Tissue-Like 3D Standard and Protocols for Microscope Quality Management.

This article outlines a global study conducted by the Association of Biomedical Resource Facilities (ABRF) Light Microscopy Research Group (LMRG). The results present a novel 3D tissue-like biologically relevant standard sample that is affordable and straightforward to prepare. Detailed sample preparation, instrument-specific image acquisition protocols and image analysis methods are presented and made available to the community. The standard consists of sub-resolution and large well characterized relative intensity fluorescence microspheres embedded in a 120 µm thick 3D gel with a refractive index of 1.365. The standard allows the evaluation of several properties as a function of depth. These include the following: 1) microscope resolution with automated analysis of the point-spread function (PSF), 2) automated signal-to-noise ratio analysis, 3) calibration and correction of fluorescence intensity loss, and 4) quantitative relative intensity. Results demonstrate expected refractive index mismatch dependent losses in intensity and resolution with depth, but the relative intensities of different objects at similar depths are maintained. This is a robust standard showing reproducible results across laboratories, microscope manufacturers and objective lens types (e.g., magnification, immersion medium). Thus, these tools will be valuable for the global community to benchmark fluorescence microscopes and will contribute to improved scientific rigor and reproducibility.

Differences in amygdala cell proliferation between adolescent and young adult rats.

Adolescence is characterized by changes in both behavior and neural organization. During this period, the amygdala, a structure that mediates social and emotional behaviors, is changing in terms of neural and glia density. We examined cell proliferation within the amygdala of adolescent (post natal day (PND) 31) and adult (PND 70) male Sprague-Dawley rats using BrdU (bromodeoxyuridine) to label dividing cells. BrdU-labeled cells were distributed throughout the amygdala, often found in fibers surrounding major nuclei. Using two independent cell counting strategies under light and confocal microcopy, respectively, we found significantly more labeled cells in the amygdala in adolescent compared to adult animals (239.3 ± 87.18 vs. 44.75 ± 13.68; n=4/group; p<.05). BrdU/doublecortin (DCX) positive cells constitute approximately 30% of all dividing cells in the amygdala in both adolescents and adults. These data suggest that compared to young adulthood, adolescence is a relatively active period of cell proliferation in the amygdala. Moreover, the normal decline in dividing cells with age does not preferentially affect cells co-containing DCX-immunoreactivity.

Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl.

The Bcr-Abl tyrosine kinase oncogene causes chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL). We describe a novel selective inhibitor of Bcr-Abl, AMN107 (IC50 <30 nM), which is significantly more potent than imatinib, and active against a number of imatinib-resistant Bcr-Abl mutants. Crystallographic analysis of Abl-AMN107 complexes provides a structural explanation for the differential activity of AMN107 and imatinib against imatinib-resistant Bcr-Abl. Consistent with its in vitro and pharmacokinetic profile, AMN107 prolonged survival of mice injected with Bcr-Abl-transformed hematopoietic cell lines or primary marrow cells, and prolonged survival in imatinib-resistant CML mouse models. AMN107 is a promising new inhibitor for the therapy of CML and Ph+ ALL.

Perspectives on Access to Novel Therapeutics Through Clinical Trials Among Adolescents and Young Adults with Advanced Cancer: Implications for Patient-Centered Clinical Trials.

Purpose: Adolescents and young adults (AYA) with advanced cancer have unequal access to and enrollment in clinical trials. Many AYA use online platforms to share their treatment experiences. The purpose of this analysis was to explore how AYA discuss clinical trials and their access to novel therapeutics through their blogs. Methods: We studied illness blogs from 22 AYA (ages 16-38 years old) with advanced cancer who specifically discussed experiences enrolling in a clinical trial. Nearly 500 excerpts were abstracted from their blogs, and we used qualitative descriptive methodology and thematic analysis to explore their longitudinal perspectives. Results: We describe three themes: (1) "Blinded", which represents the uncertainty in treatment pathway and underrepresentation of AYA in clinical trials, (2) "Totally healthy except for the damn cancer", which represents the numerous challenges associated with meeting eligibility criteria and lack of available clinical trials, and (3) "Go ahead and send me the bill!", which represents the precarious financial challenges associated with participating with clinical trials (both direct costs and indirect costs associated with travel, time away from work) as well as the costs of novel therapeutics. Conclusions: By studying AYA online narratives, we can outline several gaps in accessing clinical trials and generate future research priorities. AYA with advanced cancer are known to have aggressive trajectories, and there are opportunities to integrate patient-reported outcomes and supportive care frameworks embedded within clinical trial study design.

