Evaluation and quantification of spectral information in tissue by confocal microscopy

A confocal imaging and image processing scheme is introduced to visualize and evaluate the spatial distribution of spectral information in tissue. The image data are recorded using a confocal laser-scanning microscope equipped with a detection unit that provides high spectral resolution. The processing scheme is based on spectral data, is less error-prone than intensity-based visualization and evaluation methods, and provides quantitative information on the composition of the sample. The method is tested and validated in the context of the development of dermal drug delivery systems, introducing a quantitative uptake indicator to compare the performances of different delivery systems is introduced. A drug penetration study was performed in vitro. The results show that the method is able to detect, visualize and measure spectral information in tissue. In the penetration study, uptake efficiencies of different experiment setups could be discriminated and quantitatively described. The developed uptake indicator is a step towards a quantitative assessment and, in a more general view apart from pharmaceutical research, provides valuable information on tissue composition. It can potentially be used for clinical in vitro and in vivo applications.

Impact of del32-71-GH (exon 3 skipped GH) on intracellular GH distribution, secretion and cell viability: a quantitative confocal microscopy analysis

BACKGROUND: Familial isolated growth hormone deficiency (IGHD) is a disorder with about 5-30% of patients having affected relatives. Among those familial types, IGHD type II is an autosomal dominant form of short stature, associated in some families with mutations that result in missplicing to produce del32-71-GH, a GH peptide which cannot fold properly. The mechanism by which this mutant GH may alter the controlled secretory pathway and therefore suppress the secretion of the normal 22-kDa GH product of the normal allele is not known in detail. Previous studies have shown variance depending on cell type, transfection technique used, as well as on the method of analysis performed. AIM: The aim of our study was to analyse and compare the subcellular distribution/localization of del32-71-GH or wild-type (wt)-GH (22-kDa GH), each stably transfected into AtT-20, a mouse pituitary cell line endogenously producing ACTH, employed as the internal control for secretion assessment. METHODS: Colocalization of wt- and del32-71 mutant GH form was studied by quantitative confocal microscopy analysis. Using the immunofluorescent technique, cells were double stained for GH plus one of the following organelles: endoplasmic reticulum (ER anti-Grp94), Golgi (anti-betaCOP) or secretory granules (anti-Rab3a). In addition, GH secretion and cell viability were analysed in detail. RESULTS/CONCLUSIONS: Our results show that in AtT-20 neuroendocrine cells, in comparison to the wt-GH, the del32-71-GH has a major impact on the secretory pathway not only affecting GH but also other peptides such as ACTH. The del32-71-GH is still present at the secretory vesicles' level, albeit in reduced quantity when compared to wt-GH but, importantly, was secretion-deficient. Furthermore, while focusing on cell viability an additional finding presented that the various splice site mutations, even though leading eventually to the same end product, namely del32-71-GH, have different and specific consequences on cell viability and proliferation rate.

Search for a heavy particle decaying into an electron and a muon with the ATLAS detector in sqrt[s] = 7 TeV pp collisions at the LHC

This Letter presents the first search for a heavy particle decaying into an e ± μ(-/+) final state in sqrt[s] = 7 TeV pp collisions at the LHC. The data were recorded by the ATLAS detector during 2010 and correspond to a total integrated luminosity of 35 pb(-1). No excess above the standard model background expectation is observed. Exclusions at 95% confidence level are placed on two representative models. In an R-parity violating supersymmetric model, tau sneutrinos with a mass below 0.75 TeV are excluded, assuming all R-parity violating couplings are zero except λ(311)' = 0.11 and λ312 = 0.07. In a lepton flavor violating model, a Z'-like vector boson with masses of 0.70-1.00 TeV and corresponding cross sections times branching ratios of 0.175-0.183 pb is excluded. These results extend to higher mass R-parity violating sneutrinos and lepton flavor violating Z's than previous constraints from the Tevatron.

