Etoposide differentially affects bone marrow and dermal derived endothelial cells

Chemotherapy alteration of the bone marrow microenvironment has the potential to influence hematopoietic recovery following transplantation. To discern the effect of specific drugs on components of the complex marrow microenvironment, in vitro models have significant utility. In the current study we sought to determine whether dermal (HMEC-1) and marrow derived endothelial cells (BMEC-1) respond differently to identical chemotherapy exposure. BMEC-1 cells were consistently more sensitive to etoposide exposure than HMEC-1 cells, measured as reduced viability. BMEC-1 also had reduced focal adhesion kinase (FAK) and VCAM-1 protein expression following chemotherapy, in contrast to dermal derived endothelial cells in which neither protein was influenced dramatically by etoposide. The two endothelial cell lines had markedly different levels of baseline VE-Cadherin protein, which was modestly altered by treatment. These data indicate that marrow derived endothelial cells have disruption of specific proteins following chemotherapy that may influence their ability to facilitate hematopoietic cell entry or egress from the marrow. In addition, these observations suggest that while BMEC-1 and HMEC-1 share a variety of characteristics, they differ significantly in their response to stress and should be incorporated into specific models with this consideration

Human Cytomegalovirus Intrahost Evolution--A New Avenue for Understanding and Controlling Herpesvirus Infections [preprint]

Human cytomegalovirus (HCMV) is exquisitely adapted to the human host, and much research has focused on its evolution over long timescales spanning millennia. Here, we review recent data exploring the evolution of the virus on much shorter timescales, on the order of days or months. We describe the intrahost genetic diversity of the virus isolated from humans, and how this diversity contributes to HCMV spatiotemporal evolution. We propose mechanisms to explain the high levels of intrahost diversity and discuss how this new information may shed light on HCMV infection and pathogenesis

Extensive Genome-Wide Variability of Human Cytomegalovirus in Congenitally Infected Infants

Research has shown that RNA virus populations are highly variable, most likely due to low fidelity replication of RNA genomes. It is generally assumed that populations of DNA viruses will be less complex and show reduced variability when compared to RNA viruses. Here, we describe the use of high throughput sequencing for a genome wide study of viral populations from urine samples of neonates with congenital human cytomegalovirus (HCMV) infections. We show that HCMV intrahost genomic variability, both at the nucleotide and amino acid level, is comparable to many RNA viruses, including HIV. Within intrahost populations, we find evidence of selective sweeps that may have resulted from immune-mediated mechanisms. Similarly, genome wide, population genetic analyses suggest that positive selection has contributed to the divergence of the HCMV species from its most recent ancestor. These data provide evidence that HCMV, a virus with a large dsDNA genome, exists as a complex mixture of genome types in humans and offer insights into the evolution of the virus

The Cepheid Period-Luminosity Relation at Mid-Infrared Wavelengths: I. First-Epoch LMC Data

We present the first mid-infrared Period-Luminosity (PL) relations for Large Magellanic Cloud (LMC) Cepheids. Single-epoch observations of 70 Cepheids were extracted from Spitzer IRAC observations at 3.6, 4.5, 5.8 and 8.0 microns, serendipitously obtained during the SAGE (Surveying the Agents of a Galaxy's Evolution) imaging survey of the LMC. All four mid-infrared PL relations have nearly identical slopes over the period range 6 - 88 days, with a small scatter of only +/-0.16 mag independent of period for all four of these wavelengths. We emphasize that differential reddening is not contributing significantly to the observed scatter, given the nearly two orders of magnitude reduced sensitivity of the mid-IR to extinction compared to the optical. Future observations, filling in the light curves for these Cepheids, should noticeably reduce the residual scatter. These attributes alone suggest that mid-infrared PL relations will provide a practical means of significantly improving the accuracy of Cepheid distances to nearby galaxies.Comment: 19 pages, 4 figures, 1 table, Accepted for publication in the Astrophysical Journa

Rapid intrahost evolution of human cytomegalovirus is shaped by demography and positive selection