Epithelial ablation of Bcl-XL increases sensitivity to oxygen without disrupting lung development.

Recent studies indicate that the antiapoptotic Bcl-X(L), one of five isoforms expressed by the Bcl-X gene, protects a variety of cell lines exposed to hyperoxia. However, its role in lung development and protection against oxidative stress in vivo is not known. Here, we show Bcl-X(L) is the predominant isoform expressed in the lung, and the only isoform detected in respiratory epithelium. Because loss of Bcl-X(L) is embryonically lethal, Bcl-X(L) was ablated throughout the respiratory epithelium by mating mice with a floxed exon II of the Bcl-X gene with mice expressing Cre under control of the surfactant protein-C promoter. Interestingly, the loss of Bcl-X(L) in respiratory epithelium was perinatally lethal in approximately 50% of the expected offspring. However, some adult mice lacking the gene were obtained. The epithelial-specific ablation of Bcl-X(L) did not disrupt pulmonary function, the expression of epithelial cell-specific markers, or lung development. However, it shifted the lung toward a proapoptotic state, defined by a reduction in antiapoptotic Mcl-1, an increase in proapoptotic Bak, and increased sensitivity of the respiratory epithelium to hyperoxia. Intriguingly, increased 8-oxoguanine lesions seen during hyperoxia were also evident as lungs transitioned to room air at birth, a time when perinatal lethality in some mice lacking Bcl-X(L) was observed. These findings reveal that the epithelial-specific expression of Bcl-X(L) is not required for proper lung development, but functions to protect respiratory epithelial cells against oxygen-induced toxicity, such as during hyperoxia and the lung's first exposure to ambient air.

Chronic neuron-specific tumor necrosis factor-alpha expression enhances the local inflammatory environment ultimately leading to neuronal death in 3xTg-AD mice.

Inflammatory mediators, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta, appear integral in initiating and/or propagating Alzheimer's disease (AD)-associated pathogenesis. We have previously observed a significant increase in the number of mRNA transcripts encoding the pro-inflammatory cytokine TNF-alpha, which correlated to regionally enhanced microglial activation in the brains of triple transgenic mice (3xTg-AD) before the onset of overt amyloid pathology. In this study, we reveal that neurons serve as significant sources of TNF-alpha in 3xTg-AD mice. To further define the role of neuronally derived TNF-alpha during early AD-like pathology, a recombinant adeno-associated virus vector expressing TNF-alpha was stereotactically delivered to 2-month-old 3xTg-AD mice and non-transgenic control mice to produce sustained focal cytokine expression. At 6 months of age, 3xTg-AD mice exhibited evidence of enhanced intracellular levels of amyloid-beta and hyperphosphorylated tau, as well as microglial activation. At 12 months of age, both TNF receptor II and Jun-related mRNA levels were significantly enhanced, and peripheral cell infiltration and neuronal death were observed in 3xTg-AD mice, but not in non-transgenic mice. These data indicate that a pathological interaction exists between TNF-alpha and the AD-related transgene products in the brains of 3xTg-AD mice. Results presented here suggest that chronic neuronal TNF-alpha expression promotes inflammation and, ultimately, neuronal cell death in this AD mouse model, advocating the development of TNF-alpha-specific agents to subvert AD.

Impact of Cosmetic Lotions on Nanoparticle Penetration through ex vivo C57BL/6 Hairless Mouse and Human Skin: A Comparison Study.

