A Translational Model for Venous Thromboembolism: MicroRNA Expression in Hibernating Black Bears.

BACKGROUND: Hibernating American black bears have significantly different clotting parameters than their summer active counterparts, affording them protection against venous thromboembolism during prolonged periods of immobility. We sought to evaluate if significant differences exist between the expression of microRNAs in the plasma of hibernating black bears compared with their summer active counterparts, potentially contributing to differences in hemostasis during hibernation. MATERIALS AND METHODS: MicroRNA sequencing was assessed in plasma from 21 American black bears in summer active (n = 11) and hibernating states (n = 10), and microRNA signatures during hibernating and active state were established using both bear and human genome. MicroRNA targets were predicted using messenger RNA (mRNA) transcripts from black bear kidney cells. In vitro studies were performed to confirm the relationship between identified microRNAs and mRNA expression, using artificial microRNA and human liver cells. RESULTS: Using the bear genome, we identified 15 microRNAs differentially expressed in the plasma of hibernating black bears. Of these microRNAs, three were significantly downregulated (miR-141-3p, miR-200a-3p, and miR-200c-3p), were predicted to target SERPINC1, the gene for antithrombin, and demonstrated regulatory control of the gene mRNA expression in cell studies. CONCLUSIONS: Our findings suggest that the hibernating black bears' ability to maintain hemostasis and achieve protection from venous thromboembolism during prolonged periods of immobility may be due to changes in microRNA signatures and possible upregulation of antithrombin expression.

Hospitalizations in elderly glioblastoma patients.

BACKGROUND: Elderly glioblastoma (GB) patients are at risk of hospitalizations due to the morbidity of the disease and possible treatment toxicity. METHODS: In this observational cohort study, 255 newly diagnosed GB patients age 65 years and older were included. Survival, emergency room visits and admissions to an acute care hospital were determined. Mean and median total health care costs were calculated. Risk factors for Emergency room visits and acute care hospital admissions were determined. RESULTS: Median overall survival was 6 months. The majority of patients (68%) had at least one visit to the emergency department and 77% had at least one admission to acute care. The mean and median total costs (hospital, ambulatory, physician billing, other health care costs) per patient were $162,479.78 (CAN) and $125,511.00 (CAN), respectively. Treatment with radiation or treatment with radio-chemotherapy was associated with a relative risk (RR) of 2.31 (95% CI: 1.44-3.7; P=0.0005) and 2.19 (95% CI: 1.28-3.74; P=0.004), respectively for emergency department visits as compared to patients who were managed with comfort measures only. Patients with a baseline ECOG 0 had a RR of 1.71 (95% CI: 1.06-2.77; P=0.0289) and patients with baseline ECOG 1 had a RR of 1.49 (0.98-2.26; P=0.0623) for hospital admission as compared to patients with ECOG 4. CONCLUSIONS: A large proportion of elderly GB patients (particularly those with good baseline performance status who underwent active treatment) presented to the emergency department and had at least one admission to acute care.

Self-Complementary Adeno-Associated Virus Vectors Improve Transduction Efficiency of Corneal Endothelial Cells.

Transplantation of a donor cornea to restore vision is the most frequently performed transplantation in the world. Corneal endothelial cells (CEC) are crucial for the outcome of a graft as they maintain corneal transparency and avoid graft failure due to corneal opaqueness. Given the characteristic of being a monolayer and in direct contact with culture medium during cultivation in eye banks, CEC are specifically suitable for gene therapeutic approaches prior to transplantation. Recombinant adeno-associated virus 2 (rAAV2) vectors represent a promising tool for gene therapy of CEC. However, high vector titers are needed to achieve sufficient gene expression. One of the rate-limiting steps for transgene expression is the conversion of single-stranded (ss-) DNA vector genome into double-stranded (ds-) DNA. This step can be bypassed by using self-complementary (sc-) AAV2 vectors. Aim of this study was to compare for the first time transduction efficiencies of ss- and scAAV2 vectors in CEC. For this purpose AAV2 vectors containing enhanced green fluorescent protein (GFP) as transgene were used. Both in CEC and in donor corneas, transduction with scAAV2 resulted in significantly higher transgene expression compared to ssAAV2. The difference in transduction efficiency decreased with increasing vector titer. In most cases, only half the vector titer of scAAV2 was required for equal or higher gene expression rates than those of ssAAV2. In human donor corneas, GFP expression was 64.7±11.3% (scAAV) and 38.0±8.6% (ssAAV) (p<0.001), respectively. Furthermore, transduced cells maintained their viability and showed regular morphology. Working together with regulatory authorities, a translation of AAV2 vector-mediated gene therapy to achieve a temporary protection of corneal allografts during cultivation and transplantation could therefore become more realistic.

