Optimization and regeneration kinetics of lymphatic-specific photodynamic therapy in the mouse dermis.

Lymphatic vessels transport fluid, antigens, and immune cells to the lymph nodes to orchestrate adaptive immunity and maintain peripheral tolerance. Lymphangiogenesis has been associated with inflammation, cancer metastasis, autoimmunity, tolerance and transplant rejection, and thus, targeted lymphatic ablation is a potential therapeutic strategy for treating or preventing such events. Here we define conditions that lead to specific and local closure of the lymphatic vasculature using photodynamic therapy (PDT). Lymphatic-specific PDT was performed by irradiation of the photosensitizer verteporfin that effectively accumulates within collecting lymphatic vessels after local intradermal injection. We found that anti-lymphatic PDT induced necrosis of endothelial cells and pericytes, which preceded the functional occlusion of lymphatic collectors. This was specific to lymphatic vessels at low verteporfin dose, while higher doses also affected local blood vessels. In contrast, light dose (fluence) did not affect blood vessel perfusion, but did affect regeneration time of occluded lymphatic vessels. Lymphatic vessels eventually regenerated by recanalization of blocked collectors, with a characteristic hyperplasia of peri-lymphatic smooth muscle cells. The restoration of lymphatic function occurred with minimal remodeling of non-lymphatic tissue. Thus, anti-lymphatic PDT allows control of lymphatic ablation and regeneration by alteration of light fluence and photosensitizer dose

Characteristics of myosin profile in human vastus lateralis muscle in relation to training background.

Twenty-four male volunteers (mean +/- SD: age 25.4+/-5.8 years, height 178.6+/-5.5 cm, body mass 72.1+/-7.7 kg) of different training background were investigated and classified into three groups according to their physical activity and sport discipline: untrained students (group A), national and sub-national level endurance athletes (group B, 7.8+/-2.9 years of specialised training) and sprint-power athletes (group C, 12.8+/-8.7 years of specialised training). Muscle biopsies of vastus lateralis were analysed histochemically for mATPase and SDH activities, immunohistochemically for fast and slow myosin, and electrophoretically followed by Western immunoblotting for myosin heavy chain (MyHC) composition. Significant differences (

Photoactivation of lysosomally sequestered sunitinib after angiostatic treatment causes vascular occlusion and enhances tumor growth inhibition

The angiogenesis inhibitor sunitinib is a tyrosine kinase inhibitor that acts mainly on the VEGF and PDGF pathways. We have previously shown that sunitinib is sequestered in the lysosomes of exposed tumor and endothelial cells. This phenomenon is part of the drug-induced resistance observed in the clinic. Here, we demonstrate that when exposed to light, sequestered sunitinib causes immediate destruction of the lysosomes, resulting in the release of sunitinib and cell death. We hypothesized that this photoactivation of sunitinib could be used as a vaso-occlusive vascular-targeting approach to treating cancer. Spectral properties of sunitinib and its lysosomal accumulation were measured in vitro. The human A2780 ovarian carcinoma transplanted onto the chicken chorioallantoic membrane (CAM) and the Colo-26 colorectal carcinoma model in Balb/c mice were used to test the effects of administrating sunitinib and subsequently exposing tumor tissue to light. Tumors were subsequently resected and subject to immunohistochemical analysis. In A2780 ovarian carcinoma tumors, treatment with sunitinib+light resulted in immediate specific angio-occlusion, leading to a necrotic tumor mass 24 h after treatment. Tumor growth was inhibited by 70% as compared with the control group (**P= 4; P = 0.0002). Histology revealed that photoactivation of sunitinib resulted in a change in tumor vessel architecture. The current results suggest that the spectral properties of sunitinib can be exploited for application against certain cancer indications

Rare variants of the 3'-5' DNA exonuclease TREX1 in early onset small vessel stroke