Understanding the interactions of nanoparticles (NPs) with skin is important from a consumer and occupational health and safety perspective, as well as for the design of effective NP-based transdermal therapeutics. Despite intense efforts to elucidate the conditions that permit NP penetration, there remains a lack of translatable results from animal models to human skin. The objectives of this study are to investigate the impact of common skin lotions on NP penetration and to quantify penetration differences of quantum dot (QD) NPs between freshly excised human and mouse skin. QDs were mixed in 7 different vehicles, including 5 commercial skin lotions. These were topically applied to skin using two exposure methods; a petri dish protocol and a Franz diffusion cell protocol. QD presence in the skin was quantified using Confocal Laser Scanning Microscopy. Results show that the commercial vehicles can significantly impact QD penetration in both mouse and human skin. Lotions that contain alpha hydroxyl acids (AHA) facilitated NP penetration. Lower QD signal was observed in skin studied using a Franz cell. Freshly excised human skin was also studied immediately after the sub-cutaneous fat removal process, then after 24 hours rest ex vivo. Resting human skin 24 hours prior to QD exposure significantly reduced epidermal presence. This study exemplifies how application vehicles, skin processing and the exposure protocol can affect QD penetration results and the conclusions that maybe drawn between skin models.

Dysregulation of gene expression in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mouse substantia nigra.

Parkinson's disease pathogenesis proceeds through several phases, culminating in the loss of dopaminergic neurons of the substantia nigra (SN). Although the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of oxidative SN injury is frequently used to study degeneration of dopaminergic neurons in mice and non-human primates, an understanding of the temporal sequence of molecular events from inhibition of mitochondrial complex 1 to neuronal cell death is limited. Here, microarray analysis and integrative data mining were used to uncover pathways implicated in the progression of changes in dopaminergic neurons after MPTP administration. This approach enabled the identification of small, yet consistently significant, changes in gene expression within the SN of MPTP-treated animals. Such an analysis disclosed dysregulation of genes in three main areas related to neuronal function: cytoskeletal stability and maintenance, synaptic integrity, and cell cycle and apoptosis. The discovery and validation of these alterations provide molecular evidence for an evolving cascade of injury, dysfunction, and cell death.

Cryotherapy for treatment of ECT-induced headache.

Because headache is a common side effect of electroconvulsive therapy (ECT), this study sought to determine the effectiveness of cryotherapy (i.e., a frozen gel band) in relieving pain in patients with post-ECT headaches, and whether headache intensity and physiological measurements could predict use of an alternative analgesic (rescue medication). We used a quasi-experimental, crossover design to collect data from 31 patients ages 24 to 85 who had been referred for ECT at two medical facilities in San Diego, California. Measurements of patients' pain intensity were made at three intervals: upon perceiving headache, and at 30 and 60 minutes following the cryotherapy or acetaminophen interventions, based on the order of the crossover design. Data were analyzed using Hotelling's T2 and logistic regression. No significant difference was found between cryotherapy and acetaminophen in relieving ECT-induced headaches (p = .420). There was no influence due to the crossover design (p = .313), nor where there significant changes in physiological measures from treatment (p = .420). Logistic regression showed that 50% of patients required rescue medication after 60 minutes for both treatments (R2 = .498, p = .001), and 66% required rescue medication based on pain level and physiological measures (R2 = .662, p < .008). Based on these results, cryotherapy is an alternative treatment that may be helpful to some patients with ECT-induced headaches.

Development and characterization of antibody reagents for detecting nanoparticles.

The increasing use of nanoparticles (NPs) in technological applications and in commercial products has escalated environmental health and safety concerns. The detection of NPs in the environment and in biological systems is challenged by limitations associated with commonly used analytical techniques. In this paper we report on the development and characterization of NP binding antibodies, termed NProbes. Phage display methodology was used to discover antibodies that bind NPs dispersed in solution. We present a proof-of-concept for the generation of NProbes and their use for detecting quantum dots and titanium dioxide NPs in vitro and in an ex vivo human skin model. Continued development and refinement of NProbes to detect NPs that vary in composition, shape, size, and surface coating will comprise a powerful tool kit that can be used to advance nanotechnology research particularly in the nanotoxicology and nanotherapeutics fields.