Tumor recovery by angiogenic switch from sprouting to intussusceptive angiogenesis after treatment with PTK787/ZK222584 or ionizing radiation

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after treatment cessation. To assess the responses to irradiation and vascular endothelial growth factor-receptor tyrosine kinase inhibition (by the vascular endothelial growth factor tyrosine kinase inhibitor PTK787/ZK222854), mammary carcinoma allografts were investigated by vascular casting; electron, light, and confocal microscopy; and immunoblotting. Irradiation and anti-angiogenic therapy had similar effects on the tumor vasculature. Both treatments reduced tumor vascularization, particularly in the tumor medulla. After cessation of therapy, the tumor vasculature expanded predominantly by intussusception with a plexus composed of enlarged sinusoidal-like vessels containing multiple transluminal tissue pillars. Tumor revascularization originated from preserved alpha-smooth muscle actin-positive vessels in the tumor cortex. Quantification revealed that recovery was characterized by an angiogenic switch from sprouting to intussusception. Up-regulated alpha-smooth muscle actin-expression during recovery reflected the recruitment of alpha-smooth muscle actin-positive cells for intussusception as part of the angio-adaptive mechanism. Tumor recovery was associated with a dramatic decrease (by 30% to 40%) in the intratumoral microvascular density, probably as a result of intussusceptive pruning and, surprisingly, with only a minimal reduction of the total microvascular (exchange) area. Therefore, the vascular supply to the tumor was not severely compromised, as demonstrated by hypoxia-inducible factor-1alpha expression. Both irradiation and anti-angiogenic therapy cause a switch from sprouting to intussusceptive angiogenesis, representing an escape mechanism and accounting for the development of resistance, as well as rapid recovery, after cessation of therapy.

Size-Dependent Uptake of Particles by Pulmonary Antigen-Presenting Cell Populations and Trafficking to Regional Lymph Nodes

The respiratory tract is an attractive target organ for novel diagnostic and therapeutic applications with nano-sized carriers, but their immune effects and interactions with key resident antigen-presenting cells (APCs) such as dendritic cells (DCs) and alveolar macrophages (AMs) in different anatomical compartments remain poorly understood. Polystyrene particles ranging from 20 nm to 1,000 nm were instilled intranasally in BALB/c mice, and their interactions with APC populations in airways, lung parenchyma, and lung-draining lymph nodes (LDLNs) were examined after 2 and 24 hours by flow cytometry and confocal microscopy. In the main conducting airways and lung parenchyma, DC subpopulations preferentially captured 20-nm particles, compared with 1,000-nm particles that were transported to the LDLNs by migratory CD11blow DCs and that were observed in close proximity to CD3+ T cells. Generally, the uptake of particles increased the expression of CD40 and CD86 in all DC populations, independent of particle size, whereas 20-nm particles induced enhanced antigen presentation to CD4+ T cells in LDLNs in vivo. Despite measurable uptake by DCs, the majority of particles were taken up by AMs, irrespective of size. Confocal microscopy and FACS analysis showed few particles in the main conducting airways, but a homogeneous distribution of all particle sizes was evident in the lung parenchyma, mostly confined to AMs. Particulate size as a key parameter determining uptake and trafficking therefore determines the fate of inhaled particulates, and this may have important consequences in the development of novel carriers for pulmonary diagnostic or therapeutic applications.

Th17 cells and tissue remodeling in atopic and contact dermatitis

BACKGROUND: Eczematous skin lesions of atopic dermatitis (AD) as well as allergic and irritant contact dermatitis (ACD, ICD) are characterized by the same typical clinical signs, although due to different causes. In both AD and ACD, the presence of T helper 17 cells which play an important role in host defense, has been reported. Furthermore, IL-17 is involved in tissue repair and remodeling. This study aimed to investigate IL-17 expression in acute eczematous skin lesions and correlate it with markers of remodeling in AD, ACD, and ICD. METHODS: Skin specimens were taken from positive patch test reactions to aeroallergens, contact allergens, and irritants at days 2, 3, and 4. Inflammatory cells as well as the expression of cytokines and extracellular matrix proteins were evaluated by immunofluorescence staining and confocal microscopy. RESULTS: Allergic contact dermatitis and ICD were characterized by IFN-γ expression, whereas in AD lesions, IL-13 expression and high numbers of eosinophils were the prominent phenotype. Expression of IL-17, but also IL-21 and IL-22, was observed in all eczema subtypes. The number of IL-22+ T cells correlated with the number of eosinophils. Markers of remodeling such as MMP-9, procollagen-3, and tenascin C were observed in all acute eczematous lesions, while a correlation of IL-17+ T cell numbers with tenascin C-expressing cells and MMP-9+ eosinophils was apparent. CONCLUSION: The expression of IL-17 and related cytokines, such as IL-22, was demonstrated in acute eczematous lesions independent of their pathogenesis. Our results suggest a potential role for IL-17 in remodeling of the skin.

