The musculature and associated innervation of adult and intramolluscan stages of Echinostoma caproni (Trematoda) visualised by confocal microscopy

Gross anatomy of muscle and sensory/motor innervation of adult and intramolluscan developmental stages of Echinostoma caproni have been investigated to ascertain the organisation and the functional correlates of any stage-specific patterns of staining. Using indirect immunocytochemistry to demonstrate neuroactive substances and the phalloidin-fluorescence technique for staining myofibril F-actin, the muscle systems and aminergic and peptidergic innervation of daughter rediae, cercariae, metacercariae, and pre- and post-ovigerous adults were examined and compared using confocal scanning laser microscopy. A complex arrangement of specific muscle fibre systems occurs within the body wall (composed of circular, longitudinal and diagonal fibres), suckers (radial, equatorial, meridional), pharynx (radial, circular), gut caeca (mainly circular), cercarial tail (circular, pseudo-striated longitudinal), and ducts of the reproductive system (circular, longitudinal), presumed to serve locomotor, adhesive, alimentary and reproductive functions. Immunostaining for serotonin (5-HT) and FMRFamide-related peptides (FaRPs) was evident throughout the central (CNS) and peripheral (PNS) nervous systems of all stages, and use of dual-labelling techniques demonstrated separate neuronal pathways for 5-HT and FaRP in both CNS and PNS. FaRP expression in the innervation of the ootype wall was demonstrated only in post-ovigerous worms and not in pre-ovigerous worms, suggesting an involvement of FaRP neuropeptides in the process of egg assembly. Comparison of the present findings with those recorded for other digeneans suggests that muscle organisation and innervation patterns in trematodes are highly conserved.

Immunocolloidal Targeting of the Endocytotic Siglec-7 Receptor Using Peripheral Attachment of Siglec-7 Antibodies to Poly(Lactide-co-Glycolide) Nanoparticles

Purpose: To prepare a nanoparticulate formulation expressing variable peripheral carboxyl density using non-endcapped and endcapped poly(lactide-co-glycolide), conjugated to antibodies recognising the siglec-7 receptor, which is expressed on most acute myeloid leukaemias. The aim is to exploit this receptor as a therapeutic target by constructing an internalising drug-loaded nanoparticle able to
translocate into cytoplasm by siglec receptor-mediated internalisation.

Materials and Methods: Antibodies to the siglec-7 (CD33-like) receptor were conjugated to dye-loaded nanoparticles using carbodiimide chemistry, giving 32.6 mg protein per mg of nanoparticles using 100% of the non-endcapped PLGA. Binding studies using cognate antigen were used to verify preservation of antibody function following conjugation.

Results: Mouse embryonic fibroblasts expressing recombinant siglec-7 receptor and exposed to NileRed-loaded nanoparticles conjugated to antibody accumulated intracellular fluorescence, which was not observed if either antibody or siglec-7 receptor was absent. Confocal microscopy revealed internalised perinuclear cytoplasmic staining, with an Acridine Orange-based analysis showing red staining in localised foci, indicating localisation within acidic endocytic compartments.

Conclusions: Results show antibody-NP constructs are internalised via siglec-7 receptor-mediated internalisation. If loaded with a therapeutic agent, antibody-NP constructs can cross into cytoplasmic
space and delivery drugs intracellularly to cells expressing CD33-like receptors, such as natural killer cells and monocytes.

The effect of treatment of cystic fibrosis pulmonary exacerbations on airways and systemic inflammation

Background: Chronic infection in cystic fibrosis (CF) and airway inflammation leads to progressive lung injury Neutrophils are considered to be responsible for the onset and promotion of the inflammatory response within the CF lung. The relationship between infection and inflammation is complex but circulating inflammatory markers may not truly reflect the local inflammatory response in the lung. The aims of this study were to investigate the change of inflammatory biomarkers and cells within sputum and blood before and after intravenous antibiotics for a pulmonary exacerbation of CF Methods: Assays included neutrophil elastase (NE) and complex, interleukin-8 (IL-8) and soluble intercellular adhesion molecule-1 (sICAM-1), fas ligand (FAS-L), and TNFr-1. Analysis of sputum cell differential and absolute cell counts and immunocytochemistry (CD11b and CD95) on sputum and isolated blood neutrophils were carried out. Results: There were no significant differences in absolute or differential sputum cell counts or sputum sol measurements following antibiotics. There was a significant increase in the percentage of blood neutrophils with minimal CD11b staining, 28 (4.1) mean percentage (SEM) versus41 (2.9) and a decrease in the percentage showing maximal staining 30 (0.5) versus 15 (2.5). There was a significant increase in the percentage of blood neutrophils without CD95 staining, 43 (5.4) mean percentage versus 52 (5.1). Conclusion: These data suggest a modifiable systemic response to IV antibiotics but a local sustained inflammatory response in the lung.