The advanced LIGO input optics.

The advanced LIGO gravitational wave detectors are nearing their design sensitivity and should begin taking meaningful astrophysical data in the fall of 2015. These resonant optical interferometers will have unprecedented sensitivity to the strains caused by passing gravitational waves. The input optics play a significant part in allowing these devices to reach such sensitivities. Residing between the pre-stabilized laser and the main interferometer, the input optics subsystem is tasked with preparing the laser beam for interferometry at the sub-attometer level while operating at continuous wave input power levels ranging from 100 mW to 150 W. These extreme operating conditions required every major component to be custom designed. These designs draw heavily on the experience and understanding gained during the operation of Initial LIGO and Enhanced LIGO. In this article, we report on how the components of the input optics were designed to meet their stringent requirements and present measurements showing how well they have lived up to their design.

Human C9ORF72 Hexanucleotide Expansion Reproduces RNA Foci and Dipeptide Repeat Proteins but Not Neurodegeneration in BAC Transgenic Mice.

A non-coding hexanucleotide repeat expansion in the C9ORF72 gene is the most common mutation associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathological role of C9ORF72 in these diseases, we generated a line of mice carrying a bacterial artificial chromosome containing exons 1 to 6 of the human C9ORF72 gene with approximately 500 repeats of the GGGGCC motif. The mice showed no overt behavioral phenotype but recapitulated distinctive histopathological features of C9ORF72 ALS/FTD, including sense and antisense intranuclear RNA foci and poly(glycine-proline) dipeptide repeat proteins. Finally, using an artificial microRNA that targets human C9ORF72 in cultures of primary cortical neurons from the C9BAC mice, we have attenuated expression of the C9BAC transgene and the poly(GP) dipeptides. The C9ORF72 BAC transgenic mice will be a valuable tool in the study of ALS/FTD pathobiology and therapy.

Observation of Parametric Instability in Advanced LIGO.

Parametric instabilities have long been studied as a potentially limiting effect in high-power interferometric gravitational wave detectors. Until now, however, these instabilities have never been observed in a kilometer-scale interferometer. In this Letter, we describe the first observation of parametric instability in a gravitational wave detector, and the means by which it has been removed as a barrier to progress.

Critical role of monocytes to support normal B cell and diffuse large B cell lymphoma survival and proliferation.

Large B cell lymphomas can comprise numerous CD14+ cells in the tumor stroma, which raises the question of whether monocytes can support B cell survival and proliferation. We show that the coculture of monocytes with B cells from peripheral blood or from diffuse large B cell lymphoma enabled prolonged B cell survival. Under these conditions, diffuse large lymphoma B cells proliferated, and addition of B cell-activating factor of the TNF family (BAFF) and IL-2 enhanced cell division. Monocytes and dendritic cells (DC) had similar antiapoptotic activity on healthy B cells but displayed differences with respect to B cell proliferation. Monocytes and cord blood-derived CD14+ cells promoted B cell proliferation in the presence of an anti-CD40 stimulus, whereas DC supported B cell proliferation when activated through the BCR. DC and CD14+ cells were able to induce plasmocyte differentiation. When B cells were activated via the BCR or CD40, they released the leukocyte attractant CCL5, and this chemokine is one of the main chemokines expressed in diffuse large B cell lymphoma. The data support the notion that large B cell lymphoma recruit monocytes via CCL5 to support B cell survival and proliferation.

LPS induces rapid IL-10 release by M-CSF-conditioned tolerogenic dendritic cell precursors.

Dendritic cells (DC) obtained by culturing myeloid precursors in GM-CSF undergo maturation and induce an efficient T cell response when stimulated with microbial products. DC precursors themselves also recognize microbial products, and it remains unclear how these stimulated DC precursors modulate the immune response. We show here that M-CSF-conditioned human DC precursors responded to LPS, Mycobacteria bovis, and inflammatory cytokines by a rapid and robust production of IL-10, largely superior to that observed with immature DC or monocytes. The endogenous IL-10 restrained the DC precursors from converting into professional APC, as blocking the IL-10 receptor in the presence of LPS resulted in the formation of efficient T cell stimulators. LPS stimulation concomitant with DC differentiation gave rise to immature DC, which were tolerant to a secondary LPS exposure. Furthermore, the LPS-activated DC precursors reduced bystander DC maturation and anti-CD3/CD28-triggered T cell activation. These data suggest that when exposed to inflammatory or microbial signals, M-CSF-conditioned DC precursors can participate in the modulation of inflammation and immune response by rapid release of IL-10.

