Microbial Co-Infection Alters Macrophage Polarization, Phagosomal Escape, and Microbial Killing

Macrophages are important innate immune cells that respond to microbial insults. In response to multi-bacterial infection, the macrophage activation state may change upon exposure to nascent mediators, which results in different bacterial killing mechanism(s). In this study, we utilized two respiratory bacterial pathogens, Mycobacterium bovis (Bacillus Calmette Guẻrin, BCG) and Francisella tularensis live vaccine strain (LVS) with different phagocyte evasion mechanisms, as model microbes to assess the influence of initial bacterial infection on the macrophage response to secondary infection. Non-activated (M0) macrophages or activated M2-polarized cells (J774 cells transfected with the mouse IL-4 gene) were first infected with BCG for 24–48 h, subsequently challenged with LVS, and the results of inhibition of LVS replication in the macrophages was assessed. BCG infection in M0 macrophages activated TLR2-MyD88 and Mincle-CARD9 signaling pathways, stimulating nitric oxide (NO) production and enhanced killing of LVS. BCG infection had little effect on LVS escape from phagosomes into the cytosol in M0 macrophages. In contrast, M2-polarized macrophages exhibited enhanced endosomal acidification, as well as inhibiting LVS replication. Pre-infection with BCG did not induce NO production and thus did not further reduce LVS replication. This study provides a model for studies of the complexity of macrophage activation in response to multi-bacterial infection

Spectral Identification of Lighting Type and Character

We investigated the optimal spectral bands for the identification of lighting types and the estimation of four major indices used to measure the efficiency or character of lighting. To accomplish these objectives we collected high-resolution emission spectra (350 to 2,500 nm) for forty-three different lamps, encompassing nine of the major types of lamps used worldwide. The narrow band emission spectra were used to simulate radiances in eight spectral bands including the human eye photoreceptor bands (photopic, scotopic, and “meltopic”) plus five spectral bands in the visible and near-infrared modeled on bands flown on the Landsat Thematic Mapper (TM). The high-resolution continuous spectra are superior to the broad band combinations for the identification of lighting type and are the standard for calculation of Luminous Efficacy of Radiation (LER), Correlated Color Temperature (CCT) and Color Rendering Index (CRI). Given the high cost that would be associated with building and flying a hyperspectral sensor with detection limits low enough to observe nighttime lights we conclude that it would be more feasible to fly an instrument with a limited number of broad spectral bands in the visible to near infrared. The best set of broad spectral bands among those tested is blue, green, red and NIR bands modeled on the band set flown on the Landsat Thematic Mapper. This set provides low errors on the identification of lighting types and reasonable estimates of LER and CCT when compared to the other broad band set tested. None of the broad band sets tested could make reasonable estimates of Luminous Efficacy (LE) or CRI. The photopic band proved useful for the estimation of LER. However, the three photoreceptor bands performed poorly in the identification of lighting types when compared to the bands modeled on the Landsat Thematic Mapper. Our conclusion is that it is feasible to identify lighting type and make reasonable estimates of LER and CCT using four or more spectral bands with minimal spectral overlap spanning the 0.4 to 1.0 um region

Corrigendum to “Embryogenesis and blastocyst development after somatic cell nuclear transfer in nonhuman primates: overcoming defects caused by meiotic spindle extraction” [Dev. Biol. 276 (2004) 237–252]

AbstractTherapeutic cloning or nuclear transfer for stem cells (NTSC) seeks to overcome immune rejection through the development of embryonic stem cells (ES cells) derived from cloned blastocysts. The successful derivation of a human embryonic stem cell (hESC) line from blastocysts generated by somatic cell nuclear transfer (SCNT) provides proof-of-principle for “therapeutic cloning,” though immune matching of the differentiated NT-hES remains to be established. Here, in nonhuman primates (NHPs; rhesus and cynomologus macaques), the strategies used with human SCNT improve NHP-SCNT development significantly. Protocol improvements include the following: enucleation just prior to metaphase-II arrest; extrusion rather than extraction of the meiotic spindle-chromosome complex (SCC); nuclear transfer by electrofusion with simultaneous cytoplast activation; and sequential media. Embryo transfers (ET) of 135 SCNT-NHP into 25 staged surrogates did not result in convincing evidence of pregnancies after 30 days post-ET. These results demonstrate that (i) protocols optimized in humans generate preimplantation embryos in nonhuman primates; (ii) some, though perhaps not yet all, hurdles in deriving NT-nhpES cells from cloned macaque embryos (therapeutic cloning) have been overcome; (iii) reproductive cloning with SCNT-NHP embryos appears significantly less efficient than with fertilized embryos; (iv) therapeutic cloning with matured metaphase-II oocytes, aged oocytes, or “fertilization failures” might remain difficult since enucleation is optimally performed prior to metaphase-II arrest; and (v) challenges remain for producing reproductive successes since NT embryos appear inferior to fertilized ones due to spindle defects resulting from centrosome and motor deficiencies that produce aneuploid preimplantation embryos, among other anomalies including genomic imprinting, mitochondrial and cytoplasmic heterogeneities, cell cycle asynchronies, and improper nuclear reprogramming

