Ion imaging during axolotl tail regeneration in vivo

Several studies have reported that endogenous ion currents are involved in a wide range of biological processes from single cell and tissue behavior to regeneration. Various methods are used to assess intracellular and local ion dynamics in biological systems, e.g., patch clamping and vibrating probes. Here, we introduce an approach to detect ion kinetics in vivo using a noninvasive method that can electrophysiologically characterize an entire experimental tissue region or organism. Ion‐specific vital dyes have been successfully used for live imaging of intracellular ion dynamics in vitro. Here, we demonstrate that cellular pH, cell membrane potential, calcium, sodium and potassium can be monitored in vivo during tail regeneration in the axolotl (Ambystoma mexicanum) using ion‐specific vital dyes. Thus, we suggest that ion‐specific vital dyes can be a powerful tool to obtain electrophysiological data during crucial biological events in vivo. Developmental Dynamics 239:2048–2057, 2010. © 2010 Wiley‐Liss, Inc.     

Classification of a large microarray data set: algorithm comparison and analysis of drug signatures

A large gene expression database has been produced that characterizes the gene expression and physiological effects of hundreds of approved and withdrawn drugs, toxicants, and biochemical standards in various organs of live rats. In order to derive useful biological knowledge from this large database, a variety of supervised classification algorithms were compared using a 597-microarray subset of the data. Our studies show that several types of linear classifiers based on Support Vector Machines (SVMs) and Logistic Regression can be used to derive readily interpretable drug signatures with high classification performance. Both methods can be tuned to produce classifiers of drug treatments in the form of short, weighted gene lists which upon analysis reveal that some of the signature genes have a positive contribution (act as "rewards" for the class-of-interest) while others have a negative contribution (act as "penalties") to the classification decision. The combination of reward and penalty genes enhances performance by keeping the number of false positive treatments low. The results of these algorithms are combined with feature selection techniques that further reduce the length of the drug signatures, an important step towards the development of useful diagnostic biomarkers and low-cost assays. Multiple signatures with no genes in common can be generated for the same classification end-point. Comparison of these gene lists identifies biological processes characteristic of a given class.     

Quantum dots light up pathology

Quantum dots (QDs) are novel nanocrystal fluorophores with extremely high fluorescence efficiency and minimal photobleaching. They also possess a constant excitation wavelength together with sharp and symmetrical tunable emission spectra. These unique optical properties make them near-perfect fluorescent markers and there has recently been rapid development of their use for bioimaging. QDs can be conjugated to a wide range of biological targets, including proteins, antibodies, and nucleic acid probes, rendering them of particular interest to pathology researchers. They have been used in multiplex immunohistochemistry and in situ hybridization, which when combined with multispectral imaging, has enabled quantitative measurement of gene expression in situ. QDs have also been used for live in vivo animal imaging and are now being applied to an ever-increasing range of biological problems. These are detailed in this review, which also acts to outline the important advances that have been made in their range of applications. The relative novelty of QDs can present problems in their practical use and guidelines for their application are given.     

Intrahepatic MxA expression is correlated with interferon-alpha expression in chronic and fulminant hepatitis

Interferon-alpha (IFN-alpha) has potent pro-inflammatory and anti-viral functions. It exerts its effects by inducing intracellular proteins such as MxA. To analyse the role of intrahepatic interferon activation, IFN-alpha and MxA expression was studied by immunohistochemistry in explant livers of 20 patients with fulminant hepatic failure (FHF), 41 patients with chronic liver disease (CLD), and ten normal controls (NCs). In NCs only small numbers of Kupffer cells, but no hepatocytes, showed IFN-alpha and MxA expression. In contrast, significantly enhanced numbers of IFN-alpha- and MxA-positive Kupffer cells, along with small numbers of MxA-positive and larger numbers of IFN-alpha-positive lymphocytes, were found in CLD and in FHF. MxA protein was also expressed on hepatocytes and bile ducts in the vicinity of IFN-alpha-positive inflammatory infiltrates (hepatocytes: NCs: 0%, CLD: 8%, FHF: 68%; bile ducts: NCs: 19%, CLD: 46%, FHF: 83%). A significant correlation was found between the numbers of IFN-alpha- and MxA-positive cells (r=0.67, p<0.001). Thus, large amounts of IFN-alpha are released in the livers of patients with FHF, which is likely to contribute to immune-mediated liver cell damage. Intrahepatic MxA expression corresponds to IFN-alpha produced particularly by infiltrating inflammatory cells, rather than by hepatocytes themselves.     