The Densford Clinical Scholars Program: improving patient care through research partnerships.

The Densford Clinical Scholars Program at the University of Minnesota School of Nursing partners advanced practice nurses and faculty members to design and conduct clinical studies for improving patient care. Benefits have included changes in nursing practice and, on occasion, the practice of other members of the healthcare team; enhanced research skills for clinicians; an enriched professional practice environment; access to clinical facilities for faculty; funding for research; and an expanded network for professional development. The authors describe this innovative partnership.

Tumor necrosis factor-alpha- and IL-4-independent development of Langerhans cell-like dendritic cells from M-CSF-conditioned precursors.

GM-CSF and transforming growth factor beta (TGFbeta ) are required for the generation of Langerhans cells (LC), members of the dendritic cell (DC) family. Tumor necrosis factor alpha (TNFalpha) and IL-4 can enhance LC differentiation from human monocytes or CD34(+) progenitors. Here, we show that M-CSF-cultured DC precursors derived from CD34(+) progenitors resemble dermal CD14(+) cells and readily convert to LC-like DC in GM-CSF/TGFbeta. The cells express Langerin, CD1a, and CCR6, migrate in response to CCR6 ligand CCL20, and contain Birbeck granules. TNFalpha and IL-4, added separately or together, have an inhibitory effect on LC differentiation. Cells differentiated in the presence of IL-4 and TNFalpha express low levels of CCR7. This suggests that M-CSF-conditioned DC precursors retain the capacity to efficiently undergo a differentiation program, giving rise to LC-like DC solely through the effect of GM-CSF and TGFbeta.

CD14 and CD169 expression in human lymph nodes and spleen: specific expansion of CD14+CD169- monocyte-derived cells in diffuse large B-cell lymphomas.

The mononuclear phagocyte system of human lymphoid tissue comprises macrophages and dendritic cells (DCs). The heterogeneity of the non-DC mononuclear phagocyte population in human lymphoid tissue has been little addressed. Here, we studied the expression of 2 monocyte-derived markers, CD14 and CD169 (sialoadhesin), in reactive human lymphoid tissue as well as in a series of 51 B-cell lymphomas by immunohistochemistry on paraffin-embedded tissue. We confirmed that lymph node sinusoidal monocyte-derived cells were the only population staining for CD169. Although most sinusoidal histiocytes also expressed CD14, monocyte-derived cells with phagocytosis such as erythrophagocytosis, anthracosis, or tingible bodies macrophage lacked CD14 and CD169. Among B-cell lymphomas, splenic marginal zone lymphoma was the only one associated with an expansion of the CD14(+)CD169(+) cells in the cords. With respect to nodal B-cell lymphomas, CD14(+) cells were rare among B-chronic lymphocytic leukemia, follicular lymphoma (FL), mantle cell lymphoma (MCL). However, strikingly, we found a strong expansion of CD14(+)CD169(-) cells in numerous diffuse large B-cell lymphomas (DLBCLs), except in cases associated with numerous mitoses, apoptotic bodies, and tingible bodies macrophages. When cultivated in granulocyte/macrophage colony stimulating factor/interleukin 4, DLBCL purified CD14(+) cells differentiate into plasmacytoid cells, expressing DC-specific intercellular adhesion molecule 3-grabbing nonintegrin, suggesting dendritic cell differentiation potential. Our observation fits well with the lymph node and host response cluster signatures described in the gene profiling signatures of DLBCL. However, the role of this CD14(+) population that may constitute a microenvironment-related marker of this subgroup of DLBCL remains to be determined.

Solving the advection-diffusion equations in biological contexts using the cellular Potts model.

The cellular Potts model (CPM) is a robust, cell-level methodology for simulation of biological tissues and morphogenesis. Both tissue physiology and morphogenesis depend on diffusion of chemical morphogens in the extra-cellular fluid or matrix (ECM). Standard diffusion solvers applied to the cellular potts model use finite difference methods on the underlying CPM lattice. However, these methods produce a diffusing field tied to the underlying lattice, which is inaccurate in many biological situations in which cell or ECM movement causes advection rapid compared to diffusion. Finite difference schemes suffer numerical instabilities solving the resulting advection-diffusion equations. To circumvent these problems we simulate advection diffusion within the framework of the CPM using off-lattice finite-difference methods. We define a set of generalized fluid particles which detach advection and diffusion from the lattice. Diffusion occurs between neighboring fluid particles by local averaging rules which approximate the Laplacian. Directed spin flips in the CPM handle the advective movement of the fluid particles. A constraint on relative velocities in the fluid explicitly accounts for fluid viscosity. We use the CPM to solve various diffusion examples including multiple instantaneous sources, continuous sources, moving sources, and different boundary geometries and conditions to validate our approximation against analytical and established numerical solutions. We also verify the CPM results for Poiseuille flow and Taylor-Aris dispersion.

