Use of a Thin Layer Assay for Assessing the Angiogenic Potential of Endothelial Cells In Vitro.

Angiogenesis is essential for wound healing and regeneration and plays a significant role in several pathologies including cancer and atherosclerosis. In vitro assays offer simple and powerful tools for investigating the regulation of the angiogenic functions of primary endothelial cells (ECs) before moving to in vivo studies. The classic in vitro two-dimensional angiogenesis assay utilizes Basement Membrane Extract (BME) to study the differentiation and sprouting of ECs over a 24-h period. The protocol described here details a thin layer BME adaptation of the angiogenesis assay requiring significantly less BME and carried out in 96-well plates, allowing for a larger data yield at a greatly reduced cost, while maintaining the robustness of an assay used extensively over the past three decades.

Simultaneous Measurement of Endothelial Cell Proliferation and Cell Cycle Stage Using Flow Cytometry.

Endothelial cell proliferation rate is an important indicator of vascular health. Being able to detect the rate of endothelial cell proliferation, or cell cycle disturbances after intervention is a valuable tool for analysing any beneficial or detrimental effects of treatments in vitro. Here, we describe a straightforward flow cytometric-based method of proliferation and cell cycle tracking that can be performed on human endothelial cells in culture over several days.

Replicatively senescent human fibroblasts reveal a distinct intracellular metabolic profile with alterations in NAD+ and nicotinamide metabolism.

Cellular senescence occurs by proliferative exhaustion (PEsen) or following multiple cellular stresses but had not previously been subject to detailed metabolomic analysis. Therefore, we compared PEsen fibroblasts with proliferating and transiently growth arrested controls using a combination of different mass spectroscopy techniques. PEsen cells showed many specific alterations in both the NAD+ de novo and salvage pathways including striking accumulations of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) in the amidated salvage pathway despite no increase in nicotinamide phosphoribosyl transferase or in the NR transport protein, CD73. Extracellular nicotinate was depleted and metabolites of the deamidated salvage pathway were reduced but intracellular NAD+ and nicotinamide were nevertheless maintained. However, sirtuin 1 was downregulated and so the accumulation of NMN and NR was best explained by reduced flux through the amidated arm of the NAD+ salvage pathway due to reduced sirtuin activity. PEsen cells also showed evidence of increased redox homeostasis and upregulated pathways used to generate energy and cellular membranes; these included nucleotide catabolism, membrane lipid breakdown and increased creatine metabolism. Thus PEsen cells upregulate several different pathways to sustain their survival which may serve as pharmacological targets for the elimination of senescent cells in age-related disease.

Biomechanical strain as a trigger for pore formation in Schlemm's canal endothelial cells.

The bulk of aqueous humor passing through the conventional outflow pathway must cross the inner wall endothelium of Schlemm's canal (SC), likely through micron-sized transendothelial pores. SC pore density is reduced in glaucoma, possibly contributing to obstructed aqueous humor outflow and elevated intraocular pressure (IOP). Little is known about the mechanisms of pore formation; however, pores are often observed near dome-like cellular outpouchings known as giant vacuoles (GVs) where significant biomechanical strain acts on SC cells. We hypothesize that biomechanical strain triggers pore formation in SC cells. To test this hypothesis, primary human SC cells were isolated from three non-glaucomatous donors (aged 34, 44 and 68), and seeded on collagen-coated elastic membranes held within a membrane stretching device. Membranes were then exposed to 0%, 10% or 20% equibiaxial strain, and the cells were aldehyde-fixed 5 min after the onset of strain. Each membrane contained 3-4 separate monolayers of SC cells as replicates (N = 34 total monolayers), and pores were assessed by scanning electron microscopy in 12 randomly selected regions (∼65,000 µm(2) per monolayer). Pores were identified and counted by four independent masked observers. Pore density increased with strain in all three cell lines (p < 0.010), increasing from 87 ± 36 pores/mm(2) at 0% strain to 342 ± 71 at 10% strain; two of the three cell lines showed no additional increase in pore density beyond 10% strain. Transcellular "I-pores" and paracellular "B-pores" both increased with strain (p < 0.038), however B-pores represented the majority (76%) of pores. Pore diameter, in contrast, appeared unaffected by strain (p = 0.25), having a mean diameter of 0.40 µm for I-pores (N = 79 pores) and 0.67 µm for B-pores (N = 350 pores). Pore formation appears to be a mechanosensitive process that is triggered by biomechanical strain, suggesting that SC cells have the ability to modulate local pore density and filtration characteristics of the inner wall endothelium based on local biomechanical cues. The molecular mechanisms of pore formation and how they become altered in glaucoma may be studied in vitro using stretched SC cells.