Antigen presentation by Leishmania mexicana-infected macrophages: activation of helper T cells by a model parasite antigen secreted into the parasitophorous vacuole or expressed on the amastigote surface

Leishmania are protozoan parasites which invade mammalian macrophages and multiply as amastigotes in phagolysosomes (parasitophorous vacuoles). Using L. mexicana and bone marrow-derived macrophages (BMM), the question is addressed whether infected BMM induced to express major histocompatibility complex class II molecules can present defined antigens to specific T helper type 1 cells. As a model antigen, a membrane-bound acid phosphatase (MAP), a minor protein associated with intracellular vesicles in amastigotes, was either overexpressed at the surface of the parasites or overexpressed in a soluble form leading to antigen secretion into the parasitophorous vacuole. Presentation of MAP epitopes by these three types of amastigotes was then compared for macrophages containing live parasites or amastigotes inactivated by drug treatment. It is shown that surface-exposed and secreted MAP can be efficiently presented to T cells by macrophages harboring live amastigotes. Therefore, the parasitophorous vacuole communicates by vesicular membrane traffic with the plasmalemma of the host cell. The intracellular MAP of wild-type cells or the abundant lysosomal cysteine proteinases are not or only inefficiently presented, respectively. After killing of the parasites, abundant proteins such as overexpressed MAP and the cysteine proteinases efficiently stimulate T cells, while wild-type MAP levels are not effective. We conclude that intracellular proteins of intact amastigotes are not available for presentation, while after parasite inactivation, presentation depends on antigen abundance and possibly stability. The cell biological and possible immunological consequences of these results are discussed.     

Antibacterial polyurethane nanocomposites using chlorhexidine diacetate as an organic modifier

Polymer nanocomposites (NCs) are hypothesised to have enhanced barrier properties compared with pristine polymer, allowing more sustained drug release from the materials. In these NC systems active agents are typically incorporated into the polymer matrix and the release kinetics are theoretically perturbed by well dispersed nanoparticle inclusions. An alternative approach is to exploit active agent interactions with the nanoinclusion. In the proposed NC system, the driving hypothesis is that active agents can have dual functionality, acting as both drug and dispersant. Polyurethane-montmorillonite (PEU-MMT) NCs were prepared in which the antimicrobial agent chlorhexidine diacetate (CHX) was evaluated as an organic modifier for silicate dispersion. CHX was incorporated at various concentrations through organic modification of MMT or within the bulk polymer. X-ray diffraction and transmission electron microscopy analysis suggested that intercalated and partially exfoliated NCs were achieved, with better dispersion occurring in the presence of free CHX within the bulk. Tensile testing results showed that variations in the level of organic modification and nanoparticle loading modulated the mechanical properties. Material stiffness increased with nanoparticle loading relative to pristine PEU, and the ultimate properties decreased with nanoparticle and free CHX incorporation. Antibacterial activity against Staphylococcus epidermidis was significant in materials with higher exchanged MMT and NCs containing free CHX, for which 2-log reductions in adherent bacteria were found after 24 h. CHX was successfully used to modulate the material properties in its dual role as a dispersant and antimicrobial agent, suggesting that alternative biocides of similar structure may behave comparably within PEU-MMT NC systems.     