Populations of human cytomegalovirus (HCMV), a large DNA virus, are highly polymorphic in patient samples, which may allow for rapid evolution within human hosts. To understand HCMV evolution, longitudinally sampled genomic populations from the urine and plasma of 5 infants with symptomatic congenital HCMV infection were analyzed. Temporal and compartmental variability of viral populations were quantified using high throughput sequencing and population genetics approaches. HCMV populations were generally stable over time, with ~88% of SNPs displaying similar frequencies. However, samples collected from plasma and urine of the same patient at the same time were highly differentiated with approximately 1700 consensus sequence SNPs (1.2% of the genome) identified between compartments. This inter-compartment differentiation was comparable to the differentiation observed in unrelated hosts. Models of demography (i.e., changes in population size and structure) and positive selection were evaluated to explain the observed patterns of variation. Evidence for strong bottlenecks (\u3e90% reduction in viral population size) was consistent among all patients. From the timing of the bottlenecks, we conclude that fetal infection occurred between 13-18 weeks gestational age in patients analyzed, while colonization of the urine compartment followed roughly 2 months later. The timing of these bottlenecks is consistent with the clinical histories of congenital HCMV infections. We next inferred that positive selection plays a small but measurable role in viral evolution within a single compartment. However, positive selection appears to be a strong and pervasive driver of evolution associated with compartmentalization, affecting \u3e/= 34 of the 167 open reading frames (~20%) of the genome. This work offers the most detailed map of HCMV in vivo evolution to date and provides evidence that viral populations can be stable or rapidly differentiate, depending on host environment. The application of population genetic methods to these data provides clinically useful information, such as the timing of infection and compartment colonization

Three-Dimensional Microfluidic Tri-Culture Model of the Bone Marrow Microenvironment for Study of Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) initiates and progresses in the bone marrow, and as such, the marrow microenvironment is a critical regulatory component in development of this cancer. However, ALL studies were conducted mainly on flat plastic substrates, which do not recapitulate the characteristics of marrow microenvironments. To study ALL in a model of in vivo relevance, we have engineered a 3-D microfluidic cell culture platform. Biologically relevant populations of primary human bone marrow stromal cells, osteoblasts and human leukemic cells representative of an aggressive phenotype were encapsulated in 3-D collagen matrix as the minimal constituents and cultured in a microfluidic platform. The matrix stiffness and fluidic shear stress were controlled in a physiological range. The 3-D microfluidic as well as 3-D static models demonstrated coordinated cell-cell interactions between these cell types compared to the compaction of the 2-D static model. Tumor cell viability in response to an antimetabolite chemotherapeutic agent, cytarabine in tumor cells alone and tri-culture models for 2-D static, 3-D static and 3-D microfluidic models were compared. The present study showed decreased chemotherapeutic drug sensitivity of leukemic cells in 3-D tri-culture models from the 2-D models. The results indicate that the bone marrow microenvironment plays a protective role in tumor cell survival during drug treatment. The engineered 3-D microfluidic tri-culture model enables systematic investigation of effects of cell-cell and cell-matrix interactions on cancer progression and therapeutic intervention in a controllable manner, thus improving our limited comprehension of the role of microenvironmental signals in cancer biology

Three-Dimensional Microfluidic Tri-Culture Model of the Bone Marrow Microenvironment for Study of Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) initiates and progresses in the bone marrow, and as such, the marrow microenvironment is a critical regulatory component in development of this cancer. However, ALL studies were conducted mainly on flat plastic substrates, which do not recapitulate the characteristics of marrow microenvironments. To study ALL in a model of in vivo relevance, we have engineered a 3-D microfluidic cell culture platform. Biologically relevant populations of primary human bone marrow stromal cells, osteoblasts and human leukemic cells representative of an aggressive phenotype were encapsulated in 3-D collagen matrix as the minimal constituents and cultured in a microfluidic platform. The matrix stiffness and fluidic shear stress were controlled in a physiological range. The 3-D microfluidic as well as 3-D static models demonstrated coordinated cell-cell interactions between these cell types compared to the compaction of the 2-D static model. Tumor cell viability in response to an antimetabolite chemotherapeutic agent, cytarabine in tumor cells alone and tri-culture models for 2-D static, 3-D static and 3-D microfluidic models were compared. The present study showed decreased chemotherapeutic drug sensitivity of leukemic cells in 3-D tri-culture models from the 2-D models. The results indicate that the bone marrow microenvironment plays a protective role in tumor cell survival during drug treatment. The engineered 3-D microfluidic tri-culture model enables systematic investigation of effects of cell-cell and cell-matrix interactions on cancer progression and therapeutic intervention in a controllable manner, thus improving our limited comprehension of the role of microenvironmental signals in cancer biology

