Culture of mouse amniotic fluid-derived cells on irradiated STO feeders results in the generation of primitive endoderm cell lines capable of self-renewal in vitro.

The cells present in amniotic fluid (AF) are currently used for prenatal diagnosis of fetal anomalies but are also a potential source of cells for cell therapy. To better characterize putative progenitor cell populations present in AF, we used culture conditions that support self-renewal to determine if these promoted the generation of stable cell lines from AF-derived cells (AFC). Cells isolated from E11.5 mouse were cultured on irradiated STO fibroblast feeder layers in human embryonic germ cell derivation conditions. The cultures grew multicellular epithelial colonies that could be repropagated from single cells. Reverse transcription semiquantitative polymerase chain reaction of established cell lines revealed that they belonged to the extraembryonic endoderm (ExEn) expressing high levels of Gata6, Gata4, Sox17, Foxa2 and Sox7 mRNA. Hierarchical clustering based on the whole transcriptome expression profile of the AFC lines (AFCL) shows significant correlation between transcription profiles of AFCL and blastocyst-derived XEN, an ExEn cell line. In vitro differentiation of AFCL results in the generation of cells expressing albumin and α-fetoprotein (AFP), while intramuscular injection of AFCL into immunodeficient mice produced AFP-positive tumors with primitive endodermal appearance. Hence, E11.5 mouse AF contains cells that efficiently produce XEN lines. These AF-derived XEN lines do not spontaneously differentiate into embryonic-type cells but are phenotypically stable and have the capacity for extensive expansion. The lack of requirement for reprogramming factors to turn AF-derived progenitor cells into stable cell lines capable of massive expansion together with the known ability of ExEn to contribute to embryonic tissue suggests that this cell type may be a candidate for banking for cell therapies.

Extracorporeal photopheresis: Lighting the way to immunomodulation.

Photopheresis, initially established as an effective treatment of cutaneous T-cell lymphoma, has in recent years also been used to treat chronic graft vs. host disease, heart transplant rejection, and several other conditions requiring immunosuppression. Despite reported beneficial results of this procedure in treatment of various conditions, randomized controlled clinical trials are lacking for the majority of suggested indications. Furthermore, the mechanisms of action of this procedure are still unclear. Deeper understanding of the molecular basis of photopheresis-based immunomodulation will allow better selection of patients to be treated and will facilitate development of novel, minimally toxic immunomodulatory treatments.

In vitro function and phagocytosis of galactosylated platelet concentrates after long-term refrigeration.

BACKGROUND: Short-term refrigeration of platelets (PLTs) in the absence of plasma results in their rapid clearance after transfusion. Blocking beta-N-acetylglucosamine (beta-GlcNAc) residues of glycoprotein Ibalpha (GPIbalpha) with galactose prevents binding of refrigerated human and mouse PLTs to macrophages and prolongs the circulation times of refrigerated mouse PLTs. PLT-associated galactosyltransferase efficiently galactosylates chilled PLTs in the presence of its substrate UDP-galactose is added to PLT-rich plasma. STUDY DESIGN AND METHODS: To characterize the hemostatic function of refrigerated and galactosylated human PLTs processed in the blood bank, PLT aggregation was studied in vitro under static and flow conditions and expression of integrin beta3 (CD61), CD62P (P-selectin), GPIbalpha (CD42b), annexin V binding, and integrin alphaIIbeta3 activation with flow cytometry. Affinity of macrophages for galactosylated refrigerated PLTs was evaluated with THP-1 cells, which recognize and phagocytize refrigerated PLTs. RESULTS: PLTs refrigerated and galactosylated for 14 days 1) maintained their ability to aggregate when exposed to agonists in a standard aggregometry assay, 2) showed less pronounced changes in surface expression of GPIbalpha compared with room temperature (RT)-stored PLTs, 3) increased P-selectin expression, and 4) were poorly phagocytized by differentiated THP-1 cells in vitro. In addition, it is shown that refrigeration of PLTs does not affect their adhesive properties under in vitro flow conditions. CONCLUSION: It is shown that refrigerated human PLTs retain in vitro function better than RT PLTs during storage and demonstrate that galactosylation prevents recognition of stored refrigerated PLTs by macrophages in vitro.

Elevated soluble ICAM-1 levels induce immune deficiency and increase adiposity in mice.

Elevated soluble intercellular adhesion molecule-1 (sICAM-1) levels have been found in many pathological conditions, including obesity. To determine the effects of elevated sICAM-1 on immune responses and metabolism, we generated a transgenic mouse model overexpressing the extracellular domain of mouse ICAM-1 in the liver. The mice, showing 10-fold higher sICAM-1 levels than wild-type mice, presented elevated neutrophil count. Despite this, after intraperitoneal injection of thioglycollate, neutrophil recruitment into the peritoneal cavity was reduced, and the delayed macrophage recruitment was also affected in the transgenic mice compared with wild-type mice. Inhibition of contact hypersensitivity response in the sICAM-1 transgenic mice was comparable to ICAM-1-deficient mice and characterized by significantly less ear swelling and inflammatory cell infiltration than in wild-type mice. sICAM-1transgenic mice were more susceptible to weight gain on a Western-type diet than wild-type mice, and older animals showed excessive fat accumulation, again reminiscent of ICAM-1-deficient mice. Together, these data indicate that sICAM-1 interferes with ICAM-1-mediated cell-cell interactions, which could produce immune-suppressant effects and alteration of metabolism in persons with high levels of this soluble adhesion receptor.

ICAM-1 and beta2 integrin deficiency impairs fat oxidation and insulin metabolism during fasting.

Intercellular adhesion molecule 1 (ICAM-1) and beta2 integrins play critical roles in immune responses. ICAM-1 may also participate in regulation of energy balance because ICAM-1-deficient mice become obese on a high-fat diet. We show that mice deficient in these adhesion receptors are unable to respond to fasting by up-regulation of fatty acid oxidation. Normal mice, when fasted, exhibit reduced circulating neutrophil counts and increased ICAM-1 expression and neutrophil recruitment in liver. Mice lacking ICAM-1 or beta2 integrins fail to show these responses--instead they become hypoglycemic with steatotic livers. Fasting ICAM-1-deficient mice reduce insulin more slowly than wild-type mice. This produces fasting hyperinsulinemia that prevents activation of adenosine mono-phosphate (AMP)-activated protein kinase in muscles and liver, which results in decreased import of long chain fatty acids into mitochondria. Thus, we show a new role for immune cells and their adhesion receptors in regulating metabolic response to fasting.