splenic B cells had increased proliferative capacity and survival after stimulation ex vivo with lipopolysaccharides, B-cell receptor cross-linking or CD40 activation, consistent with enhanced NF-κB activity in these cells. Interestingly, the increase in plasma cells was not observed in compound Iμ HABCL6/ A20Cγ1HET and Iμ HABCL6/ A20Cγ1KO animals, presumably due to the known role of BCL6 in blocking plasma cell differentiation (Tunyaplin et al., J. of Immunol., 2004). In contrast, these animals displayed a marked increase in the B220+PNAhi GC B cell compartment, as compared to both A20 knockout and Iμ HABCL6 mice (2.5% and 3.9% respectively, versus 1.9% for wild-type littermates). Overall, these findings document that A20 acts as a negative regulator of B cell proliferation and survival as well as of plasma cell differentiation in vivo, and support a model by which loss of A20 synergizes with BCL6 deregulation to promote the expansion of GC B cells while preventing terminal differentiation. Long-term follow-up of these cohorts will provide critical information on the role of A20 as a tumor suppressor gene in vivo and on its cooperative activity with BCL6 deregulation in the pathogenesis of DLBCL. Disclosures:No relevant conflicts of interest to declare.

Prospective Identification and Skeletal Localization of Cells Capable of Multilineage Differentiation In Vivo

A prospective in vivo assay was used to identify cells with potential for multiple lineage differentiation. With this assay, it was first determined that the 5-fluorouracil resistant cells capable of osseous tissue formation in vivo also migrated toward stromal derived factor-1 (SDF-1) in vitro. In parallel, an isolation method based on fluorescence-activated cell sorting was employed to identify a very small cell embryonic-like Lin-/Sca-1+CD45- cell that with as few as 500 cells was capable of forming bone-like structures in vivo. Differential marrow fractionation studies determined that the majority of the Lin-Sca-1+CD45- cells reside in the subendosteal regions of marrow. To determine whether these cells were capable of differentiating into multiple lineages, stromal cells harvested from Col2.3 Delta TK mice were implanted with a gelatin sponge into SCID mice to generate thymidine kinase sensitive ossicles. At 1.5 months, 2,000 green fluorescent protein (GFP)+ Lin-Sca-1+CD45- cells were injected into the ossicles. At harvest, colocalization of GFP-expressing cells with antibodies to the osteoblast-specific marker Runx-2 and the adipocyte marker PPAP gamma were observed. Based on the ability of the noncultured cells to differentiate into multiple mesenchymal lineages in vivo and the ability to generate osseous tissues at low density, we propose that this population fulfills many of the characteristics of mesenchymal stem cells.

Heme Oxygenase-1 Expression in Murine Dendritic Cell Subpopulations: Effect on CD8 + Dendritic Cell Differentiation in Vivo

Heme oxygenase-1 (HO-1) is a microsomal enzyme with antioxidant, antiapoptotic, and immunoregulatory functions. We studied the expression of HO-1 by bone marrow-derived dendritic cells (BMDCs) and splenic DC subpopulations under quiescent conditions or following lipopolysaccharide (LPS) stimulation. The kinetics of HO-1 expression by BMDCs depended on the conditions under which they were propagated. Expression of HO-1 in mouse BMDCs in 100 U/ml GM-CSF peaked at 16 hours after LPS treatment and maintained expression for at least 48 hours. But cultures in 800 U/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) showed peak expression by 16 hours that disappeared by 48 hours after LPS stimulation, similar to BMDCs cultured in both 100 U/ml GM-CSF and IL-4 (10 ng/ml). By flow cytometry, a large proportion of CD8(+) splenic DCs strongly expressed HO-1, and this population significantly increased following LPS administration in vivo. In HO-1(-/-) mice, the proportion of splenic CD8(+) DCs was significantly decreased in comparison with HO-1(+/+) mice. In addition, a unique subpopulation of MHC II(-)CD11b(+)CD11c(+) cells was prominent in HO-1(-/-) spleens. Injection of GFP-labeled HO-1(+/+) splenic DC precursors into HO-1(+/+) mice resulted in the generation of GFP(+)CD8(+) DCs in the spleen after 5 days, but GFP(+) CD8(+) DCs failed to appear in HO-1(-/-) spleens. Conversely, GFP(+)HO-1(-/-) splenic cells also generated GFP(+)CD8(+) DCs in HO-1(+/+) mice. These results show that HO-1 is involved in splenic DC differentiation, and/or the homing of CD8(+) splenic DC precursors appears to be dependent on HO-1 expression by the host.