Background: Monoallelic and biallelic mutations in the exonuclease TREX1 cause monogenic small vessel diseases (SVD). Given recent evidence for genetic and pathophysiological overlap between monogenic and polygenic forms of SVD, evaluation of TREX1 in small vessel stroke is warranted. Methods: We sequenced the TREX1 gene in an exploratory cohort of patients with lacunar stroke (Edinburgh Stroke Study, n=290 lacunar stroke cases). We subsequently performed a fully blinded case-control study of early onset MRI-confirmed small vessel stroke within the UK Young Lacunar Stroke Resource (990 cases, 939 controls). Results: No patients with canonical disease-causing mutations of TREX1 were identified in cases or controls. Analysis of an exploratory cohort identified a potential association between rare variants of TREX1 and patients with lacunar stroke. However, subsequent controlled and blinded evaluation of TREX1 in a larger and MRI-confirmed patient cohort, the UK Young Lacunar Stroke Resource, identified heterozygous rare variants in 2.1% of cases and 2.3% of controls. No association was observed with stroke risk (odds ratio = 0.90; 95% confidence interval, 0.49-1.65 p=0.74). Similarly no association was seen with rare TREX1 variants with predicted deleterious effects on enzyme function (odds ratio = 1.05; 95% confidence interval, 0.43-2.61 p=0.91). Conclusions: No patients with early-onset lacunar stroke had genetic evidence of a TREX1-associated monogenic microangiopathy. These results show no evidence of association between rare variants of TREX1 and early onset lacunar stroke. This includes rare variants that significantly affect protein and enzyme function. Routine sequencing of the TREX1 gene in patients with early onset lacunar stroke is therefore unlikely to be of diagnostic utility, in the absence of syndromic features or family history

Intravital Immunofluorescence for Visualizing the Microcirculatory and Immune Microenvironments in the Mouse Ear Dermis

Visualizing the dynamic behaviors of immune cells in living tissue has dramatically increased our understanding of how cells interact with their surroundings, contributing important insights into mechanisms of leukocyte trafficking, tumor cell invasion, and T cell education by dendritic cells, among others. Despite substantial advances with various intravital imaging techniques including two-photon microscopy and the generation of multitudes of reporter mice, there is a growing need to assess cell interactions in the context of specific extracellular matrix composition and microvascular functions, and as well, simpler and more widely accessible methods are needed to image cell behaviors in the context of living tissue physiology. Here we present an antibody-based method for intravital imaging of cell interactions with the blood, lymphatic, and the extracellular matrix compartments of the living dermis while simultaneously assessing capillary permeability and lymphatic drainage function. Using the exposed dorsal ear of the anesthetized mouse and a fluorescence stereomicroscope, such events can be imaged in the context of specific extracellular matrix proteins, or matrix-bound chemokine stores. We developed and optimized the method to minimize tissue damage to the ear, rapidly immunostain for multiple extracellular or cell surface receptors of interest, minimize immunotoxicity with pre-blocking Fc gamma receptors and phototoxicity with extracellular antioxidants, and highlight the major dermal tissue structures with basement membrane markers. We demonstrate differential migration behaviors of bone marrow-derived dendritic cells, blood-circulating leukocytes, and dermal dendritic cells, with the latter entering sparse CCL21-positive areas of pre-collecting lymphatic vessels. This new method allows simultaneous imaging of cells and tissue structures, microvascular function, and extracellular microenvironment in multiple skin locations for 12 hours or more, with the flexibility of immunolabeling in addition to genetic-based fluorescent reporters

Flow cytometric determination of genome size in European sunbleak Leucaspius delineatus (Heckel, 1843)