Progressive reduction of synaptophysin message in single neurons in Alzheimer disease.

The data presented here examine 2 hypotheses: 1) that viable but vulnerable single neurons remaining in the Alzheimer brain lose synaptic markers, and 2) that the extent of this loss is related to the disease state of these single neurons when disease state is defined by immunoreactivity. We used double immunohistochemistry (IHC) to define neurofibrillary tangle (NFT) and phosphorylation status of tau at selected defined epitopes. This double IHC was combined with quantitative in situ hybridization for message for the synaptic marker, synaptophysin, in 1,127 single hippocampal CA1 pyramidal neurons from 15 Alzheimer disease (AD) and 4 control cases. We found that there is a graded, progressive, decrease of synaptophysin message expressed by single neurons related to immunohistochemical markers of tau status, and that neurons in similar immunohistochemically defined classes show similar losses of synaptophysin message regardless of whether they were sampled from clinical control brains or advanced AD. The resulting conclusions are consistent with a suggestion that differences among clinically defined AD and control status are defined by the numbers of neurons in various disease states.

The ligand-mediated nuclear mobility and interaction with estrogen-responsive elements of estrogen receptors are subtype specific.

17ß-Estradiol (E(2)) plays important roles in functions of many tissues. E(2) effects are mediated by estrogen receptor (ER) α and ß. ERs regulate transcriptions through estrogen-responsive element (ERE)-dependent and ERE-independent modes of action. ER binding to ERE constitutes the basis of the ERE-dependent pathway. Direct/indirect ER interactions with transcription complexes define ERE-independent signaling. ERs share functional features. Ligand-bound ERs nevertheless induce distinct transcription profiles. Live cell imaging indicates a dynamic nature of gene expressions by highly mobile ERs. However, the relative contribution of ER mobility at the ERE-independent pathway to the overall kinetics of ER mobility remains undefined. We used fluorescent recovery after a photo-bleaching approach to assess the ligand-mediated mobilities of ERE binding-defective ERs, ER(EBD). The decrease in ERα mobility with E(2) or the selective ER modulator 4-hydroxyl-tamoxifen (4HT) was largely due to the interaction of the receptor with ERE. Thus, ERα bound to E(2) or 4HT mediates transcriptions from the ERE-independent pathway with remarkably fast kinetics that contributes fractionally to the overall motility of the receptor. The antagonist Imperial Chemical Industries 182 780 immobilized ERαs. The mobilities of ERß and ERß(EBD) in the presence of ligands were indistinguishable kinetically. Thus, ERß mobility is independent of the nature of ligands and the mode of interaction with target sites. Chimeric ERs indicated that the carboxyl-termini are critical regions for subtype-specific mobility. Therefore, while ERs are highly mobile molecules interacting with target sites with fast kinetics, an indication of the hit-and-run model of transcription, they differ mechanistically to modulate transcriptions.

A neurotoxic phosphoform of Elk-1 associates with inclusions from multiple neurodegenerative diseases.

Neurodegenerative diseases are characterized by a number of features including the formation of inclusions, early synaptic degeneration and the selective loss of neurons. Molecules serving as links between these shared features have yet to be identified. Identifying candidates within the diseased microenvironment will open up novel avenues for therapeutic intervention. The transcription factor Elk-1 resides within multiple brain areas both in nuclear and extranuclear neuronal compartments. Interestingly, its de novo expression within a single dendrite initiates neuronal death. Given this novel regionalized function, we assessed whether extranuclear Elk-1 and/or phospho-Elk-1 (pElk-1) protein might be associated with a spectrum of human neurodegenerative disease cases including Lewy body Disease (e.g. Parkinson's), Alzheimer's disease, and Huntington's Disease. We first determined the importance of Elk-1 post-translational modifications on its ability to initiate regionalized cell death. We next screened human cases from three major neurodegenerative diseases to look for remarkable levels of Elk-1 and/or pElk-1 protein as well as their association with inclusions characteristic of these diseases. We compared our findings to age-matched control cases. We find that the ability of Elk-1 to initiate regionalized neuronal death depends on a specific phosphosite, T417. Furthermore, we find that T417(+) Elk-1 uniquely associates with several types of inclusions present in cases of human Lewy body Disease, Alzheimer's disease, and Huntington's Disease. These results suggest a molecular link between the presence of inclusions and neuronal loss that is shared across a spectrum of neurodegenerative disease.

