Cancer-cell-secreted extracellular vesicles suppress insulin secretion through miR-122 to impair systemic glucose homeostasis and contribute to tumour growth.

Epidemiological studies demonstrate an association between breast cancer (BC) and systemic dysregulation of glucose metabolism. However, how BC influences glucose homeostasis remains unknown. We show that BC-derived extracellular vesicles (EVs) suppress pancreatic insulin secretion to impair glucose homeostasis.

Discovery and preclinical evaluation of anti-miR-17 oligonucleotide RGLS4326 for the treatment of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in either PKD1 or PKD2 genes, is one of the most common human monogenetic disorders and the leading genetic cause of end-stage renal disease. Unfortunately, treatment options for ADPKD are limited. Here we report the discovery and characterization of RGLS4326, a first-in-class, short oligonucleotide inhibitor of microRNA-17 (miR-17), as a potential treatment for ADPKD.

Impact of a patient-derived hepatitis C viral RNA genome with a mutated microRNA binding site.

Hepatitis C virus (HCV) depends on liver-specific microRNA miR-122 for efficient viral RNA amplification in liver cells. This microRNA interacts with two different conserved sites at the very 5' end of the viral RNA, enhancing miR-122 stability and promoting replication of the viral RNA. Treatment of HCV patients with oligonucleotides that sequester miR-122 resulted in profound loss of viral RNA in phase II clinical trials.

Immune phenotype and function of natural killer and T cells in chronic hepatitis C patients who received a single dose of anti-MicroRNA-122, RG-101.

Unlabelled: MicroRNA-122 is an important host factor for the hepatitis C virus (HCV). Treatment with RG-101, an N-acetylgalactosamine-conjugated anti-microRNA-122 oligonucleotide, resulted in a significant viral load reduction in patients with chronic HCV infection. Here, we analyzed the effects of RG-101 therapy on antiviral immunity.

Safety, tolerability, and antiviral effect of RG-101 in patients with chronic hepatitis C: a phase 1B, double-blind, randomised controlled trial.

Background: miR-122 is an important host factor for hepatitis C virus (HCV) replication. The aim of this study was to assess the safety and tolerability, pharmacokinetics, and antiviral effect of a single dose of RG-101, a hepatocyte targeted N-acetylgalactosamine conjugated oligonucleotide that antagonises miR-122, in patients with chronic HCV infection with various genotypes.

Methods: In this randomised, double-blind, placebo-controlled, multicentre, phase 1B study, patients were randomly assigned to RG-101 or placebo (7:1).

An integrated approach to extreme thermostabilization and affinity maturation of an antibody.

Antibodies are important tools for a broad range of applications due to their high specificity and ability to recognize virtually any target molecule. However, in order to be practically useful, antibodies must be highly stable and bind their target antigens with high affinity. We present a combinatorial approach to generate high-affinity, highly stable antibodies through the design of stable frameworks, specificity grafting and maturation via somatic hypermutation in vitro.

Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies.

A novel approach has been developed for the isolation and maturation of human antibodies that replicates key features of the adaptive immune system by coupling in vitro somatic hypermutation (SHM) with mammalian cell display. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID), and can be replicated in non-B cells through expression of recombinant AID. A library of human antibodies, based on germline V-gene segments with recombined human regions was used to isolate low-affinity antibodies to human β nerve growth factor (hβNGF).

Keratinocyte growth factor and the transcription factors C/EBP alpha, C/EBP delta, and SREBP-1c regulate fatty acid synthesis in alveolar type II cells.

Strategies to stimulate endogenous surfactant production require a detailed understanding of the regulation of lipogenesis in alveolar type II cells. We developed culture conditions in which keratinocyte growth factor (KGF) stimulates fatty acid and phospholipid synthesis. KGF stimulated acetate incorporation into phosphatidylcholine, disaturated phosphatidylcholine, and phosphatidylglycerol more than 5% rat serum alone.

Recombinant human interleukin-11 synergizes with steel factor and interleukin-3 to promote directly the early stages of murine megakaryocyte development in vitro.

The authors studied the role that interleukin (IL)-11 plays during the early stages of megakaryocyte (MK) development by investigating its in vitro effects on cell subpopulations enriched for bone marrow primitive progenitor cells and early and late committed progenitor cells. Progenitor subpopulations were isolated from bone marrow of normal or 5-fluorouracil (5FU)-treated mice and separated by sorting based on the surface antigens Sca-1, c-kit, and CD34. Functional analysis of the cell subpopulations, 5FU Lin(-)Sca-1(+)c-kit(+) or normal bone marrow (NBM) Lin(-)Sca-1(+)c-kit(+)CD34(-)cells, indicated that exposure of these cells to recombinant human (rh)IL-11 in combination with steel factor (SF) stimulates the formation of colonies in methylcellulose and their proliferation in single cell-containing liquid cultures.