Neural Progenitors of the Postnatal and Adult Mouse Forebrain Retain the Ability to Self-Replicate, Form Neurospheres, and Undergo Multipotent Differentiation In Vivo

Somatic stem cells are reservoirs to replace lost cells or damaged tissue. Cells with neural stem cell (NSC) characteristics can be isolated from the postnatal mammalian brain into adulthood and expanded as neurospheres. We addressed the ability of these in vitro expanded putative NSCs to retain progenitor characteristics in vivo, in analogy to hematopoietic stem cells. When transplanted in utero, both postnatal and adult neural progenitors colonize host brains and contribute neurons and glia. In stark contrast to what has been reported when transplanted in postnatal hosts, epidermal growth factor-expanded cells also remain self-replicating and multipotent in vivo over many months and can be serially transplanted into multiple hosts. Surprisingly, embryonically transplanted NSCs remain in the neurogenic regions in adult hosts, where they express progenitor cell markers and continue to proliferate even after 6 months without tumor formation. These data indicate that spherogenic cells of the postnatal and adult mammalian brain retain their potential in vitro and in vivo throughout the life of the organism and beyond transplantation, which has important implications for cell replacement strategies.

Stage-Restricted Non-Metabolic Function for Aconitase Enzymes in Erythropoietin Programming of Erythroid Growth and Differentiation in Vivo

Erythropoietin (EPO) is the main cytokine responsible for red blood cell production in the human marrow. Signal transduction through the EPO receptor consists of multiple limbs that promote the proliferation, differentiation, and survival of erythroid progenitors. Regulatory loops that modulate EPO signaling are not fully understood, but could prove critical in providing new therapeutic approaches to EPO-refractory anemias. One such poorly understood loop is the regulation of EPO-driven erythropoiesis by iron. Previously presented work has established that iron deprivation acts in a lineage specific manner to block erythroid development and inhibit the activity of aconitase, a Krebs cycle enzyme that interconverts citrate and isocitrate. In the current studies, the causal relationship between aconitase inhibition and erythroid blockade was characterized in normal donor-derived mobilized human primary hematopoietic CD34+ cells and in adult wild type C57BL/6 mice. We hypothesized that critical thresholds of aconitase activity are required for specific facets of EPO signaling during discrete phases of erythroid development. CD34+ cells in erythroid-promoting culture conditions (EPO and iron) were strongly growth inhibited in a dose-dependent manner when treated with fluoroacetate, a reversible aconitase inhibitor. Interestingly, there was no increase in overall cell death in the growth impaired cultures, providing the first evidence that growth and survival signals in primary erythroid precursors can be dissociated. Aconitase blockade also inhibited EPO-dependent erythroid maturation, with decreased glycophorin A upregulation, diminished CD34 downregulation, and a sharp decrease in globin chain protein levels after 5 days of culture. Biochemical analysis of known EPO targets showed no alterations in the phosphorylation status of STAT5, Akt and PKCalpha, effectively ruling out a role for the corresponding pathways in the impaired growth and differentiation. Moreover, intracellular ATP levels were unaffected by aconitase inhibition, and alterations in AMPK activation, an intracellular sensor of the ATP:AMP ratio, could not be detected. These results argue against energy starvation as the cause of the observed developmental defects. Cell cycle analysis using propidium iodide showed no evidence for phase-specific arrest, excluding standard checkpoint mechanisms. Delayed addition or washout of fluoroacetate at various time points during cultures identified the existence of aconitase-dependent and subsequent aconitase-independent phases of human erythroid development. To extend these studies to in vivo erythropoiesis, C57BL/6 mice underwent fluoroacetate (n=10) or saline (n=10) treatment with continuous infusion pumps at a drug dose of 4 mg/kg/day. In vivo blockade of aconitase over a two-week period resulted in an anemia characterized by a significant decrease in mean hemoglobin (11.6 vs. 14.7 g/dL, P<0.001), hematocrit (37.3 vs. 44%, P<0.001) and red cell number (7.49 vs. 9.32 × 1012 cells/liter, P<0.001). No thrombocytopenia or neutropenia was noted. Reticulocytes were diminished (6.6 vs. 7.9%, P<0.001), and mean serum EPO levels were 3 fold higher in the treated animals (533.2 vs. 172.3 pg/ml, P<0.001). Flow cytometric analysis of the marrow erythroid compartment consistently showed accumulation of cells at a Ter119-bright C71-intermediate stage (n=3 for each group). In summary, our data establish a new function for aconitase in EPO-driven erythropoiesis that is distinct from its metabolic role in cellular energy homeostasis. Sustained aconitase activity is required for the proliferation and maturation of erythroid precursors, but does not impact survival or specific cell cycle phases. Furthermore, this requirement for aconitase activity is stage specific and restricted to an early phase of EPO-dependent erythroid development. Taken together, these results suggest the existence of a new regulatory loop in which levels of aconitase activity modulate EPO-mediated growth and maturation. This novel checkpoint could provide new targets in the treatment of many disorders of red cell production.