The HST Key Project on the Extragalactic Distance Scale XXVI. The Calibration of Population II Secondary Distance Indicators and the Value of the Hubble Constant

A Cepheid-based calibration is derived for four distance indicators that utilize stars in the old stellar populations: the tip of the red giant branch (TRGB), the planetary nebula luminosity function (PNLF), the globular cluster luminosity function (GCLF) and the surface brightness fluctuation method (SBF). The calibration is largely based on the Cepheid distances to 18 spiral galaxies within cz =1500 km/s obtained as part of the HST Key Project on the Extragalactic Distance Scale, but relies also on Cepheid distances from separate HST and ground-based efforts. The newly derived calibration of the SBF method is applied to obtain distances to four Abell clusters in the velocity range between 3800 and 5000 km/s, observed by Lauer et al. (1998) using the HST/WFPC2. Combined with cluster velocities corrected for a cosmological flow model, these distances imply a value of the Hubble constant of H0 = 69 +/- 4 (random) +/- 6 (systematic) km/s/Mpc. This result assumes that the Cepheid PL relation is independent of the metallicity of the variable stars; adopting a metallicity correction as in Kennicutt et al. (1998), would produce a (5 +/- 3)% decrease in H0. Finally, the newly derived calibration allows us to investigate systematics in the Cepheid, PNLF, SBF, GCLF and TRGB distance scales.Comment: Accepted for publication in the Astrophysical Journal. 48 pages (including 13 figures and 4 tables), plus two additional tables in landscape format. Also available at http://astro.caltech.edu/~lff/pub.htm K' SBF magnitudes have been update

Neurotrophins Regulate Bone Marrow Stromal Cell IL-6 Expression through the MAPK Pathway

The host's response to infection is characterized by altered levels of neurotrophins and an influx of inflammatory cells to sites of injured tissue. Progenitor cells that give rise to the differentiated cellular mediators of inflammation are derived from bone marrow progenitor cells where their development is regulated, in part, by cues from bone marrow stromal cells (BMSC). As such, alteration of BMSC function in response to elevated systemic mediators has the potential to alter their function in biologically relevant ways, including downstream alteration of cytokine production that influences hematopoietic development.In the current study we investigated BMSC neurotrophin receptor expression by flow cytometric analysis to determine differences in expression as well as potential to respond to NGF or BDNF. Intracellular signaling subsequent to neurotrophin stimulation of BMSC was analyzed by western blot, microarray analysis, confocal microscopy and real-time PCR. Analysis of BMSC Interleukin-6 (IL-6) expression was completed using ELISA and real-time PCR.BMSC established from different individuals had distinct expression profiles of the neurotrophin receptors, TrkA, TrkB, TrkC, and p75(NTR). These receptors were functional, demonstrated by an increase in Akt-phosphorylation following BMSC exposure to recombinant NGF or BDNF. Neurotrophin stimulation of BMSC resulted in increased IL-6 gene and protein expression which required activation of ERK and p38 MAPK signaling, but was not mediated by the NFkappaB pathway. BMSC response to neurotrophins, including the up-regulation of IL-6, may alter their support of hematopoiesis and regulate the availability of inflammatory cells for migration to sites of injury or infection. As such, these studies are relevant to the growing appreciation of the interplay between neurotropic mediators and the regulation of hematopoiesis