CD25+ natural regulatory T cells are critical in limiting innate and adaptive immunity and resolving disease following respiratory syncytial virus infection.

Regulatory CD4(+) T cells have been shown to be important in limiting immune responses, but their role in respiratory viral infections has received little attention. Here we observed that following respiratory syncytial virus (RSV) infection, CD4(+) Foxp3(+) CD25(+) natural regulatory T-cell numbers increased in the bronchoalveolar lavage fluid, lung, mediastinal lymph nodes, and spleen. The depletion of CD25(+) natural regulatory T cells prior to RSV infection led to enhanced weight loss with delayed recovery that was surprisingly accompanied by increased numbers of activated natural killer cells in the lung and bronchoalveolar lavage fluid on day 8 postinfection. Increased numbers of neutrophils were also detected within the bronchoalveolar lavage fluid and correlated with elevated levels of myeloperoxidase as well as interleukin-6 (IL-6) and gamma interferon (IFN-gamma). CD25(+) natural regulatory T-cell depletion also led to enhanced numbers of proinflammatory T cells producing IFN-gamma and tumor necrosis factor alpha (TNF-alpha) in the lung. Despite these increases in inflammatory responses and disease severity, the viral load was unaltered. This work highlights a critical role for natural regulatory T cells in regulating the adaptive and innate immune responses during the later stages of lung viral infections.

Safety, risk acceptability, and morality.

The primary aim of this article is to develop and defend a conceptual analysis of safety. The article begins by considering two previous analyses of safety in terms of risk acceptability. It is argued that these analyses fail because the notion of risk acceptability is more subjective than safety, as risk acceptability takes into account potential benefits in a way that safety does not. A distinction is then made between two different kinds of safety--safety qua cause and safety qua recipient--and both are defined in terms of the probability of a loss of value, though the relationship between safety and the probability of loss varies in each case. It is then shown that although this analysis is less subjective than the previously considered analyses, subjectivity can still enter into judgments of safety via the notions of probability and value. In the final section of this article, it is argued that the difference between safety and risk acceptability is important because it corresponds in significant ways to the difference between consequentialist and deontological moral viewpoints.

Electronic spectroscopy of the E<--X transition of NO-Kr and shielding/penetration effects in Rydberg states of NO-Rg complexes.

We report the results of a (2+1) resonance-enhanced multiphoton ionization (REMPI) study of the E2Sigma+(4ssigma) Rydberg state of NO-Kr. We present an assignment of the two-photon spectrum based on a simulation, and discuss it in the context of previously-reported spectra of NO-Ne and NO-Ar. In addition, we report on spectra in the region of the vNO=1 level of the E, F and H' 4s and 3d Rydberg states of NO-Rg (Rg=Ne-Kr). Since the NO vibrational frequency is affected by electron donation from the rare-gas (Rg) atom to the NO+ core, as well as by the penetration of the Rydberg electron, the fundamental NO-stretch frequency reflects the interactions in the complex. The results indicate that the 4s Rydberg state has a strong interaction between the NO+ core and the Kr atom, as was the case for NO-Ar and NO-Ne. For the 3d Rydberg states, although penetration is not as significant as for the 4s Rydberg states, it does play an important role, with subtle angular effects being notable.

BRE is an antiapoptotic protein in vivo and overexpressed in human hepatocellular carcinoma.