Electron and photon energy calibration with the ATLAS detector using LHC Run 1 data

This paper presents the electron and photon energy calibration achieved with the ATLAS detector using about 25 fb−1 of LHC proton–proton collision data taken at centre-of-mass energies of √s = 7 and 8 TeV. The reconstruction of electron and photon energies is optimised using multivariate algorithms. The response of the calorimeter layers is equalised in data and simulation, and the longitudinal profile of the electromagnetic showers is exploited to estimate the passive material in front of the calorimeter and reoptimise the detector simulation. After all corrections, the Z resonance is used to set the absolute energy scale. For electrons from Z decays, the achieved calibration is typically accurate to 0.05% in most of the detector acceptance, rising to 0.2% in regions with large amounts of passive material. The remaining inaccuracy is less than 0.2–1% for electrons with a transverse energy of 10 GeV, and is on average 0.3% for photons. The detector resolution is determined with a relative inaccuracy of less than 10% for electrons and photons up to 60 GeV transverse energy, rising to 40% for transverse energies above 500 GeV.

Phosphorylation of sst2 receptors in neuroendocrine tumors after octreotide treatment of patients

Somatostatin analogues, which are used to treat neuroendocrine tumors, target the high levels of somatostatin receptor subtype 2 (SSTR1; alias sst2) expressed in these cancers. However, some tumors are resistant to somatostatin analogues, and it is unknown whether the defect lies in sst2 activation or downstream signaling events. Because sst2 phosphorylation occurs rapidly after receptor activation, we examined whether sst2 is phosphorylated in neuroendocrine tumors. The sst2 receptor phosphorylation was evaluated by IHC and Western blot analysis with the new Ra-1124 antibody specific for the sst2 receptor phosphorylated at Ser341/343 in receptor-positive neuroendocrine tumors obtained from 10 octreotide-treated and 7 octreotide-naïve patients. The specificity, time course, and subcellular localization of sst2 receptor phosphorylation were examined in human embryo kinase-sst2 cell cultures by immunofluorescence and confocal microscopy. All seven octreotide-naïve tumors displayed exclusively nonphosphorylated cell surface sst2 expression. In contrast, 9 of the 10 octreotide-treated tumors contained phosphorylated sst2 that was predominantly internalized. Western blot analysis confirmed the IHC data. Octreotide treatment of human embryo kinase-sst2 cells in culture demonstrated that phosphorylated sst2 was localized at the plasma membrane after 10 seconds of stimulation and was subsequently internalized into endocytic vesicles. These data show, for the first time to our knowledge, that phosphorylated sst2 is present in most gastrointestinal neuroendocrine tumors from patients treated with octreotide but that a striking variability exists in the subcellular distribution of phosphorylated receptors among such tumors.

The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.

Empirical chemosensitivity testing in a spheroid model of ovarian cancer using a microfluidics-based multiplex platform.

The use of biomarkers to infer drug response in patients is being actively pursued, yet significant challenges with this approach, including the complicated interconnection of pathways, have limited its application. Direct empirical testing of tumor sensitivity would arguably provide a more reliable predictive value, although it has garnered little attention largely due to the technical difficulties associated with this approach. We hypothesize that the application of recently developed microtechnologies, coupled to more complex 3-dimensional cell cultures, could provide a model to address some of these issues. As a proof of concept, we developed a microfluidic device where spheroids of the serous epithelial ovarian cancer cell line TOV112D are entrapped and assayed for their chemoresponse to carboplatin and paclitaxel, two therapeutic agents routinely used for the treatment of ovarian cancer. In order to index the chemoresponse, we analyzed the spatiotemporal evolution of the mortality fraction, as judged by vital dyes and confocal microscopy, within spheroids subjected to different drug concentrations and treatment durations inside the microfluidic device. To reflect microenvironment effects, we tested the effect of exogenous extracellular matrix and serum supplementation during spheroid formation on their chemotherapeutic response. Spheroids displayed augmented chemoresistance in comparison to monolayer culturing. This resistance was further increased by the simultaneous presence of both extracellular matrix and high serum concentration during spheroid formation. Following exposure to chemotherapeutics, cell death profiles were not uniform throughout the spheroid. The highest cell death fraction was found at the center of the spheroid and the lowest at the periphery. Collectively, the results demonstrate the validity of the approach, and provide the basis for further investigation of chemotherapeutic responses in ovarian cancer using microfluidics technology. In the future, such microdevices could provide the framework to assay drug sensitivity in a timeframe suitable for clinical decision making.