Modification of smooth muscle Ca2+-sparks by tetracaine: evidence for sequential RyR activation.

Spontaneous Ca(2+)-sparks were imaged using confocal line scans of fluo-4 loaded myocytes in retinal arterioles. Tetracaine produced concentration-dependent decreases in spark frequency, and modified the spatiotemporal characteristics of residual sparks. Tetracaine (10 microM) reduced the rate of rise but prolonged the average rise time so that average spark amplitude was unaltered. The mean half-time of spark decay was also unaffected, suggesting that spark termination, although delayed, remained well synchronized. Sparks spread transversely across the myocytes in these vessels, and the speed of spread within individual sparks was slowed by approximately 60% in 10 microM tetracaine, as expected if the spark was propagated across the cell but the average P(o) for RyRs was reduced. Staining of isolated vessels with BODIPY-ryanodine and di-4-ANEPPS showed that RyRs were located both peripherally, adjacent to the plasma membrane, and in transverse extensions of the SR from one side of the cell to the other. Immuno-labelling of retinal flat mounts demonstrated the presence RyR(2) in arteriole smooth muscle but not RyR(1). We conclude that Ca(2+)-sparks in smooth muscle can result from sequential activation of RyRs distributed over an area of several microm(2), rather than from tightly clustered channels as in striated muscle.

Neuroactive substances and associated major muscle systems in Bucephaloides gracilescens (Trematoda : Digenea) metacercaria and adult

Cholinergic, serotoninergic and neuropeptidergic components of the nervous system were examined and compared in the progenetic metacercaria and adult gasterostome trematode, Bucephaloides gracilescens in order to provide baseline information on neuronal control of the musculature involved in egg-assembly. Enzyme cytochemistry and indirect immunocytochemical techniques interfaced with confocal scanning laser microscopy demonstrated all three classes of neuroactive substance throughout the central and peripheral nervous systems. A comparable orthogonal arrangement of the central nervous system (CNS) and peripheral array of nerve plexuses was observed in both metacercaria and adult. Staining patterns for cholinergic and peptidergic substances showed significant overlap, while the serotoninergic system was confined to a separate set of neurons. Immunostaining for FMRFamide-related peptides (FaRPs) was strong in the CNS and peripheral innervation to the attachment apparatus of metacercaria and adult but was only found in the innervation of the ootype in ovigerous adults, implicating FaRPs in neuronal control of the muscle of the female reproductive tract during egg-assembly.

Organization of the musculature of schistosome cercariae

Phalloidin-fluorescein isothiocyanate staining of filamentous actin was used to identify muscle systems within the cercariae of Schistosoma mansoni. Examination of labeled cercariae by confocal scanning laser microscopy revealed distinct organizational levels of myofiber arrangements within the body wall, anterior cone, acetabulum, and esophagus. The body wall throughout showed a typical latticelike arrangement of outer circular and inner longitudinal myofibers, with an additional innermost layer of diagonal fibers in the anterior portion of the body. Circular and longitudinal fibers were also evident in the anterior organ and esophagus and. to some extent, the ventral acetabulum. Most striking was the striation of the cercarial tail musculature.

Organisation of the nervous system in the Acoela: an immunocytochemical study

In order to broaden the information about the organisation of the nervous system in taxon Acoela, an immunocytochemical study of an undetermined Acoela from Cape Kartesh, Faerlea glomerata, Avagina incola and Paraphanostoma crassum has been performed. Antibodies to 5-HT and the native flatworm neuropeptide GYIRFamide were used. As in earlier studies, the pattern of 5-HT immunoreactivity revealed an anterior structure composed mainly of commissures, a so-called commissural brain. Three types of brain shapes were observed. No regular orthogon was visualised. GYIRFamide immunoreactive cell clusters were observed peripherally to the 5-HT immunoreactive commissural brain. Staining with anti-GYIRFamide revealed more nerve processes than did staining with anti-FMRFamide. As no synapomorphies were found in the organisation of the nervous system of the Acoela and that of the Platyhelminthes, the results support the view that the Acoela is not a member of the Platyhelminthes. (C) 2001 Harcourt Publishers Ltd.