BRE binds to the cytoplasmic domains of tumor necrosis factor receptor-1 and Fas, and in cell lines can attenuate death receptor-initiated apoptosis by inhibiting t-BID-induced activation of the mitochondrial apoptotic pathway. Overexpression of BRE by transfection can also attenuate intrinsic apoptosis and promote growth of the transfected Lewis lung carcinoma line in mice. There is, however, a complete lack of in vivo data about the protein. Here, we report that by using our BRE-specific monoclonal antibody on the immunohistochemistry of 123 specimens of human hepatocellular carcinoma (HCC), significant differences in BRE expression levels between the paired tumoral and non-tumoral regions (P<2.2e-16) were found. Marked overexpression of BRE was detected in majority of the tumors, whereas most non-tumoral regions expressed the same low level of the protein as in normal livers. To investigate whether BRE overexpression could promote cell survival in vivo, liver-specific transgenic BRE mice were generated and found to be significantly resistant to Fas-mediated lethal hepatic apoptosis. The transgenic model also revealed post-transcriptional regulation of Bre level in the liver, which was not observed in HCC and non-HCC cell lines. Indeed, all cell lines analysed express high levels of BRE. In conclusion, BRE is antiapoptotic in vivo, and may promote tumorigenesis when overexpressed.

Electronic spectroscopy of NO-(Rg)x complexes (Rg = Ne,Ar) via the 4s and 3d Rydberg states.

We have employed (2 + 1) resonance enhanced multiphoton ionization spectroscopy to investigate the 3d and 4s Rydberg states of the NO molecule when bound to the surface of Rg(x) clusters (Rg = rare gas). We observe that the spectra of the NO-Ar(x) species converge in appearance as x increases, and this is discussed in terms of two Rg atoms interacting with the NO+ core, with other Rg atoms being "outside" the Rydberg orbital. We show that the interaction of each of the Rg atoms with the NO is essentially independent for the NO-Rg2 complexes: both by comparing our spectra for Rydberg states of NO-Rg and NO-Rg2, and from the results of ab initio calculations on NO+ - Rg and NO+ - Rg2. In addition, we discuss the disappearance of some electronic bands upon complexation in terms of Franck-Condon factors that are very sensitive to the angular coordinate. We relate our results to those of the bulk by comparing to the previously reported electronic spectroscopy of NO in both Rg matrices and He nanodroplets.

Electronic spectroscopy of the 3d Rydberg states of NO-Rg (Rg=Ne,Ar,Kr,Xe) van der Waals complexes.

We have employed (2+1) resonance-enhanced multiphoton ionization spectroscopy to record electronic absorption spectra of NO-Rg (Rg=Ne,Ar,Kr) van der Waals complexes. The nitric oxide molecule is the chromophore, and the excitation corresponds to an electron being promoted from the 2ppi* orbital to 3dsigma, 3dpi, and 3ddelta Rydberg states. We review the ordering of the 3dlambda states of NO and use this as a basis for discussing the 3d components in the NO-Rg complexes, in terms of the interactions between the Rydberg electron, the core, and the Rg atom. Predissociation of the H' 2Pi state occurs through the F2Delta state for NO-Ar and NO-Kr, and this will be considered. We shall also outline problems encountered when trying to record similar spectra for NO-Xe, related to the presence of atomic Xe resonances.

Multipotent neural precursors express neural and hematopoietic factors, and enhance ex vivo expansion of cord blood CD34+ cells, colony forming units and NOD/SCID-repopulating cells in contact and noncontact cultures.

In view of the possible crosstalks between hematopoiesis and neuropoiesis, we evaluated two microenvironments, murine neonatal neural cell line C17.2 and primary embryonic aorta-gonad-mesonephros (AGM) stromal cells, on the ex vivo expansion of CD34+ cells from human cord blood. In a contact culture system, C17.2 or AGM cells significantly enhanced the expansion of CD34+ cells to a panel of early and committed hematopoietic progenitor cells. In a noncontact transwell system, pre-established C17.2 cells significantly increased the expansion of total nucleated cells, CD34+ cells and multilineage colony forming cells (P<0.01). Expanded cells were infused into nonobese diabetic/severe-combined immunodeficient mice. The engraftment of human (hu)CD45+ cells in the bone marrow of these mice was consistently higher in all the 10 experiments conducted with the support of C17.2 cells when compared with those in respective control groups (11.9 vs 2.43%, P=0.03). Using RT-PCR and Southern blot analysis, we showed that AGM and C17.2 cells expressed a panel of hematopoietic, bone morphogenetic and neurotrophic factors. Our data provided the first evidence on the promoting effects of a neural progenitor cell line on hematopoiesis at a noncontact condition. The mechanism could be mediated by the expression of multilineage regulatory factors.