谷胱甘肽保护的金纳米团簇对HeLa细胞毒性研究

目的 探究由谷胱甘肽作为表面保护剂的金纳米团簇(GSH-Au NCs)对宫颈癌HeLa细胞株的毒性影响.方法 利用荧光分光光度计测定用含有GSH-Au NCs的培养基处理HeLa细胞后不同时间点的荧光强度,观察HeLa细胞对GSH-Au NCs在1、2、6、12、24 h内的摄取情况.同时采用BALB/c荷瘤小鼠进行体内实验,分别腹腔注射0.2 ml浓度为3 mmol/L的GSH-Au NCs和等体积的蒸馏水(对照组)后24 h取出肿瘤组织,通过电感耦合等离子体质谱(ICP-MS)检测组织中的金元素含量以探究纳米团簇在肿瘤处随时间的摄取情况.最后用噻唑蓝(MTT)比色法研究不同浓度(0.003～0.3 mmol/L)的GSH-Au NCs处理HeLa细胞24、48 h的细胞毒性.结果 HeLa细胞对GSH-Au NCs的摄取率在24 h内不断升高,24h时达峰值73.13％.荷瘤小鼠实验表明,腹腔注射GSH-Au NCs 24 h后,肿瘤组织对GSH-Au NCs的摄取量(320±15) ng/g较对照组(腹腔注射蒸馏水)高,差异具有统计学意义(P＜0.05).用不同浓度的GSH-Au NCs处理HeLa细胞24 h,对细胞存活率有轻微影响,随浓度升高对细胞的抑制作用更为明显,GSH-Au NCs浓度为0.3 mmol/L时的HeLa细胞存活率降为对照组(GSH-Au NCs浓度为0)的86％(P＜0.05);处理48 h时,各浓度组的细胞存活率与对照组间差异均无统计学意义(P＞0.05).结论 虽然GSH-Au NCs在体外和体内均易被细胞和肿瘤组织摄取,但其本身对HeLa细胞并无明显细胞毒性,可安全应用于影像、载药及靶向给药等生物医药领域.     

Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4 signaling to prevent differentiation

Corneal epithelial stem cells (SCs) are an ideal model for investigating how adult lineage-committed epithelial SCs are regulated by an anatomically defined and accessible niche, that is, limbal palisades of Vogt, located between the cornea and the conjunctiva. We have used collagenase digestion to isolate the entire limbal epithelial SCs and subjacent mesenchymal cells, and we have demonstrated that their close association is crucial for promoting epithelial clonal growth, implying that the latter serves as niche cells (NCs). After their close association was disrupted by trypsin/EDTA, single SCs and NCs could reunite to generate sphere growth in three-dimensional Matrigel in the embryonic SC medium, and that such sphere growth initiated by SC-NC reunion was mediated by SDF-1 uniquely expressed by limbal epithelial progenitor cells and its receptor CXCR4, but not CXCR7, strongly expressed by limbal stromal NCs. Inhibition of CXCR4 by AMD3100 or a blocking antibody to CXCR4 but not CXCR7 disrupted their reunion and yielded separate spheres with a reduced size, while resultant epithelial spheres exhibited more corneal differentiation and a notable loss of holoclones. For the first time, these results provide strong evidence supporting that limbal SC function depends on close physical association with their native NCs via SDF-1/CXCR4 signaling. This novel in vitro model of sphere growth with NCs can be used for investigating how limbal SC self-renewal and fate decision might be regulated in the limbal niche.     