Multiresolution identification of germ layer components in teratomas derived from human and nonhuman primate embryonic stem cells

We propose a system for identification of germ layer components in teratomas derived from human and nonhuman primate embryonic stem cells. Tissue regeneration and repair, drug testing and discov-ery, the cure of genetic and developmental syndromes all may rest on the understanding of the biology and behavior of embryonic stem (ES) cells. Within the field of stem cell biology, an ES cell is not con-sidered an ES cell until it can produce a teratoma tumor (the ”gold” standard test); a seemingly disorganized mass of tissue derived from all three embryonic germ layers; ectoderm, mesoderm, and endo-derm. Identification and quantification of tissue types within ter-atomas derived from ES cells may expand our knowledge of abnor-mal and normal developmental programming and the response of ES cells to genetic manipulation and/or toxic exposures. In addition, because of the tissue complexity, identifying and quantifying the tis-sue is tedious and time consuming, but in turn the teratoma provides an excellent biological platform to test robust image analysis algo-rithms. We use a multiresolution (MR) classification system with texture features, as well as develop novel nuclear texture features to recognize germ layer components. With redundant MR transform, we achieve a classification accuracy of approximately 88%. Index Terms — Stem cell biology, multiresolution, classifica-tion, feature extractio

Cloud Coverage Acts as an Amplifier for Ecological Light Pollution in Urban Ecosystems

The diurnal cycle of light and dark is one of the strongest environmental factors for life on Earth. Many species in both terrestrial and aquatic ecosystems use the level of ambient light to regulate their metabolism, growth, and behavior. The sky glow caused by artificial lighting from urban areas disrupts this natural cycle, and has been shown to impact the behavior of organisms, even many kilometers away from the light sources. It could be hypothesized that factors that increase the luminance of the sky amplify the degree of this “ecological light pollution”. We show that cloud coverage dramatically amplifies the sky luminance, by a factor of 10.1 for one location inside of Berlin and by a factor of 2.8 at 32 km from the city center. We also show that inside of the city overcast nights are brighter than clear rural moonlit nights, by a factor of 4.1. These results have important implications for choronobiological and chronoecological studies in urban areas, where this amplification effect has previously not been considered

Differential localization and high expression of SURVIVIN splice variants in human embryonic stem cells but not in differentiated cells implicate a role for SURVIVIN in pluripotency

The BIRC5 gene encodes the oncofetal protein SURVIVIN, as well as four additional splice variants (ΔEx3, 2B, 3B and 2α). SURVIVIN, an inhibitor of apoptosis, is also a chromosomal passenger protein (CPP). Previous results have demonstrated that SURVIVIN is expressed at high levels in embryonic stem cells and inhibition of SURVIVIN function results in apoptosis, however these studies have not investigated the other four splice variants. In this study, we demonstrate that all variants are expressed at significantly higher levels in human embryonic stem (hES) cells than in differentiated cells. We examined the subcellular localization of the three most highly expressed variants. SURVIVIN displayed canonical CPP localization in mitotic cells and cytoplasmic localization in interphase cells. In contrast, SURVIVIN–ΔEx3 and SURVIVIN–2B did not localize as a CPP; SURVIVIN–ΔEx3 was found constitutively in the nucleus while SURVIVIN–2B was distributed along the chromosomes during mitosis and also to the mitotic spindle poles. We used inducible shRNA against SURVIVIN to inhibit expression in a titratable fashion. Using this system, we reduced the mRNA levels of these three variants to approx. 40%, resulting in a concomitant reduction of OCT4 and NANOG mRNA, suggesting a role for the SURVIVIN variants in pluripotency