Utility of white cell count and ultrasound in the diagnosis of acute appendicitis.

BACKGROUND: Despite considerable advances in imaging, the diagnosis of acute appendicitis remains a clinical one. Nonetheless, knowledge of the characteristics of commonly used investigations for appendicitis may aid surgical decision-making. METHODS: The pathology, full blood counts and ultrasounds of 1013 patients who underwent appendicectomy were reviewed and subjected to statistical analysis in order to determine diagnostic characteristics of various white cell count and ultrasound results. RESULTS: Total white cell count was found to be higher among patients with complicated appendicitis than those with simple appendicitis or normal appendices. Ultrasound was found to be less accurate than white cell count and neutrophil count in the diagnosis of acute appendicitis by estimation of area under the receiver operator characteristic curve. CONCLUSION: Knowledge of the meaning of various white cell count values may be invaluable in clinical decision-making with regard to the diagnosis of acute appendicitis. Ultrasound is of limited utility in the diagnosis of appendicitis and should only be used in selected clinical situations.

Cobblestone area-forming cells, long-term culture-initiating cells and NOD/SCID repopulating cells in human neonatal blood: a comparison with umbilical cord blood.

Our prior study demonstrated that neonatal blood (NB) contained hematopoietic stem and progenitor cells that declined rapidly after birth. To validate that NB is a source of functional stem cells, we characterized this population in terms of cobblestone area-forming cells (CAFC), long-term culture-initiating cells (LTC-IC) and NOD/SCID mouse repopulating cells (SRC) in NB and umbilical cord blood (CB). Our data demonstrated that the frequencies of CAFC (30.2 vs 37.1, P = 0.14) and LTC-IC (28.6 vs 31.0, P = 0.49) in 1 x 10(5) mononuclear cells (MNC) of NB and CB were similar, suggesting that these cells were preserved in the circulation of the neonates shortly after birth. Sublethally irradiated NOD/SCID mice were transplanted with CD34(+) cells enriched from thawed NB and CB. At 6 weeks post transplant, human (hu)CD45(+) cells were detected in the bone marrow (BM), spleen and peripheral blood (PB) of the mice as demonstrated by flow cytometric and DNA analysis. Levels of huCD45(+)cells and colony forming units (CFU) appeared to be dependent on the infusion cell dose and were higher in animals receiving CB cells when compared with those of the NB group. The transplanted cells were capable of differentiation into multi-lineage progenitor cells (CD34(+) cells and differential CFU), as well as mature myeloid (CD14(+), CD33(+)), B lymphoid (CD19(+)) and megakaryocytic (CD61(+)) cells in the recipients. NB cells, subjected to ex vivo culture in an optimized preclinical condition, were significantly expanded to early and committed progenitor cells. Expanded NB contained SRC at a reduced quantity but with high proportions of CD14(+) cells and CD33(+) cells. Our study confirms that NB contains pluripotent hematopoietic stem and progenitor cells capable of homing and engrafting the NOD/SCID mice.

Delivery and scavenging system for small animal inhalational anesthesia.

BACKGROUND: Inhalational agents have been widely used for anesthesia in laboratory animals. However, the safe use of inhalational agents in small laboratory animals has been limited by the lack of a suitable and effective scavenging system for the removal of waste anesthetic gases. The aim of the present study is to develop an anesthetic system that can be manufactured using common household and laboratory items. MATERIALS AND METHODS: An anesthetic system was designed for rats weighing from 300 to 350 g. A face mask for the rat was made by cutting off the distal part of a 50-ml centrifuge tube. A scavenging hood was made from a transparent plastic food storage box. Exhaust of anesthetic gases from the scavenger hood was facilitated by fitting an outlet connected to a pump. Four experienced researchers or technicians tested the scavenger hood. RESULTS: In 79.2% of the trials the participants could smell halothane when the pump from the scavenger system was not operational. However, when the pump was switched on, halothane was detected only 16.7% of the time (P < 0.001). CONCLUSION: We have developed a simple and effective method of delivering inhalational anesthesia to small laboratory animals and of removing waste anesthetic gases.