Basiepidermal nervous system in Nemertoderma westbladi (Nemertodermatida): GYIRFamide immunoreactivity

The Nemertodermatida are a small group of microscopic marine worms. Recent molecular studies have demonstrated that they are likely to be the earliest extant bilaterian animals. What was the nervous system (NS) of a bilaterian ancestor like? In order to answer that question, the NS of Nemertoderma westbladi was investigated by means of indirect immunofluorescence technique and confocal scanning laser microscopy. The antibodies to a flatworm neuropeptide GYIRFamide were used in combination with anti-serotonin antibodies and phalloidin-TRITC staining. The immunostaining revealed an entirely basiepidermal NS. A ring lying outside the body wall musculature at the level of the statocyst forms the only centralisation, the

A tip-high, Ca2+-interdependent, reactive oxygen species gradient is associated with polarized growth in Fucus serratus zygotes

We report the existence of a tip-high reactive oxygen species (ROS) gradient in growing Fucus serratus zygotes, using both 5-(and 6-) chloromethyl-2',7'-dichlorodihydrofluorescein and nitroblue tetrazolium staining to report ROS generation. Suppression of the ROS gradient inhibits polarized zygotic growth; conversely, exogenous ROS generation can redirect zygotic polarization following inhibition of endogenous ROS. Confocal imaging of fluo-4 dextran distributions suggests that the ROS gradient is interdependent on the tip-high [Ca2+](cyt) gradient which is known to be associated with polarized growth. Our data support a model in which localized production of ROS at the rhizoid tip stimulates formation of a localized tip-high [Ca2+](cyt) gradient. Such modulation of intracellular [Ca2+](cyt) signals by ROS is a common motif in many plant and algal systems and this study extends this mechanism to embryogenesis.

Clinical and immunohistochemical assessment of vulval intraepithelial neoplasia following photodynamic therapy using a novel bioadhesive patch-type system loaded with 5-aminolevulinic acid

Background: The work in this study appraised photodynamic treatment (PDT) as a treatment method for vulval intraepithelial neoplasia (VIN) using a novel bioadhesive patch to deliver aminolevulinic acid. An analysis of changes in expression of apoptotic and cell cycle proteins (p53, p21, Mdm2, Blc-2, Bax, Ki-67) in response to PDT was evaluated. Methods: PDT was performed using non-laser light, either as a one or two-cycle treatment, with clinical and pathological assessment following after 6 weeks. Twenty-three patients with 25 VIN lesions underwent 49 cycles of PDT Patches were designed to conform to uneven vulval skin and contained 38 mg cm(-2) aminolevulinic acid. Assessment was carried out at 6 weeks post-treatment. Patient-based treatment assessment, along with clinical and pathological changes, were monitored. Immunohistochemical staining was used to elucidate a possible biomolecular basis for induced cellular changes. Results: Most patients (52%) reported a symptomatic response, with normal pathology restored in 38% of lesions. The patch was easy to apply and remove, causing minimal discomfort. Fluorescence inspection confirmed protoporphyrin accumulation. Pain during implementation of PDT was problematic, necessitating some form of local analgesia. Changes in expression of cell cycle and apoptotic-related proteins suggested involvement of apoptotic pathways. Down regulation of p21 and inverse changes in Bcl-2 and Bax were key findings. Conclusion: Treatment of VIN lesions using a novel bioadhesive patch induced changes in cell cycle and apoptotic proteins in response to PDT with possible utilisation of apoptotic pathways. The efficacy of PDT in treating VIN could be improved by a better understanding of these apoptotic mechanisms, the influence of factors, such as HPV status, and of the need for effective pain management.

A novel method for isolation of human lung T cells from lung resection tissue reveals increased expression of GAPDH and CXCR6

Lung T lymphocytes are important in pulmonary immunity and inflammation. it has been difficult to study these cells due to contamination with other cell types, mainly alveolar macrophages. We have developed a novel method for isolating lung T cells from lung resection tissue, using a combination of approaches. Firstly the lung tissue was finely chopped and filtered through a nylon mesh. Lymphocytic cells were enriched by Percoll density centrifugation and the T cells purified using human CD3 microbeads, resulting in 90.5% +/- 1.9% (n = 11) pure lymphocytes. The T cell yield from the crude cell preparation was 10.8 +/- 2.1% and viability, calculated using propidium iodide (PI) staining and trypan blue, was typically over 95%. The purification process did not affect expression of CD69 or CD103, nor was there a difference in the proportion of CD4 and CD8 cells between the starting population and the purified cells. Microarray analysis and real time RT-PCR revealed upregulation of GAPDH and CXCR6 of the lung T cells as compared to blood-derived T cells. This technique highly enriches lung T cells to allow detailed investigation of the biology of these cells. (C) 2008 Elsevier B.V. All rights reserved.