Differentiation In Vivo of Cardiac Committed Human Embryonic Stem Cells in Postmyocardial Infarcted Rats

Human embryonic stem (HES) cells can give rise to cardiomyocytes in vitro. However, whether undifferentiated HES cells also feature a myocardial regenerative capacity after in vivo engraftment has not been established yet. We compared two HES cell lines (HUES-1 and I6) that were specified toward a cardiac lineage by exposure to bone morphogenetic protein-2 (BMP2) and SU5402, a fibroblast growth factor receptor inhibitor. Real-time polymerase chain reaction (PCR) revealed that the cardiogenic inductive factor turned on expression of mesodermal and cardiac genes (Tbx6, Isl1, FoxH1, Nkx2.5, Mef2c, and alpha-actin). Thirty immunosuppressed rats underwent coronary artery ligation and, 2 weeks later, were randomized and received in-scar injections of either culture medium (controls) or BMP2 (+/-SU5402)-treated HES cells. After 2 months, human cells were detected by anti-human lamin immunostaining, and their cardiomyocytic differentiation was evidenced by their expression of cardiac markers by reverse transcription-PCR and immunofluorescence using an anti-beta myosin antibody. No teratoma was observed in hearts or any other organ of the body. The ability of cardiac-specified HES cells to differentiate along the cardiomyogenic pathway following transplantation into infarcted myocardium raises the hope that these cells might become effective candidates for myocardial regeneration.

Role of the Cldn6 Cytoplasmic Tail Domain in Membrane Targeting and Epidermal Differentiation In Vivo

It is widely recognized that the claudin (Cldn) family of four tetraspan transmembrane proteins is crucial for tight junction assembly and permeability barrier function; however, the precise role of the tail and loop domains in Cldn function is not understood. We hypothesized that the cytoplasmic tail domain of Cldn6 is crucial for membrane targeting and hence epidermal permeability barrier (EPB) formation. To test this hypothesis via a structure-function approach, we generated a tail deletion of Cldn6 (CDelta187) and evaluated its role in epidermal differentiation and EPB formation through its forced expression via the involucrin (Inv) promoter in the suprabasal compartment of the transgenic mouse epidermis. Even though a functional barrier formed, Inv-CDelta187 mice displayed histological and biochemical abnormalities in the epidermal differentiation program and stimulation of epidermal cell proliferation in both the basal and suprabasal compartments of the interfolliclar epidermis, leading to a thickening of the epidermis after 1 week of age that persisted throughout life. Although some membrane localization was evident, our studies also revealed a significant amount of not only Cldn6 but also Cldn10, Cldn11, and Cldn18 in the cytoplasm of transgenic epidermal cells as well as the activation of a protein-unfolding pathway. These findings demonstrate that the overexpression of a tail truncation mutant of Cldn6 mislocalizes Cldn6 and other Cldn proteins to the cytoplasm and triggers a postnatal increase in proliferation and aberrant differentiation of the epidermis, emphasizing the importance of the Cldn tail domain in membrane targeting and function in vivo.