The aim of this study was to compare DNA content in hepatocyte and erythrocyte nuclei of the European sunbleak, Leucaspius delineatus, in relation to nuclear and cell size by means of flow cytometry and fluorescence microscopy. The DNA standards, chicken and rainbow trout erythrocytes, were prepared in parallel with both cell types, with initial separation of liver cells in pepsin solution followed by cell filtering. Standards and investigated cells were stained with a mixture of propidium iodide, citric acid, and Nonidet P40 in the presence of RNAse, and fluorescence of at least 50,000 nuclei was analyzed by flow cytometry. Average cell size was determined by flow cytometry, using fresh cell suspension in relation to latex beads of known diameter. The size of nuclei was examined on the basis of digital micrographs obtained by fluorescence microscopy after nuclei staining with DAPI. The sunbleak’s erythrocyte nuclei contain 2.25 ± 0.06 pg of DNA, whereas the hepatocyte nuclei contain 2.46 ± 0.06 pg of DNA. This difference in DNA content was determined spectroscopically using isolated DNA from the two cell types. The modal diameters of the erythrocytes and hepatocytes were estimated to be 5.1 ± 0.2 and 22.3 ± 5.0 μm, respectively, and the corresponding modal dimensions of their nuclei (measured as surface area) were 15.2 and 21.4 μm2, respectively. The nucleoplasmic index, as calculated from diameters estimated from surface area of nuclear profiles, was 2.51 for the erythrocytes compared with 0.08 for hepatocytes

Endocrine disruptors and spontaneous premature labor: a case control study

<p>Abstract</p> <p>Background</p> <p>Premature labor is a poorly understood condition. Estrogen is thought to play a key role and therefore the labor process may be affected by endocrine disruptors. We sought to determine whether or not an environmental toxicant, DDE, or dietary derived endocrine disruptors, daidzein and genistein, are associated with spontaneous preterm labor.</p> <p>Methods</p> <p>Cases were defined as primiparous patients having a preterm delivery at or before 35 weeks following the spontaneous onset of labor. Controls were defined as primiparous women who delivered on the same day as the cases but at term gestation.</p> <p>Over approximately 1 year, 26 cases and 52 controls were recruited. Subjects agreed to have blood tests on day one postpartum for DDE and for the phytoestrogens genistein and daidzein.</p> <p>Results</p> <p>The mean concentration of DDE was similar in the case and control groups: 4.29 vs 4.32 ng/g lipid p = .85. In the case group, 13/26 had detectable levels of daidzein (range 0.20 – 1.56 ng/ml) compared to 25/52 controls (range 0.21 – 3.26 ng/ml). The mean concentration of daidzein was similar in cases compared to controls: 0.30 vs .34 ng/ml p = 0.91. Of the case group,14/26 had detectable levels of genistein (range 0.20 – 2.19 ng/ml) compared to 32/52 controls (range 0.21 – 2.55 ng/ml). The mean concentration of genistein was similar in cases compared to controls: 0.39 vs 0.31 ng/ml, p = 0.61.</p> <p>Conclusion</p> <p>The serum levels of DDE in this population were found to be low.</p> <p>There appears to be no relationship between serum concentrations of DDE, daidzein, and genistein and spontaneous preterm labor in our population. The inability to identify an effect may be related to the comparatively low concentrations of DDE in our population and the rapid and variable reduction of phytoestrogens from women in labor.</p

Mechanisms and treatment of ischaemic stroke: insights from genetic associations

The precise pathophysiology of ischaemic stroke is unclear, and a greater understanding of the different mechanisms that underlie large-artery, cardioembolic and lacunar ischaemic stroke subtypes would enable the development of more-effective, subtype-specific therapies. Genome-wide association studies (GWASs) are identifying novel genetic variants that associate with the risk of stroke. These associations provide insight into the pathophysiological mechanisms, and present opportunities for novel therapeutic approaches. In this Review, we summarize the genetic variants that have been linked to ischaemic stroke in GWASs to date and discuss the implications of these associations for both our understanding and treatment of ischaemic stroke. The majority of genetic variants identified are associated with specific subtypes of ischaemic stroke, implying that these subtypes have distinct genetic architectures and pathophysiological mechanisms. The findings from the GWASs highlight the need to consider whether therapies should be subtype-specific. Further GWASs that include large cohorts are likely to provide further insights, and emerging technologies will complement and build on the GWAS findings