Phagocytic uptake of Encephalitozoon cuniculi by nonprofessional phagocytes

Encephalitozoon cuniculi is an obligate intracellular, spore-forming parasite belonging to the microsporidia that can cause disseminated infection in immunocompromised persons. E. cuniculi spores infect host cells by germination, i.e., by explosively everting the polar filament, through which the spore contents (sporoplasms) are subsequently injected into the cytoplasm. In addition, we observed intracellular, nongerminated spores in various nonprofessional phagocytes. In MRC5 cells, the number of internalized spores was approximately 10-fold higher than the number of injected sporoplasms. Compared to the rate of uptake by human monocyte-derived macrophages, internalization rates by A549 cells, MRC5 cells, and 293 cells were 0.6, 4.4, and 22.2%, respectively. The mechanism of uptake was studied in MRC5 cells. Killed spores were internalized at the same rate as live spores, indicating that nongerminated parasites do not actively participate in cell entry. Cytochalasin D inhibited uptake of spores by 95%, demonstrating an actin-dependent process. By electron and epifluorescence microscopy, intracellular spores were found in a tightly fitting membrane-bound compartment. The vacuole containing the spores was positive for the lysosomal membrane protein LAMP-1 and colocalized with the late endosomal-lysosomal content marker rhodamine dextran. Our results show that, in addition to the unique way in which microsporidia infect cells, E. cuniculi spores enter nonprofessional phagocytes by phagocytosis and traffic into a late endosomal-lysosomal compartment.

Increased nuclear suppressor of cytokine signaling 1 in asthmatic bronchial epithelium suppresses rhinovirus induction of innate interferons.

BACKGROUND Rhinovirus infections are the dominant cause of asthma exacerbations, and deficient virus induction of IFN-α/β/λ in asthmatic patients is important in asthma exacerbation pathogenesis. Mechanisms causing this interferon deficiency in asthmatic patients are unknown. OBJECTIVE We sought to investigate the expression of suppressor of cytokine signaling (SOCS) 1 in tissues from asthmatic patients and its possible role in impaired virus-induced interferon induction in these patients. METHODS We assessed SOCS1 mRNA and protein levels in vitro, bronchial biopsy specimens, and mice. The role of SOCS1 was inferred by proof-of-concept studies using overexpression with reporter genes and SOCS1-deficient mice. A nuclear role of SOCS1 was shown by using bronchial biopsy staining, overexpression of mutant SOCS1 constructs, and confocal microscopy. SOCS1 levels were also correlated with asthma-related clinical outcomes. RESULTS We report induction of SOCS1 in bronchial epithelial cells (BECs) by asthma exacerbation-related cytokines and by rhinovirus infection in vitro. We found that SOCS1 was increased in vivo in bronchial epithelium and related to asthma severity. SOCS1 expression was also increased in primary BECs from asthmatic patients ex vivo and was related to interferon deficiency and increased viral replication. In primary human epithelium, mouse lung macrophages, and SOCS1-deficient mice, SOCS1 suppressed rhinovirus induction of interferons. Suppression of virus-induced interferon levels was dependent on SOCS1 nuclear translocation but independent of proteasomal degradation of transcription factors. Nuclear SOCS1 levels were also increased in BECs from asthmatic patients. CONCLUSION We describe a novel mechanism explaining interferon deficiency in asthmatic patients through a novel nuclear function of SOCS1 and identify SOCS1 as an important therapeutic target for asthma exacerbations.