Primary T Cell Expansion and Differentiation In Vivo Requires Antigen Presentation by B Cells

B cells are well documented as APC; however, their role in supporting and programming the T cell response in vivo is still unclear. Studies using B cell-deficient mice have given rise to contradictory results. We have used mixed BM chimeric mice to define the contribution that B cells make as APC. When the B cell compartment is deficient in MHC class II, while other APC are largely normal, T cell clonal expansion is significantly reduced and the differentiation of T cells into cytokine-secreting effector cells is impaired (in particular, Th2 cells). The development of the memory T cell populations is also decreased. Although MHC class II-mediated presentation by B cells was crucial for an optimal T cell response, neither a B cell-specific lack of CD40 (influencing costimulation) nor lymphotoxin alpha (influencing lymphoid tissue architecture) had any effect on the T cell response. We conclude that in vivo B cells provide extra and essential Ag presentation capacity over and above that provided by dendritic cells, optimizing expansion and allowing the generation of memory and effector T cells.

Different Effects of Amlodipine and Enalapril on the Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase-Extracellular Signal-Regulated Kinase Pathway for Induction of Vascular Smooth Muscle Cell Differentiation In Vivo

Although recent clinical trials have shown that amlodipine exerts antiatherogenic effects, the mechanism of these effects remains unknown. This study was designed to examine which signal transduction pathway might be important for the antiatherogenic property of amlodipine, as assessed by aortic smooth muscle cell (SMC) phenotypes in hypertension in vivo. Stroke-prone spontaneously hypertensive rats (SHRSP) were randomly treated with a vehicle, amlodipine, or enalapril while Wistar-Kyoto rats (WKY) used as controls were treated with only the vehicle. Both drugs were equally effective at reducing systolic blood pressure, and inhibiting the progression of aortic remodeling and fibrosis in comparison to those of vehicle-treated SHRSP. In the aortas of vehicle-treated SHRSP, the level of contractile-type smooth muscle (SM) myosin heavy chain (MHC) SM2 was significantly lower, whereas the level of synthetic-type MHC NMHC-B/SMemb was significantly higher compared with those in the WKY aortas. Compared to the vehicle-treated SHRSP group, both drugs significantly and equally shifted the aortic SMC phenotype in SHRSP toward the differentiated state by reducing NMHC-B/SMemb and increasing SM2. The levels of MKK6, p38 MAPK, MEK1 and p-42/44 ERK were significantly higher in the vehicle-treated SHRSP than in the WKY. Both drugs significantly reduced these values in the SHRSP aorta. Furthermore, the levels of MEK1 and p-42/44 ERK were significantly lower in the amlodipine- than in the enalapril-treated SHRSP group, whereas enalapril was more effective than amlodipine at increasing p-Akt and endothelial NO synthase in SHRSP aortas, which were significantly lower in the vehicle SHRSP group than in the WKY group. Thus, the MEK-ERK pathway might be one of the crucial determinants of the aortic SMC phenotype activated by amlodipine treatment of hypertension in vivo.

Expression of Cholecystokinin-2/Gastrin Receptor in the Murine Pancreas Modulates Cell Adhesion and Cell Differentiation in Vivo

The presence of gastrin and cholecystokinin-2 (CCK2) receptors in human preneoplastic and neoplastic gastrointestinal lesions suggests a role in cancer development. In addition to the growth-promoting action of gastrin, recently a role of the cholecystokinin-2/gastrin receptor (CCK2-R) modulating cellular morphology in cultured epithelial cells has been shown. Here, we have investigated in transgenic (ElasCCK2) mice whether ectopic expression of human CCK2-R in the exocrine pancreas affected epithelial differentiation. Cellular localization of cell adhesion molecules, differentiation markers, and transcription factors was determined using immunofluorescence techniques. Before tumor formation, expression and subcellular localization of proteins of the adherens junction complex, differentiation markers, and transcription factors were altered in ElasCCK2 exocrine pancreas, indicating an evolution from an acinar to a ductal phenotype. Loss of cell polarity, defective secretion, and loss of intercellular adhesion in acini of ElasCCK2 mice was confirmed by ultrastructural analysis. Finally, expression of the transgene in mice treated with the carcinogen azaserine resulted in enhanced size of preneoplastic lesions as well as an increased degree of acinar-ductal transdifferentiation. Thus, these data represent the first evidence for the CCK2-R modulating intercellular adhesion and cell fate in vivo and show that these alterations may contribute to enhanced sensitivity of ElasCCK2 pancreas to chemical carcinogens.