Molecular mechanisms of paraptosis induction: implications for a non-genetically modified tumor vaccine.

Paraptosis is the programmed cell death pathway that leads to cellular necrosis. Previously, rodent and human monocytes/macrophages killed glioma cells bearing the membrane macrophage colony stimulating factor (mM-CSF) through paraptosis, but the molecular mechanism of this killing process was never identified. We have demonstrated that paraptosis of rat T9 glioma cells can be initiated through a large potassium channel (BK)-dependent process initiated by reactive oxygen species. Macrophage mediated cytotoxicity upon the mM-CSF expressing T9-C2 cells was not prevented by the addition of the caspase inhibitor, zVAD-fmk. By a combination of fluorescent confocal and electron microscopy, flow cytometry, electrophysiology, pharmacology, and genetic knock-down approaches, we demonstrated that these ion channels control cellular swelling and vacuolization of rat T9 glioma cells. Cell lysis is preceded by a depletion of intracellular ATP. Six-hour exposure to BK channel activation caused T9 cells to over express heat shock proteins (Hsp 60, 70, 90 and gp96). This same treatment forced HMGB1 translocation from the nuclear region to the periphery. These last molecules are "danger signals" that can stimulate immune responses. Similar inductions of mitochondrial swelling and increased Hsp70 and 90 expressions by BK channel activation were observed with the non-immunogenic F98 glioma cells. Rats injected with T9 cells which were killed by prolonged BK channel activation developed immunity against the T9 cells, while the injection of x-irradiated apoptotic T9 cells failed to produce the vaccinating effect. These results are the first to show that glioma cellular death induced by prolonged BK channel activation improves tumor immunogenicity; this treatment reproduces the vaccinating effects of mM-CSF transduced cells. Elucidation of strategies as described in this study may prove quite valuable in the development of clinical immunotherapy against cancer.

VIP is a transcriptional target of Nurr1 in dopaminergic cells.

The orphan nuclear receptor Nurr1 is required for the development of the ventral mesencephalic dopaminergic neurons. These are the same neurons that are invariantly lost in patients with Parkinson's disease. Nurr1 mRNA expression is not confined to the developing midbrain, and yet Nurr1 appears to be essential for either the maturation of progenitors into fully post-mitotic dopaminergic neurons and/or once formed, their survival. The function of Nurr1 in the transactivation of gene(s) important for neuronal development and/or maintenance is uncharacterized. To characterize potential downstream target genes of Nurr1, we sought to identify mRNAs that are differentially affected by Nurr1 expression. Using a dopaminergic cell line in which Nurr1 content was tightly regulated, differential display analysis identified transcripts altered by Nurr1 expression, including the mRNA encoding vasoactive intestinal peptide (VIP). Herein, we demonstrate that Nurr1 regulates VIP mRNA and protein levels, and transactivates the VIP promoter through Nurr1-responsive cis elements. In addition, dopaminergic cells release and utilize VIP to mediate survival when challenged with paraquat. Nurr1 regulation of VIP is also demonstrated in vivo as loss of Nurr1 function results in diminished VIP mRNA levels within the developing midbrain.

Generation of human innate immune responses towards membrane macrophage colony stimulating factor (mM-CSF) expressing U251 glioma cells within immunodeficient (NIH-nu/beige/xid) mice.