Cultures of Needleless Connectors Are Useful for Ruling Out Central Venous Catheter Colonization

Semiquantitative cultures of skin surrounding intravascular catheter entry sites and catheter hubs have high negative predictive values for catheter tip colonization. However, culturing samples from the inner side of the hub requires the catheter to be manipulated, thus increasing the risk of migration of microorganisms into the bloodstream. Today, hubs are closed using needleless connectors (NCs). Cultures of NCs could predict catheter colonization. Our objective was to compare the yield of NC sonicate cultures for prediction of catheter colonization with that of hub cultures. For 6 months, we prospectively collected all short-term central lines and systems removed from patients admitted to the cardiac surgery postoperative care unit, irrespective of the reason for withdrawal. Hub cultures were obtained immediately before withdrawal and were cultured using a semiquantitative method. Catheter tips were cultured using the roll-plate technique and sonication, and NCs were cultured using a semiquantitative technique after sonication. We considered NCs to be colonized when ≥1 culture was positive. We collected a total of 75 central systems. The catheter colonization rate was 10.7%. The rates for hub and NC colonization were 6.7% and 12.0%, respectively. The validity values for hubs and NCs for prediction of catheter colonization were as follows: sensitivity, 25.0% and 87.5%; specificity, 95.5% and 97.0%; positive predictive value, 40.0% and 77.8%; negative predictive value, 91.4% and 98.5%; validity index, 88.0% and 96.0%, respectively. Cultures of closed NCs can be used to rule out catheter tip colonization and are superior to hub cultures in ruling out short-term central venous catheter colonization.     

Microbial Biofilms on Needleless Connectors for Central Venous Catheters: Comparison of Standard and Silver-Coated Devices Collected from Patients in an Acute Care Hospital

Microorganisms may colonize needleless connectors (NCs) on intravascular catheters, forming biofilms and predisposing patients to catheter-associated infection (CAI). Standard and silver-coated NCs were collected from catheterized intensive care unit patients to characterize biofilm formation using culture-dependent and culture-independent methods and to investigate the associations between NC usage and biofilm characteristics. Viable microorganisms were detected by plate counts from 46% of standard NCs and 59% of silver-coated NCs (P = 0.11). There were no significant associations (P > 0.05, chi-square test) between catheter type, side of catheter placement, number of catheter lumens, site of catheter placement, or NC placement duration and positive NC findings. There was an association (P = 0.04, chi-square test) between infusion type and positive findings for standard NCs. Viable microorganisms exhibiting intracellular esterase activity were detected on >90% of both NC types (P = 0.751), suggesting that a large percentage of organisms were not culturable using the conditions provided in this study. Amplification of the 16S rRNA gene from selected NCs provided a substantially larger number of operational taxonomic units per NC than did plate counts (26 to 43 versus 1 to 4 operational taxonomic units/NC, respectively), suggesting that culture-dependent methods may substantially underestimate microbial diversity on NCs. NC bacterial communities were clustered by patient and venous access type and may reflect the composition of the patient''s local microbiome but also may contain organisms from the health care environment. NCs provide a portal of entry for a wide diversity of opportunistic pathogens to colonize the catheter lumen, forming a biofilm and increasing the potential for CAI, highlighting the importance of catheter maintenance practices to reduce microbial contamination.     

Effect of Chlamydia pneumoniae on cellular ATP content in mouse macrophages: role of Toll-like receptor 2

Chlamydiae are obligate intracellular gram-negative bacteria and are dependent on the host cell for ATP. Thus, chlamydial infection may alter the intracellular levels of ATP and affect all energy-dependent processes within the cell. We have shown that both live C. pneumoniae and inactivated C. pneumoniae induce markers of cell death prior to completion of the bacterial growth cycle. As depletion of ATP could account for the observed increase in cell death, the effects of C. pneumoniae on ATP concentrations within mouse macrophages were investigated. Live, heat-killed, and UV-inactivated C. pneumoniae cultures (at multiplicities of infection [MOIs] of 0.01, 0.1, and 1.0) were incubated with mouse bone marrow macrophages isolated from C57BL/6J mice and mice deficient in Toll-like receptors. Treatment of the macrophages with both live and inactivated C. pneumoniae increased the ATP content of the cells. In cells infected with live C. pneumoniae, the increase was inversely proportional to the MOI. In cells treated with inactivated C. pneumoniae, the increase in ATP content was smaller than that induced by infection with live organisms and was proportional to the MOI. The increase in ATP content early in the developmental cycle was independent of the growth of C. pneumoniae, while sustained induction required live organisms. The capacity of C. pneumoniae to increase the ATP content was ablated in macrophages deficient in expression of either Toll-like receptor 2 or the Toll-like receptor accessory protein MyD88. In contrast, no effect was observed in macrophages lacking expression of Toll-like receptor 4.     