Dendritic cell precursors are permissive to dengue virus and human immunodeficiency virus infection.

CD14(+) interstitial cells reside beneath the epidermis of skin and mucosal tissue and may therefore play an important role in viral infections and the shaping of an antiviral immune response. However, in contrast to dendritic cells (DC) or blood monocytes, these antigen-presenting cells (APC) have not been well studied. We have previously described long-lived CD14(+) cells generated from CD34(+) hematopoietic progenitors, which may represent model cells for interstitial CD14(+) APC. Here, we show that these cells carry DC-SIGN and differentiate into immature DC in the presence of granulocyte-macrophage colony-stimulating factor. We have compared the CD14(+) cells and the DC derived from these cells with respect to dengue virus and human immunodeficiency virus type 1 (HIV-1) infection. Both cell types are permissive to dengue virus infection, but the CD14(+) cells secrete the anti-inflammatory cytokine interleukin 10 and no tumor necrosis factor alpha. Regarding HIV, the CD14(+) cells are permissive to HIV-1, release higher p24 levels than the derived DC, and more efficiently activate HIV Pol-specific CD8(+) memory T cells. The CD14(+) DC precursors infected with either virus retain their DC differentiation potential. The results suggest that interstitial CD14(+) APC may contribute to HIV-1 and dengue virus infection and the shaping of an antiviral immune response.

Long-lived immature dendritic cells mediated by TRANCE-RANK interaction.

Immature dendritic cells (DCs) reside in interstitial tissues (int-DC) or in the epidermis, where they capture antigen and, thereafter, mature and migrate to draining lymph nodes (LNs), where they present processed antigen to T cells. We have identified int-DCs that express both TRANCE (tumor necrosis factor-related activation-induced cytokine) and RANK (receptor activator of NF-kappaB) and have generated these cells from CD34(+) human progenitor cells using macrophage colony-stimulating factor (M-CSF). These CD34(+)-derived int-DCs, which are related to macrophages, are long-lived, but addition of soluble RANK leads to significant reduction of cell viability and Bcl-2 expression. This suggests that constitutive TRANCE-RANK interaction is responsible for CD34(+)-derived int-DC longevity. Conversely, CD1a(+) DCs express only RANK and are short-lived. However, they can be rescued from cell death either by recombinant soluble TRANCE or by CD34(+)-derived int-DCs. CD34(+)-derived int-DCs mature in response to lipopolysaccharide (LPS) plus CD40 ligand (L) and become capable of CCL21/CCL19-mediated chemotaxis and naive T-cell activation. Upon maturation, they lose TRANCE, making them, like CD1a(+) DCs, dependent on exogenous TRANCE for survival. These findings provide evidence that TRANCE and RANK play important roles in the homeostasis of DCs.

The ADAMDEC1 (decysin) gene structure: evolution by duplication in a metalloprotease gene cluster on chromosome 8p12.

Members of the ADAM superfamily of metalloprotease genes are involved in a number of biological processes, including fertilization, neurogenesis, muscle development, and the immune response. These proteins have been classified into several groups. The prototypic ADAM family is comprised of a pro-domain, a metalloprotease domain, a disintegrin domain, a cysteine-rich region, a transmembrane domain, and a variable cytoplasmic tail. We recently identified a novel member of this superfamily, ADAMDEC1 (decysin). Due to the partial lack of a disintegrin domain and the total lack of a cysteine-rich domain, this protein has been placed in a novel subclass of the ADAM gene family. We have investigated the gene structure of the human and mouse ADAMDEC1 and have revealed a metalloprotease gene cluster on human Chromosome 8p12 comprising ADAMDEC1, ADAM7, and ADAM28. Our results suggest that ADAMDEC1 has arisen by partial gene duplication from an ancestral gene at this locus and has acquired a novel function. ADAMDEC1 is expressed in the immune system, by dendritic cells and macrophages. The relatedness of ADAMDEC1, ADAM7, and ADAM28 suggests that these proteases share a similar function.