The response of human peripheral blood mononuclear cells (PBMC) to cloned human HLA-A2+ U251 glioma cells (U251-2F11/TK) expressing membrane macrophage colony stimulating factor (mM-CSF) was investigated in vitro and in vivo. Enriched human monocytes derived from cancer patients produced a respiratory burst following 20min of interaction with mM-CSF expressing U251 glioma cells. This respiratory burst response was not observed in the enriched human monocytes following similar exposure to the viral vector control U251 (U251-VV) cells. Reactive oxygen species such as H(2)O(2) and HOCl produced death of the U251 cells. The U251-2F11/TK cells failed to grow in severely compromised combined immunodeficient (NIH-bg-nu-xidBR) mice that were depleted of murine monocyte/macrophages then reconstituted with human HLA-A2+ PBMC. Reactive oxygen species (ROS) were produced by PBMC, both in vitro and in vivo in response tomM-CSF expressing U251 cells. U251-2F11/TK cells failed to form subcutaneous tumors in macrophage depleted mice reconstituted with human PBMC; whereas, progressive growth of such tumors was observed with the U251-VV cells. U251-2F11/TK tumors formed if the initial inoculums of PBMC were depleted of monocytes. From this work it can be concluded that mM-CSF transduced U251-2F11/TK glioma cells can safely stimulate human innate immune responses in vivo.

Human monocytes kill M-CSF-expressing glioma cells by BK channel activation.

In this study, human monocytes/macrophages were observed to kill human U251 glioma cells expressing membrane macrophage colony-stimulating factor (mM-CSF) via a swelling and vacuolization process called paraptosis. Human monocytes responded to the mM-CSF-transduced U251 glioma cells, but not to viral vector control U251 glioma cells (U251-VV), by producing a respiratory burst within 20 min. Using patch clamp techniques, functional big potassium (BK) channels were observed on the membrane of the U251 glioma cell. It has been previously reported that oxygen indirectly regulates BK channel function. In this study, it was demonstrated that prolonged BK channel activation in response to the respiratory burst induced by monocytes initiates paraptosis in selected glioma cells. Forced BK channel opening within the glioma cells by BK channel activators (phloretin or pimaric acid) induced U251 glioma cell swelling and vacuolization occurred within 30 min. U251 glioma cell cytotoxicity, induced by using BK channel activators, required between 8 and 12 h. Swelling and vacuolization induced by phloretin and pimaric acid was prevented by iberiotoxin, a specific BK channel inhibitor. Confocal fluorescence microscopy demonstrated BK channels co-localized with the endoplasmic reticulum and mitochondria, the two targeted organelles affected in paraptosis. Iberiotoxin prevented monocytes from producing death in mM-CSF-expressing U251glioma cells in a 24 h assay. This study demonstrates a novel mechanism whereby monocytes can induce paraptosis via the disruption of internal potassium ion homeostasis.

Beneficial effects of combining nilotinib and imatinib in preclinical models of BCR-ABL+ leukemias.

Drug resistance resulting from emergence of imatinib-resistant BCR-ABL point mutations is a significant problem in advanced-stage chronic myelogenous leukemia (CML). The BCR-ABL inhibitor, nilotinib (AMN107), is significantly more potent against BCR-ABL than imatinib, and is active against many imatinib-resistant BCR-ABL mutants. Phase 1/2 clinical trials show that nilotinib can induce remissions in patients who have previously failed imatinib, indicating that sequential therapy with these 2 agents has clinical value. However, simultaneous, rather than sequential, administration of 2 BCR-ABL kinase inhibitors is attractive for many reasons, including the theoretical possibility that this could reduce emergence of drug-resistant clones. Here, we show that exposure of a variety of BCR-ABL+ cell lines to imatinib and nilotinib results in additive or synergistic cytotoxicity, including testing of a large panel of cells expressing BCR-ABL point mutations causing resistance to imatinib in patients. Further, using a highly quantifiable bioluminescent in vivo model, drug combinations were at least additive in antileukemic activity, compared with each drug alone. These results suggest that despite binding to the same site in the same target kinase, the combination of imatinib and nilotinib is highly efficacious in these models, indicating that clinical testing of combinations of BCR-ABL kinase inhibitors is warranted.