Infusion of high concentration of lactate in perfused liver,simulating in vivo hyperlactatemia,prevents the reduction of gluconeogenesis in Walker-256 tumor-bearing rats

Gluconeogenesis (GN) is increased in patients with cancer cachexia, but is reduced in liver perfusion of Walker-256 tumor-bearing cachectic rats (TB rats). The causes of these differences are unknown. We investigated the influence of circulating concentrations of lactate (NADH generator) and NADH on GN in perfused livers of TB rats. Lactate, at concentrations similar to those found on days 5 (3.0 mM), 8 (5.5 mM), and 12 (8.0 mM) of the tumor, prevented the reduction of GN from 2.0 mM lactate (lactatemia of healthy rat) in TB rats. NADH, 50 or 75 μM, but not 25 μM, increased GN from 2.0 mM lactate in TB rats to higher values than healthy rats. High concentrations of pyruvate (no NADH generator, 5.0 and 8.0 mM) did not prevent the reduction of GN from 2.0 mM pyruvate in TB rats. However, 50 or 75 μM NADH, but not 25 μM, increased GN from 2.0 mM pyruvate in TB rats to similar or higher values than healthy rats. High concentration of glutamine (NADH generator, 2.5 mM) or 50 μM NADH prevented the reduction of GN from 1 mM glutamine in TB rats. Intraperitoneal administration of pyruvate (1.0 mg/kg) or glutamine (0.5 mg/kg) similarly increased the glycemia of healthy and TB rats. In conclusion, high lactate concentration, similar to hyperlactatemia, prevented the reduction of GN in perfused livers of TB rats, an effect probably caused by the increased redox potential (NADH/NAD⁺). Thus, the decreased GN in livers from TB rats is due, at least in part, to the absence of simulation of in vivo hyperlactatemia in liver perfusion studies.     

Stem cells from human amniotic fluid exert immunoregulatory function via secreted indoleamine 2,3‐dioxygenase1

Although human amniotic fluid does contain different populations of foetal‐derived stem cells, scanty information is available on the stemness and the potential immunomodulatory activity of in vitro expanded, amniotic fluid stem cells. By means of a methodology unrequiring immune selection, we isolated and characterized different stem cell types from second‐trimester human amniotic fluid samples (human amniotic fluid stem cells, HASCs). Of those populations, one was characterized by a fast doubling time, and cells were thus designated as fHASCs. Cells maintained their original phenotype under prolonged in vitro passaging, and they were able to originate embryoid bodies. Moreover, fHASCs exhibited regulatory properties when treated with interferon (IFN)‐γ, including induction of the immunomodulatory enzyme indoleamine 2,3‐dioxygenase 1 (IDO1). On coculture with human peripheral blood mononuclear cells, IFN‐γ–treated fHASCs caused significantly decreased T‐cell proliferation and increased frequency in CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells. Both effects required an intact IDO1 function and were cell contact‐independent. An unprecedented finding in our study was that purified vesicles from IFN‐γ–treated fHASCs abundantly expressed the functional IDO1 protein, and those vesicles were endowed with an fHASC‐like regulatory function. In vivo, fHASCs were capable of immunoregulatory function, promoting allograft survival in a mouse model of allogeneic skin transplantation. This was concurrent with the expansion of CD4⁺ CD25⁺ Foxp3⁺ T cells in graft‐draining lymph nodes from recipient mice. Thus fHASCs, or vesicles thereof, may represent a novel opportunity for immunoregulatory maneuvers both in vitro and in vivo.     

A GpC-Rich Oligonucleotide Acts on Plasmacytoid Dendritic Cells To Promote Immune Suppression

Short synthetic oligodeoxynucleotides (ODNs) rich in CpG or GpG motifs have been considered as potential modulators of immunity in clinical settings. In this study, we show that a synthetic GpC-ODN conferred highly suppressive activity on mouse splenic plasmacytoid dendritic cells, demonstrable in vivo in a skin test assay. The underlying mechanism involved signaling by noncanonical NF-κB family members and TGF-β-dependent expression of the immunoregulatory enzyme IDO. Unlike CpG-ODNs, the effects of GpC-ODN required TLR7/TRIF-mediated but not TLR9/MyD88-mediated events, as do sensing of viral ssRNA and the drug imiquimod. Induction of IDO by a GpC-containing ODN could also be demonstrated in human dendritic cells, allowing those cells to assist FOXP3(+) T cell generation in vitro. Among potentially therapeutic ODNs, this study identifies GpC-rich sequences as novel activators of TLR7-mediated, IDO-dependent regulatory responses.     