Defects in hemopoietic stem cell activity in Ikaros mutant mice.

Here we provide evidence that the Ikaros family of DNA binding factors is critical for the activity of hemopoietic stem cells (HSCs) in the mouse. Mice homozygous for an Ikaros null mutation display a >30-fold reduction in long-term repopulation units, whereas mice homozygous for an Ikaros dominant negative mutation have no measurable activity. The defect in HSC activity is also illustrated by the ability of wild-type marrow to repopulate unconditioned Ikaros mutants.

Bone morphogenetic protein 9 is a potent synergistic factor for murine hemopoietic progenitor cell generation and colony formation in serum-free cultures.

The effect of the recently cloned cytokine bone morphogenetic protein 9 (BMP-9) on colony formation and generation in vitro clonable hematopoietic progenitors (CFU-C) in serum-free liquid cultures (LC) of both normal and post-5-fluorouracil murine bone marrow cells was studied in the presence of various other cytokines. In LC, BMP-9 concentrations of 100 ng or more per ml led to complete inhibition of Steel Factor (SF) + interleukin-11 (IL-11) or IL-12 supported CFU-C generation, which was partly abrogated when IL-3 was additionally included. We found this inhibitory effect of BMP-9 to be mediated by an increased TGF-beta1 elaboration and TGF-beta1 mRNA expression in bone marrow cells with increasing BMP-9 concentrations.

Differential involvement of a Fas-CPP32-like protease pathway in apoptosis of TCR/CD9-costimulated, naive T cells and TCR-restimulated, activated T cells.

Our previous study showed that CD9 costimulation of TCR-triggered naive T cells elicits activation ([3H]TdR incorporation) that is similar to CD28 costimulation; however, unlike CD28 costimulation, CD9 costimulation results in apoptosis of these previously activated T cells. Here, we investigated whether the apoptosis occurring after TCR/CD9 stimulation is associated with a death pathway involving Fas stimulation and Fas-mediated caspase activation as observed in activation-induced cell death (AICD). In contrast to AICD, the apoptosis resulting from TCR/CD9 stimulation in C57BL/6 T cells was independent of Fas, because this form of apoptosis was not prevented by anti-Fas ligand mAb and was also induced in MRL/lpr T cells.

A fundamental difference in the capacity to induce proliferation of naive T cells between CD28 and other co-stimulatory molecules.

T cell activation requires two signals: a signal from the TCR and a co-stimulatory signal provided by antigen-presenting cells (APC). In addition to CD28, multiple molecules on the T cell have been described to deliver co-stimulatory signals. Here, we investigated whether there exist quantitative or qualitative differences in the co-stimulatory capacity between CD28 and other molecules.

CD9-mediated costimulation of TCR-triggered naive T cells leads to activation followed by apoptosis.

The induction of full activation or death in TCR-triggered T cells depends largely on whether appropriate costimulatory signals are provided. In this study, we show that the costimulation of CD9 on naive T cells during TCR stimulation results in transient, albeit potent, activation followed by apoptosis, rather than full activation. Anti-CD9 mAb synergized with suboptimal doses of anti-CD3 mAb in inducing T cell activation.

Synergy between CD28 and CD9 costimulation for naive T-cell activation.

Our previous study demonstrated that CD9 is expressed on most mature naive T-cells and delivers a potent costimulatory signal that functions independently of CD28. Here, we investigated whether this CD9-mediated signal is different from the CD28-mediated signal in the mode of costimulation and whether both signals function synergistically for T-cell activation. Anti-CD9 or anti-CD28 monoclonal antibody (mAb) increased [3H]TdR incorporation of naive T-cells in the absence of antigen-presenting cells (APC) when coimmobilized with submitogenic doses of anti-CD3 mAb.

Aiolos, a lymphoid restricted transcription factor that interacts with Ikaros to regulate lymphocyte differentiation.

Development of the lymphoid system is dependent on the activity of zinc finger transcription factors encoded by the Ikaros gene. Differences between the phenotypes resulting from a dominant-negative and a null mutation in this gene suggest that Ikaros proteins act in concert with another factor with which they form heterodimers. Here we report the cloning of Aiolos, a gene which encodes an Ikaros homologue that heterodimerizes with Ikaros proteins.

In vitro differentiation of embryonic stem cells: immunophenotypic analysis of cultured embryoid bodies.