Review of circulating tumor markers: from enzyme, carcinoembryonic protein to oncogene and suppressor gene

The ease and non-invasive nature of the procedure for drawing blood has made it possible to measure circulating tumor markers for cancer screening, diagnosis, follow-up and early detection of recurrence. Even though a tumor-specific marker has not yet been found, the specificity and sensitivity of currently used tumor markers have improved over the last several decades as they have progressed from enzyme, hormone and carcinoembryonic protein to monoclonal antibody-defined epitope and finally, in recent years, to oncogene, suppressor gene and their encoded protein product. Both phenotype and genotype are included in these latest new tumor markers. Unlike earlier tumor markers, they can be identified with specific biological processes regulating cell growth, such as the cell cycle, angiogenesis, apoptosis and cell adhesion. Any elevation of these new tumor markers, therefore, can be used to identify defects in a specific metabolic pathway and facilitate the design of effective drug therapy.     

Will neuroimaging ever be used to diagnose pediatric bipolar disorder?

There is a great need for discovery of biological markers that could be used diagnostically for pediatric onset disorders, particularly those with potentially confusing phenomenology such as pediatric-onset bipolar disorder (BD). Obtaining these markers would help overcome current subjective diagnostic techniques of relying on parent and child interview and symptomatic history. Brain imaging may be the most logical choice for a diagnostic tool, and certain neurobiological abnormalities have already been found in pediatric BD. However, much work remains to be done before neuroimaging can be used reliably to diagnose this disorder, and because of the nature of BD and the limitations of imaging technology and technique, neuroimaging will likely at most be only a diagnostic aide in the near future. In this paper we discuss the characteristics of pediatric BD that complicate the use of biological markers as diagnostic tools, how neuroimaging techniques have been used to study pediatric BD so far, and the limitations and potential of such techniques for future diagnostic use.     

Human CD4+ regulatory T cells express lower levels of the IL-7 receptor alpha chain (CD127), allowing consistent identification and sorting of live cells

Although quantitative identification and viable enrichment of natural regulatory T cells (T-regs) in humans are problematic, such steps would greatly facilitate the analysis of these cells in disease states. In an attempt to identify markers that are sensitive and specific for human T-regs, we analyzed the expression of fourteen intracellular and cell surface markers on human CD4(+) cells. Many markers were partially selective for CD25(hi) T-regs, but consistent and specific discrimination of functional T-regs was only made possible by focus on CD127, the alpha chain of the IL-7 receptor. Although most CD4(+) human T cells express CD127, T-regs exhibiting suppressive activity in vitro display distinctly lower surface expression of this marker, irrespective of their level of CD25 expression. Sorted cells with the surface phenotype CD4(+)CD25(+)CD127(low) had higher levels of intracellular FOXP3 and CTLA-4 and, as determined by functional assays, were suppressive, hypoproliferative, and poorly responsive to TCR signaling. The CD4(+)CD25(+)CD127(low) phenotype was also found to be characteristic of T-regs found in mice and in rhesus macaques. This surface phenotype should allow for quantitative studies of regulatory T cells in disease states as well as for enrichment of live regulatory T cells for functional analyses and/or expansion in vitro.     