Immunity and tolerance to Aspergillus involve functionally distinct regulatory T cells and tryptophan catabolism

The inherent resistance to diseases caused by Aspergillus fumigatus suggests the occurrence of regulatory mechanisms that provide the host with adequate defense without necessarily eliminating the fungus or causing unacceptable levels of host damage. In this study, we show that a division of labor occurs between functionally distinct regulatory T cells (Treg) that are coordinately activated by a CD28/B-7-dependent costimulatory pathway after exposure of mice to Aspergillus conidia. Early in infection, inflammation is controlled by the expansion, activation and local recruitment of CD4+CD25+ Treg capable of suppressing neutrophils through the combined actions of IL-10 and CTLA-4 on indoleamine 2,3-dioxygenase. The levels of IFN-gamma produced in this early phase set the subsequent adaptive stage by conditioning the indoleamine 2,3-dioxygenase-dependent tolerogenic program of dendritic cells and the subsequent activation and expansion of tolerogenic Treg, which produce IL-10 and TGF-beta, inhibit Th2 cells, and prevent allergy to the fungus. The coordinate activation of Treg may, however, be subverted by the fungus, as germinating conidia are capable of interfering with anti-inflammatory and tolerogenic Treg programs. Thus, regulation is an essential component of the host response in infection and allergy to the fungus, and its manipulation may allow the pathogen to overcome host resistance and promote disease.     

Predatory behaviour of common kestrels (Falco tinnunculus) in the wild

The predatory behaviour of eight wild adult common kestrels (Falco tinnunculus) was recorded during predatory tests carried out in the wild under controlled conditions. Those birds were offered one laboratory mouse each, which was restrained on a base, thereby simulating natural predation. The predatory sequence was recorded directly, but also video-taped. The sequence was rather homogeneous among the kestrels, with most kestrels starting the attack glide from a perch, then capturing the prey at high speed. The mouse was grabbed directly, upon landing; in one instance, however, it disentangled from the bird’s foot and was captured after a few seconds. The target was usually grabbed at the shoulders or neck, or at the trunk. Soon after capture the kestrel flew to a distant perch, where it usually stroked the prey with one single peck, before starting ingestion, which began about 1 min time after prey grasping. Our results are the first to show the possibility of maintaining standardized conditions to study the predatory behaviour of birds of prey. As they are very similar to those obtained in previous tests carried out in captivity using rehabilitated kestrels, our results also confirm earlier ones showing that the kestrel’s predatory behaviour is rather stereotyped—i.e. performed with limited variation—and that it can be studied reliably even in captivity.     

Complex Segmental Duplications Mediate a Recurrent dup(X)(p11.22-p11.23) Associated with Mental Retardation, Speech Delay, and EEG Anomalies in Males and Females

Submicroscopic copy-number variations make a considerable contribution to the genetic etiology of human disease. We have analyzed subjects with idiopathic mental retardation (MR) by using whole-genome oligonucleotide-based array comparative genomic hybridization (aCGH) and identified familial and de novo recurrent Xp11.22-p11.23 duplications in males and females with MR, speech delay, and a peculiar electroencephalographic (EEG) pattern in childhood. The size of the duplications ranges from 0.8–9.2 Mb. Most affected females show preferential activation of the duplicated X chromosome. Carriers of the smallest duplication show X-linked recessive inheritance. All other affected individuals present dominant expression and comparable clinical phenotypes irrespective of sex, duplication size, and X-inactivation pattern. The majority of the rearrangements are mediated by recombination between flanking complex segmental duplications. The identification of common clinical features, including the typical EEG pattern, predisposing genomic structure, and peculiar X-inactivation pattern, suggests that duplication of Xp11.22-p11.23 constitutes a previously undescribed syndrome.     