Preclinical ex vivo expansion of cord blood hematopoietic stem and progenitor cells: duration of culture; the media, serum supplements, and growth factors used; and engraftment in NOD/SCID mice.

BACKGROUND: Ex vivo expansion of cord blood (CB) hematopoietic stem and progenitor cells increases cell dose and may reduce the severity and duration of neutropenia and thrombocytopenia after transplantation. This study's purpose was to establish a clinically applicable culture system by investigating the use of cytokines, serum-free media, and autologous plasma for the expansion of CB cells and the engraftment of expanded product in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. STUDY DESIGN AND METHODS: Enriched CB CD34+ cells were cultured in four media (Iscove's modified Dulbecco's medium with FCS, Gibco; X-Vivo-10, BioWhittaker; QBSF-60, Quality Biological; and StemSpan SFEM, Stem Cell Technologies) with four cytokine combinations (thrombopoietin [TPO], SCF, Flt-3 ligand [FL] with and without G-CSF, and/or IL-6). The effect of autologous CB plasma was also investigated. The read-out measures were evaluated on Days 8 and 12. After expansion at the optimized condition, cultured cells were transplanted into sublethally irradiated NOD/SCID mice. The engraftment of human CD45+ cells and subsets in the bone marrow, spleen, and peripheral blood was determined. RESULTS: QBSF-60 or StemSpan SFEM supported high yields of early progenitors (CD34+ cells,

The effect of percutaneous oestradiol on atheroma formation in ovariectomized cholesterol-fed rabbits.

OBJECTIVE: The aim of this study was to examine the effect of percutaneous oestradiol on the lipid profile and on atheroma formation using an animal model. METHODS: The study was of 12 weeks duration. Fifty sexually mature female New Zealand White rabbits were divided into five groups of equal size. Two groups acted as controls and received normal rabbit chow. Rabbits in one of these groups were ovariectomized. The remaining three groups were ovariectomized but received 1% cholesterol enriched rabbit chow. One of these cholesterol-fed groups received 0.3 mg/kg percutaneous oestradiol daily whilst another received 0.1 mg/kg oral oestradiol daily. Measurements of concentrations of total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and triglycerides (TG) were made at the beginning and end of the study. Aortic atheroma formation was measured using computerized image analysis of uptake of Sudan III staining. RESULTS: After 12 weeks there were significant increases in the mean concentrations of TC in the three cholesterol-fed groups compared with controls (P < 0.001). Changes in HDL-C and TG concentrations were less consistent. The mean area of aortic atheroma formation was significantly less in both the percutaneous oestradiol group (4.9%) and the oral oestradiol group (8.6%) compared with the non-oestrogen-treated cholesterol-fed group (19.5%) (P < 0.001, < 0.01 respectively). CONCLUSION: These results suggest that percutaneous oestradiol has a direct protective effect on atheroma formation independent of serum concentrations of total cholesterol.

A pilot study of the theoretical and technical competence and appropriate education for the use of nine physical agent modalities in occupational therapy practice.

OBJECTIVE: This study described occupational therapy practitioners' perceptions about the content and method of training or education necessary for gaining theoretical and technical competence in the use of nine physical agent modalities (PAMs). METHOD: A survey was developed and sent to 543 members of the Physical Disabilities Special Interest Section of the American Occupational Therapy Association who had identified their primary area of practice as hand therapy. One hundred and fifty-one completed surveys (28% response rate) were returned. RESULTS: The respondents indicated that theoretical and technical expertise necessary for competent use of PAMs varied according to the type of modality being considered. Continuing education courses were identified as the best method for gaining theoretical and technical competence for the use of deep thermal agents, such as ultrasound and electrical stimulation agents, whereas entry-level professional education and one-the-job training were identified as most appropriate for superficial thermal agents, such as paraffin bath and hot and cold packs. CONCLUSION: The results suggest that considerations regarding the type and amount of education necessary for gaining theoretical and technical competence in the use of PAMs depend on the type of modality being addressed. These differences should be considered in the future development of competency objectives for the use of PAMs.