Advantages of nonhuman primates as preclinical models for evaluating stem cell-based therapies for Parkinson's disease

AbstractThe derivation of dopaminergic neurons from induced pluripotent stem cells brings new hope for a patient-specific, stem cell-based replacement therapy to treat Parkinson's disease (PD) and related neurodegenerative diseases; and this novel cell-based approach has already proven effective in animal models. However, there are several aspects of this procedure that have yet to be optimized to the extent required for translation to an optimal cell-based transplantation protocol in humans. These challenges include pinpointing the optimal graft location, appropriately scaling up the graft volume, and minimizing the risk of chronic immune rejection, among others. To advance this procedure to the clinic, it is imperative that a model that accurately and fully recapitulates characteristics most pertinent to a cell-based transplantation to the human brain is used to optimize key technical aspects of the procedure. Nonhuman primates mimic humans in multiple ways including similarities in genomics, neuroanatomy, neurophysiology, immunogenetics, and age-related changes in immune function. These characteristics are critical to the establishment of a relevant model in which to conduct preclinical studies to optimize the efficacy and safety of cell-based therapeutic approaches to the treatment of PD. Here we review previous studies in rodent models, and emphasize additional advantages afforded by nonhuman primate models in general, and the baboon model in particular, for preclinical optimization of cell-based therapeutic approaches to the treatment of PD and other neurodegenerative diseases. We outline current unresolved challenges to the successful application of stem cell therapies in humans and propose that the baboon model in particular affords a number of traits that render it most useful for preclinical studies designed to overcome these challenges

Optimization of culture conditions for the derivation and propagation of baboon (Papio anubis) induced pluripotent stem cells

<div><p>Induced pluripotent stem cells (iPSCs) offer the possibility of cell replacement therapies using patient-matched cells to treat otherwise intractable diseases and debilitations. To successfully realize this potential, several factors must be optimized including i) selection of the appropriate cell type and numbers to transplant, ii) determination of the means of transplantation and the location into which the transplanted cells should be delivered, and iii) demonstration of the safety and efficacy of the cell replacement protocol to mitigate each targeted disease state. A majority of diseases or debilitations likely to be targeted by cell-based therapeutic approaches represent complex conditions or physiologies manifest predominantly in primates including humans. Nonhuman primates afford the most clinically relevant model system for biomedical studies and testing of cell-based therapies. Baboons have 92% genomic similarity with humans overall and especially significant similarities in their immunogenetic system, rendering this species a particularly valuable model for testing procedures involving cell transplants into living individuals. To maximize the utility of the baboon model, standardized protocols must be developed for the derivation of induced pluripotent stem cells from living adults and the long-term maintenance of these cells in culture. Here we tested four commercially available culture systems (ReproFF, mTeSR1, E8 and Pluristem) for competence to maintain baboon iPSCs in a pluripotent state over multiple passages, and to support the derivation of new lines of baboon iPSCs. Of these four media only Pluristem was able to maintain baboon pluripotency as assessed by morphological characteristics, immunocytochemistry and RT-qPCR. Pluristem also facilitated the derivation of new lines of iPSCs from adult baboon somatic cells, which had previously not been accomplished. We derived multiple iPS cell lines from adult baboon peripheral blood mononuclear cells cultured in Pluristem. These were validated by expression of the pluripotency markers OCT4, NANOG, SOX2, SSEA4 and TRA181, as well as the ability to differentiate into tissues from all three germ layers when injected into immunocompromised mice. These findings further advance the utility of the baboon as an ideal preclinical model system for optimizing iPS cell-based, patient-specific replacement therapies in humans.</p></div

Immunocytochemical localization of pluripotency associated factors.

<p>As evaluated by immunocytochemical staining, baboon iPSCs cultured in conditioned media and Pluristem had similar expression levels and subcellular distribution of OCT4 (A,F, Green), NANOG (B,G, Green), SOX2 (C,H, Green), TRA181 (D,I, Green) and SSEA4 (E,J, Green) Blue = DNA, Bar = 20 μm.</p