Modulation of paraoxonase 1 and 3 expression after moderate exercise training in the rat

Paraoxonases (PONs) are a small family of antioxidant enzymes whose antiatherogenic activity is well known. The aim of the present study was the evaluation of the effects of moderate aerobic training on their expression using a rat model. In order to discriminate between PON1 and PON3 enzymatic activity, we took advantage of some differences in their substrate preferences. PON1 and PON3 enzymatic activities and their protein levels were analyzed in plasma and in liver microsomes, and their mRNA levels in the liver. Exercise training did not affect PON1 expression or enzymatic activity but increased PON3 mRNA, protein levels, and enzymatic activity. Training also induced variations in plasma membrane composition, including an increase in polyunsaturated and a decrease in mono- and di-unsaturated fatty acids. On the other hand, acute exercise inhibited PON activities while increasing PON3 protein content in liver microsomes and reversing the relative composition in mono-, di-, and poly-unsaturated fatty acids, suggesting that physical stress, by altering membrane composition, may impair PON release from liver membranes. In conclusion, we documented, for the first time, the presence of PON3 in rat serum and, notably, found that the upregulation of PON3, rather than PON1, appears to be associated with physical training.     

Evaluation of Morphology and Seed Production of Alpine Cat's Tail [Phleum rhaeticum (Humph.) Rauschert] Germplasm in View of its Use for Ecological Restoration at High Altitude

"Site-specific" vegetation, that is germplasm ecologically adapted to the prevailing pedoclimatic conditions and native to the target region, is increasingly required in the revegetation of disturbed areas at high altitude. Some key site-specific species have been reported for the Alpine region. Better knowledge is needed on the existing variability of these species, while high seed yield is required to ensure the availability of commercially adequate seed supply. Natural populations of Alpine cat's tail from the Rhaetian Alps were evaluated for morphology and seed production at a mountain and at a lowland site of northern Italy aiming at (1) identifying possible superior populations for the selection of adapted high-yielding cultivars and (2) verifying whether the seed multiplication of site-specific species could also be undertaken in areas different from those of origin. The germplasm from one valley featured an interesting architecture of traits, representing a possible ideotype for seed production purposes. Individual populations across different valleys also proved to be a valuable source of variation for economically useful traits. A discussion on possible implications of use of nonlocal germplasm sources for restoration purposes is provided. The lowland site gave much lower seed production than the mountain location and experienced outstanding plant mortality after the first summer. The specific site conditions, namely the occurrence of an appreciable level of heat and drought stress in summer and a severe rust infection rather than the altitude per se, limited the suitability of this site for seed production of the alpine germplasm.     

Endothelial cells from cord blood CD133+CD34+ progenitors share phenotypic, functional and gene expression profile similarities with lymphatics

The existence of endothelial progenitor cells (EPC) with high cell-cycle rate in human umbilical cord blood has been recently shown and represents a challenging strategy for therapeutic neovascularization. To enhance knowledge for future cellular therapy, we compared the phenotypic, functional and gene expression differences between EPC-derived cells generated from cord blood CD34⁺ cells, and lymphatic and macrovascular endothelial cells (EC) isolated from human foreskins and umbilical veins, respectively. Under appropriate culture conditions, EPC developed into fully matured EC with expression of similar endothelial markers as lymphatic and macrovascular EC, including CD31, CD36, von Willebrand factor FVIII, CD54 (ICAM-1), CD105 (endoglin), CD144 (VE-cadherin), Tie-1, Tie-2, VEGFR-1/Flt-1 and VEGFR-2/Flk-1. Few EPC-derived cells became positive for LYVE-1, indicating their origin from haematopoietic stem cells. However they lacked expression of other lymphatic cell-specific markers such as podoplanin and Prox-1. Functional tests demonstrated that the cobblestone EPC-derived cells up-regulated CD54 and CD62E expression in response to TNF-α, incorporated DiI-acetylated low-density liproprotein and formed cord- and tubular-like structures with capillary lumen in three-dimensional collagen culture – all characteristic features of the vascular endothelium. Structures compatible with Weibel-Palade bodies were also found by electron microscopy. Gene microarray profiling revealed that only a small percentage of genes investigated showed differential expression in EPC-derived cells and lymphatic EC. Among them were adhesion molecules, extracellular matrix proteins and cytokines. Our data point to the close lineage relationship of both types of vascular cells and support the theory of a venous origin of the lymphatic system.