Immune activation in Retinal Aging: A Gene Expression Study.

PURPOSE
To investigate changes in gene expression during aging of the retina in the mouse.

METHODS
Total RNA was extracted from the neuroretina of young (3-month-old) and old (20-month-old) mice and processed for microarray analysis. Age-related, differentially expressed genes were assessed by the empiric Bayes shrinkagemoderated t-statistics method. Statistical significance was based on dual criteria of a ratio of change in gene expression >2 and a P < 0.01. Differential expression in 11 selected genes was further verified by real-time PCR. Functional pathways involved in retinal ageing were analyzed by an online software package (DAVID-2008) in differentially expressed gene lists. Age-related changes in differential expression in the identified retinal molecular pathways were further confirmed by immunohistochemical staining of retinal flat mounts and retinal cryosections.

RESULTS
With ageing of the retina, 298 genes were upregulated and 137 genes were downregulated. Functional annotation showed that genes linked to immune responses (Ir genes) and to tissue stress/injury responses (TS/I genes) were most likely to be modified by ageing. The Ir genes affected included those regulating leukocyte activation, chemotaxis, endocytosis, complement activation, phagocytosis, and myeloid cell differentiation, most of which were upregulated, with only a few downregulated. Increased microglial and complement activation in the aging retina was further confirmed by confocal microscopy of retinal tissues. The most strongly upregulated gene was the calcitonin receptor (Calcr; >40-fold in old versus young mice).

CONCLUSIONS
The results suggest that retinal ageing is accompanied by activation of gene sets, which are involved in local inflammatory responses. A modified form of low-grade chronic inflammation (para-inflammation) characterizes these aging changes and involves mainly the innate immune system. The marked upregulation of Calcr in ageing mice most likely reflects this chronic inflammatory/stress response, since calcitonin is a known systemic biomarker of inflammation/sepsis. © Association for Research in Vision and Ophthalmology.

Reduced Nitro-oxidative Stress and Neural Cell Death Suggests a Protective Role for Microglial Cells in TNFα−/− Mice in Ischemic Retinopathy

Purpose. Neovascularization occurs in response to tissue ischemia and growth factor stimulation. In ischemic retinopathies, however, new vessels fail to restore the hypoxic tissue; instead, they infiltrate the transparent vitreous. In a model of oxygen-induced retinopathy (OIR), TNFa and iNOS, upregulated in response to tissue ischemia, are cytotoxic and inhibit vascular repair. The aim of this study was to investigate the mechanism for this effect.

Methods. Wild-type C57/BL6 (WT) and TNFa-/- mice were subjected to OIR by exposure to 75% oxygen (postnatal days 7–12). The retinas were removed during the hypoxic phase of the model. Retinal cell death was determined by TUNEL staining, and the microglial cells were quantified after Z-series capture with a confocal microscope. In situ peroxynitrite and superoxide were measured by using the fluorescent dyes DCF and DHE. iNOS, nitrotyrosine, and arginase were analyzed by real-time PCR, Western blot analysis, and activity determined by radiolabeled arginine conversion. Astrocyte coverage was examined after GFAP immunostaining.

Results. The TNFa-/- animals displayed a significant reduction in TUNEL-positive apoptotic cells in the inner nuclear layer of the avascular retina compared with that in the WT control mice. The reduction coincided with enhanced astrocytic survival and an increase in microglial cells actively engaged in phagocytosing apoptotic debris that displayed low ROS, RNS, and NO production and high arginase activity.

Conclusions. Collectively, the results suggest that improved vascular recovery in the absence of TNFa is associated with enhanced astrocyte survival and that both phenomena are dependent on preservation of microglial cells that display an anti-inflammatory phenotype during the early ischemic phase of OIR.