The process of in vitro embryonic stem cell differentiation and embryoid body development was monitored using a panel of antibodies against surface markers traditionally associated with embryonic tissue (Forssman, SSEA-1) and hematopoietic progenitor cells (Fall-3, HSA, Sca-1, Thy-1.2, ER-MP12, CD45, AA4.1, and c-kit).

Structural identification of the hematopoietic progenitor antigen ER-MP12 as the vascular endothelial adhesion molecule PECAM-1 (CD31).

The monoclonal antibody ER-MP12 was recently described to recognize an antigen present on cell subpopulations of adult mouse bone marrow, including pluripotent hematopoietic stem cells. In an effort to understand the function of ER-MP12 antigen in hematopoiesis, we used biochemical and physical methods to determine its identity. ER-MP12 antigen was isolated by immunoprecipitation from FDCP-1 cell membrane proteins, yielding a glycosylated 113-kDa band upon analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

Molecular cloning and characterization of murine interleukin-11.

Human interleukin-11 (IL-11) has been shown to have pleiotropic action on hematopoietic, hepatic, stromal, epithelial, neural, and osteoclast cells. In the present work, the murine IL-11 cDNA has been isolated from a fetal thymic cell line, and its structure and function compared with human IL-11. The murine protein was demonstrated to have identical actions on the proliferation of a murine plasmacytoma cell line, murine primitive bone marrow progenitor cells, and megakaryocyte precursors.

A role for CD9 molecules in T cell activation.

Costimulation mediated by the CD28 molecule plays an important role in optimal activation of T cells. However, CD28-deficient mice can mount effective T cell-dependent immune responses, suggesting the existence of other costimulatory systems. In a search for other costimulatory molecules on T cells, we have developed a monoclonal antibody (mAb) that can costimulate T cells in the absence of antigen-presenting cells (APC).

Interleukin-4 (IL-4) in combination with IL-11 or IL-6 reverses the inhibitory effect of IL-3 on early B lymphocyte development.

We have previously described a two-step methylcellulose culture system in which individual primitive progenitors from 5-fluorouracil (5-FU)-treated mice were shown to have both myeloid and B lymphoid differentiation capacity. Highly enriched Lin-Sca+FU2d BM cells were cultured in methylcellulose in the presence of Steel factor (SF), interleukin-7 (IL-7), and pokeweed mitogen stimulated spleen cell conditioned medium (PWM-SCM). Primary mixed myeloid colonies were replated after 8-11 days into secondary cultures containing SF and IL-7, which supported the generation of B220+sIgM- pre-B cell colonies.

The role of recombinant interleukin 11 in megakaryocytopoiesis.

Recombinant human interleukin 11 (rHuIL-11) is a multifunctional cytokine with activities on a broad range of hematopoietic cells including primitive stem cells and mature progenitor cells. Analysis of rHuIL-11 in vitro has revealed that its hematopoietic activities are predominantly a result of synergistic interactions with other early-acting factors such as IL-3 and Steel factor. Studies indicate that rHuIL-11 acts directly on purified stem and progenitor cell populations and can support the growth of colony forming units-megakaryocyte in these cultures.

Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay.

Stroma-dependent long-term bone marrow cultures (LTBMC) assay the ability of primitive haematopoietic stem cells (HSC) for long-term production of clonable progenitors. We have developed a limiting dilution type LTBMC assay allowing frequency analysis of transiently repopulating HSC and long-term culture initiating cells (LTC-IC) without the necessity to replate large numbers of wells. Normal or 5-FU-treated Ficoll bone marrow cells (BMC), or BMCs sorted on CD34 or HLA-DR expression, or Rh123 retention, (input range 40-70,000 CFU-GM/BFU-E/10(5) cells) were plated at limiting dilution on unirradiated adherent layers formed by a novel murine preadipose cell line (FBMD-1).

Synergistic effects of interleukin-11 with other growth factors on the expansion of murine hematopoietic progenitors and maintenance of stem cells in liquid culture.

The effect of interleukin-11 (IL-11), alone and in combination with other hematopoietic growth factors, was studied in liquid suspension cultures of murine bone marrow. Stem and progenitor cell contents of input bone marrow 2 days after 5-fluorouracil (5-FU) treatment (FU2dBM) and lineage-depleted, Sca-1-positive FU2dBM (Lin-Sca+Fu2dBM) were compared with output values obtained after 6 days in different growth factor combinations. Stem and progenitor cell recoveries were measured by long-term in vivo competitive repopulation, spleen colony-forming unit (CFU-S), and in vitro limiting dilution long-term bone marrow culture (cobblestone area-forming cell [CAFC]) assays.