Characterization of Dendritic Cells that Induce Tolerance and T Regulatory 1 Cell Differentiation In Vivo

Active suppression is mediated by a subpopulation of CD4 + T cells that prevents autoimmunity. However, the mechanisms involved in their differentiation in vivo are currently under intensive research. Here we show that in vitro culture of bone marrow cells in the presence of IL-10 induces the differentiation of a distinct subset of dendritic cells with a specific expression of CD45RB. These CD11c lowCD45RB high DCs are present in the spleen and lymph nodes of normal mice and are significantly enriched in the spleen of IL-10 Tg mice. These natural or in vitro-derived DCs display plasmacytoid morphology and an immature-like phenotype, and secrete high levels of IL-10 after activation. OVA peptide-pulsed CD11c lowCD45RB high DCs specifically induce tolerance through the differentiation of Tr1 cells in vitro and in vivo. Our findings identify a natural DC subset that induces the differentiation of Tr1 cells and suggest their therapeutic use.

CTLA-4 Regulates Tolerance Induction and T Cell Differentiation In Vivo

Cytotoxic T lymphocyte Ag-4 (CTLA-4; CD152) is an important T cell regulatory molecule. In vitro experiments have shown that the blockade of signals through CTLA-4 augments T cell expansion, while CTLA-4 cross-linking results in decreased T cell proliferation due to decreased IL-2 production. However, less is known about the role of CTLA-4 in regulating an ongoing immune response. In this study, we examined the role of CTLA-4 in the expansion, decline, tolerization, and differentiation of T cells following treatment with staphylococcal enterotoxin B (SEB). Anti-CTLA-4 treatment resulted in increased numbers of SEB-reactive T cells and blockade of subsequent tolerance induction. Further examination of the SEB-reactive cells from anti-CTLA-4-treated mice demonstrated that both the CD4+ and CD8+ Vbeta8+ T cells produced IL-4, providing evidence that not only do signals through CTLA-4 regulate T cell-tolerizing events, but they also play an important role in the differentiation of T cells in vivo.

Characterization of an 80-kD Membrane Glycoprotein (GP80) of Human Keratinocytes: A Marker for Commitment to Terminal Differentiation In Vivo and In Vitro

We have characterized an 80-kD cell-surface glycoprotein (gp80) identified by monoclonal antibody BT 15, the expression of which is closely associated with a commitment to terminal squamous or follicular differentiation of keratinocytes in normal adult and fetal human epidermis. Maximum expression was found in the suprabasal layers, but basal cells located at the epidermal sulci were also clearly positive, in contrast to the virtually negative basal cells at the epidermal ridges. This protein was also present in benign hyperproliferative disorders of the epidermis (i.e., common warts, keratoacanthoma, psoriasis, and seborrhoic keratoses) with monoclonal antibody BT 15 preferentially staining suprabasal cells and some basal cells at the epidermal sulci. Gp80 was completely lacking in most basal cell carcinomas; the only exceptions were two cases of partially cornifying tumors that were strongly stained around keratotic pearls. In squamous cell carcinomas, gp80 was expressed in keratinized areas of the tumors. In organotypic keratinocyte cultures that resemble the in vivo situation, gp80 was strongly expressed in the suprabasal layers. However, unlike known markers for terminal differentiation, gp80 was weakly expressed by basal cells. Synthesis rates of gp80 were high in keratinocyte cell suspensions freshly prepared from skin, and decreased in primary cultures and first and second subcultures (ratio 10:4:2:1). Elevated concentrations of the Ca++ that increased stratification of cultured keratinocytes resulted in a two- to threefold increase of gp80 synthesis. GP80 was not synthesized at detectable levels by the immortal keratinocyte cell line HaCaT; however, it was expressed in HaCaT cultures treated with mitomycin C, indicating an association with cessation of growth. Pulse-chase experiments revealed that gp80 is synthesized from a 55-kD precursor molecule, the maturation of which was prevented by treating cells with tunicamycin. Glycosidase digestion of BT 15 immunoprecipitates from untreated cells indicated that the predominant post-translational modification of the protein is N-linked glycosylation. Our data indicate that gp80 is a glycoprotein that is expressed by growth-arrested human keratinocytes or as part of the terminal differentiation program.