Involvement of MAPKs in endostatin-mediated regulation of blood-retinal barrier function

Purpose: This study aimed to evaluate the effects of endostatin on tight junction (TJ) integrity in retinal microvascular endothelial cells (RMECs) in vitro and in vivo. Moreover, it was hypothesized that endostatin-induced occludin upregulation regulated VEGF(165)-mediated increases in endothelial cell permeability and involved activation of the MAPK signaling cascade. Endostatin is a 20-kDa fragment of collagen XVIII that has been shown to be efficacious in the eye by preventing retinal neovascularization. Endostatin is a specific inhibitor of endothelial cell proliferation, migration, and angiogenesis and has been reported to reverse VEGF-mediated increases in vasopermeability and to promote integrity of the blood-retinal barrier (BRB). In order to determine the mechanism of endostatin action on BRB integrity, we have examined the effects of endostatin on a number of intracellular pathways implicated in endothelial cell physiology. Methods: C57/Bl6 mice were injected with VEGF(165) and/or endostatin, and the distribution of occludin staining was determined using retinal flatmounts. Western blot analysis of RMECs treated with VEGF(165) and/or endostatin was used to determine changes in occludin expression and p38 MAPK and extracellular regulated kinase (ERK1/ERK2 MAPK) activation, while FD-4 flux across the RMEC monolayer was used to determine changes in paracellular permeability. Results: Endostatin prevented the discontinuous pattern of occludin staining observed at the retinal blood vessels of mice administered an intraocular injection of VEGF(165). It was shown that endostatin activated p38 MAPK 5 min after addition to RMECs and continued to do so for approximately 30 min. Endostatin was also shown to activate ERK1/ERK2 5 min after addition and continued to do so, albeit with less potency, up to and including 15 min after addition. Inhibition of p38 MAPK and ERK1/ERK2 prevented endostatin's ability to upregulate levels of occludin expression. Inhibition of these key signaling molecules was shown to prevent endostatin's ability to protect against VEGF(165)- mediated increases in paracellular permeability in vitro. However, it appears that p38 MAPK may play a more important role in VEGF-mediated permeability, as inhibition of ERK1/ERK2 will not prevent VEGF(165)- mediated permeability compared with control ( untreated) cells or cells treated with both a p38 MAPK inhibitor and VEGF(165). Conclusions: Occludin is important for the maintenance of tight junction integrity in vivo. In a p38 MAPK and ERK1/ERK2 dependent manner, endostatin was shown to upregulate the levels of expression of the tight junction protein occludin. Inhibition of these key MAPK components may prevent endostatin's ability to decrease VEGF(165)-induced paracellular permeability.

Increased levels of advanced glycation endproducts in the lenses and blood vessels of cigarette smokers.

BACKGROUND: Advanced glycation endproducts (AGEs) arise from the spontaneous reaction of reducing sugars with the amino groups of macromolecules. AGEs accumulate in tissue as a consequence of diabetes and aging and have been causally implicated in the pathogenesis of several of the end-organ complications of diabetes and aging, including cataract, atherosclerosis, and renal insufficiency. It has been recently proposed that components in mainstream cigarette smoke can react with plasma and extracellular matrix proteins to form covalent adducts with many of the properties of AGEs. We wished to ascertain whether AGEs or immunochemically related molecules are present at higher levels in the tissues of smokers.

MATERIALS AND METHODS: Lens and coronary artery specimens from nondiabetic smokers and nondiabetic nonsmokers were examined by immunohistochemistry, immunoelectron microscopy, and ELISA employing several distinct anti-AGE antibodies. In addition, lenticular extracts were tested for AGE-associated fluorescence by fluorescence spectroscopy.

RESULTS: Immunoreactive AGEs were present at significantly higher levels in the lenses and lenticular extracts of nondiabetic smokers (p < 0.003). Anti-AGE immunogold staining was diffusely distributed throughout lens fiber cells. AGE-associated fluorescence was significantly increased in the lenticular extracts of nondiabetic smokers (p = 0.005). AGE-immunoreactivity was significantly elevated in coronary arteries from nondiabetic smokers compared with nondiabetic nonsmokers (p = 0.015).

CONCLUSIONS: AGEs or immunochemically related molecules are present at higher levels in the tissues of smokers than in nonsmokers, irrespective of diabetes. In view of previous reports implicating AGEs in a causal association with numerous pathologies, these findings have significant ramifications for understanding the etiopathology of diseases associated with smoking, the single greatest preventable cause of morbidity and mortality in the United States.