Intracellular accumulation and immunological properties of fluorescent gold nanoclusters in human dendritic cells

We have studied the effect of highly fluorescent gold nanoclusters (Au NCs) (∅ < 3 nm) stabilized by different ligands on the intracellular accumulation and immune response of human derived-monocyte dendritic cells (DCs). Results indicate that the high uptake efficiency of Au NCs is strongly related to their small size and to the nature of the ligand, with zwitterionic ligands being more effective than PEGylated ones. Evidence from flow cytometry and microscopy demonstrate time and concentration-dependent Au NCs internalization by endocytic pathway(s) involving amorphous and laminar organelles, while maintaining their discrete size and photoluminescence properties. The uptake of zwitterionic ligand-stabilized Au NCs induced very low cytotoxicity and a strong immunosuppressive response (Th1/Treg pattern), associated with a DC maturation state. This behavior contrasts to the effect of bigger particles (∼12 nm size) which induced a cytotoxic response involving Natural Killer (CD56) cells. Overall, this study stresses the critical importance of particle size and ligand type on the immunostimulation of DCs and highlights the remarkable potential of this new class of nanomaterial as a novel vaccine platform.     

Live influenza vaccine: laboratory markers of attenuation

Screening for candidate reassortants is an important step in the development of live influenza vaccine (LIV). The temperature-sensitive (ts) and cold-adapted (ca) phenotypes of vaccine strains are generally determined, by employing chicken embryos, and used as ts and ca attenuation markers. However, it is difficult to use the egg-determined ts phenotypes of vaccine candidate reassortants as an attenuation marker due to a wide circulation of natural ts epidemic influenza viruses. This study used two new alternative ts and ca attenuation markers in MDCK cells. The MDCK cell line was shown to be able to differentiate cold-adapted influenza viruses from any epidemic strains whereas they were undistinguishable when using eggs. The reduced ability of influenza type A vaccine viruses to grow in the MDCK cell culture at temperatures above 37 degrees C can be successfully used as a "cell-culture" ts marker. The similar marker for influenza B viruses may serve their reduced activity in the MDCK cells at 38 degrees C. The high reproductive activity of cold-adapted viruses in the MDCK cells at 26 degrees C was shown to be a suitable ca attenuation marker. The presented attenuation markers may be included into the standard scheme of primary screening of ts reassortant candidates for commercial live influenza vaccine as additional selection factors and may be used as basic markers in the design of culture vaccine.     

Test comparisons among stress-affected,stress-resilient,and nonclassified fourth- through sixth-grade urban children

Test performance of three groups of urban 4th- through 6th-graders – highly stressed children with stress-resilient (SR) or stress-affected (SA) adjustment outcomes, and demographically matched nonclassified (NC) peers – from 2-year cohorts, was compared on measures used both to select SRs and SAs and to expand the definitional net for childhood resilience. On adjustment-selection and verification measures, SRs exceeded NCs and NCs exceeded SAs. On the criterion test measures, both SRs and NCs exceeded SAs.     

Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry

Membrane-damaged cells caused by either mechanical trauma or through normal biological processes can produce artifacts in immunophenotyping analysis by flow cytometry. Dead cells can nonspecifically bind monoclonal antibody conjugates, potentially leading to erroneous conclusions, particularly when cell frequencies are low. To date, DNA intercalating dyes (Ethidium monoazaide (EMA), Propidium Iodide, 7AAD, etc.) or Annexin V have been commonly used to exclude dead cells; however, each suffer from technical problems. The first issue with such dyes is the dependence on a consistent dead cell source for fluorescence compensation. Another major issue is the stability of the staining; except for EMA, fixation and permeablization used for intracellular staining procedures can cause loss of fluorescence. EMA requires a UV exposure to covalently bond to DNA; while this dye is effective and is not affected by intracellular treatments it is weakly fluorescent. Here we report on the optimization of a new class of viability dyes, the amine reactive viability dyes (ViD) as a dead cell exclusion marker. The inclusion of ViD into the staining panel was found to be simple, reproducible and can have a significant benefit on the accuracy of identifying appropriate cell populations. We show the fluorescence of cells stained with these dyes correlates with traditional dead cell discriminating markers, even after fixation and permeabilization. Amine reactive viability dyes are a powerful tool for fluorescence immunophenotyping experiments.