Aortic aneurysm is the most life-threatening complication in Marfan syndrome (MFS) patients. Doxycycline, a nonselective matrix metalloproteinases inhibitor, was reported to improve the contractile function and elastic fiber structure and organization in a Marfan mouse aorta using ex vivo small chamber myography. In this study, we assessed the hypothesis that a long-term treatment with doxycycline would reduce aortic root growth, improve aortic wall elasticity as measured by pulse wave velocity, and improve the ultrastructure of elastic fiber in the mouse model of MFS. In our study, longitudinal measurements of aortic root diameters using high-resolution ultrasound imaging display significantly decreased aortic root diameters and lower pulse wave velocity in doxycycline-treated Marfan mice starting at 6 months as compared to their non-treated MFS counterparts. In addition, at the ultrastructural level, our data show that long-term doxycycline treatment corrects the irregularities of elastic fibers within the aortic wall of Marfan mice to the levels similar to those observed in control subjects. Our findings underscore the key role of matrix metalloproteinases during the progression of aortic aneurysm, and provide new insights into the potential therapeutic value of doxycycline in blocking MFS-associated aortic aneurysm.

Objectives The approximately 250 years old remains of the Kwädąy Dän Ts'ìnchį man were found in a glacier in Canada. Studying the state of preservation of the corpse, we observed black deposits in his lung. Following this observation we wanted to determine: (1) location of the deposits in the lung tissue, (2) composition and origins of the deposits. Methods By light microscopy (LM) and transmission electron microscopy (TEM), we studied the deposits in the Kwädąy Dän Ts'ìnchį man’ s lung and compared it with distribution of anthracotic deposits in contemporary samples from the David Harwick Pathology Centre (DHPC). To determine chemical composition of the inclusions we used Raman spectroscopy. Scanning electron microscopy and elemental mapping was used for determine the chemical elements. Results The histopathological identification of anthracosis in the Kwädąy Dän Ts'ìnchį man's lung allowed us to distinguish crushed parenchyma from conducting airway tissue and identification of particles using LM and TEM. Crystal particles were found using TEM. Ordered carbonaceous material (graphene and graphite), disordered carbonaceous material (soot) and what might be minerals (likely conglomerates) were found with Raman spectrometry. Gold and lead particles in the lung were discovered with scanning electron microscopy and elemental mapping. Conclusions Presence of soot particles in anthracotic areas in the Kwädąy Dän Ts'ìnchį man's lung probably were due to an inhalation of particles in open fires. Gold and lead particles are most likely of an environmental origin and may have been inhaled and could have impacted his health and his Champagne and Aishihik First Nations (CAFN) contemporaries.

Survival during lung injury requires a coordinated program of damage limitation and rapid repair. CD34 is a cell surface sialomucin expressed by epithelial, vascular, and stromal cells that promotes cell adhesion, coordinates inflammatory cell recruitment, and drives angiogenesis. To test whether CD34 also orchestrates pulmonary damage and repair, we induced acute lung injury in wild-type (WT) and Cd34-/- mice by bleomycin administration. We found that Cd34-/- mice displayed severe weight loss and early mortality compared with WT controls. Despite equivalent early airway inflammation to WT mice, CD34-deficient animals developed interstitial edema and endothelial delamination, suggesting impaired endothelial function. Chimeric Cd34-/- mice reconstituted with WT hematopoietic cells exhibited early mortality compared with WT mice reconstituted with Cd34-/- cells, supporting an endothelial defect. CD34-deficient mice were also more sensitive to lung damage caused by influenza infection, showing greater weight loss and more extensive pulmonary remodeling. Together, our data suggest that CD34 plays an essential role in maintaining vascular integrity in the lung in response to chemical- and infection-induced tissue damage.

A characteristic feature of asthma is exaggerated airway narrowing, termed airway hyper-responsiveness (AHR) due to contraction of airway smooth muscle (ASM). Although smooth muscle (SM)-specific asthma susceptibility genes have been identified, it is not known whether asthmatic ASM is phenotypically different from nonasthmatic ASM in terms of subcellular structure or mechanical function. The present study is the first to systematically quantify, using electron microscopy, the ultrastructure of tracheal SM from subjects with asthma and nonasthmatic subjects. Methodological details concerning tissue sample preparation, ultrastructural quantification, and normalization of isometric force by appropriate morphometric parameters are described. We reasoned that genetic and/or acquired differences in the ultrastructure of asthmatic ASM could be associated with functional changes. We recently reported that asthmatic ASM is better able to maintain and recover active force generation after length oscillations simulating deep inspirations. The present study was designed to seek structural evidence to account for this observation. Contrary to our hypotheses, no significant qualitative or quantitative differences were found in the subcellular structure of asthmatic versus nonasthmatic tracheal SM. Specifically, there were no differences in average SM cell cross-sectional area; fraction of the cell area occupied by nonfilamentous area; amounts of mitochondria, dense bodies, and dense plaques; myosin and actin filament densities; basal lamina thickness; and the number of microtubules. These results indicate that functional differences in ASM do not necessarily translate into observable structural changes.

Electron microscopy is a powerful technique that has been used to answer numerous structure related research questions in all fields, including atherosclerotic research. Recent technology developments are expanding the capabilities of electron microscopy to address the physiology and pathology of arterial function. The purpose of this review is to describe what was known about the ultrastructure of atherosclerosis in the mid 1990s, what has been added to this knowledge basis since then, and to detail some of the recent electron microscopy techniques that could allow us to shed light on hitherto unaddressed aspects of this disease.

In April 2010, a NIH workshop was convened to discuss the current state of understanding of lung cell plasticity, including the responses of epithelial cells to injury, with the objectives of summarizing what is known, what the field needs to know, and how to get there. The proximal stimulus for this workshop is the body of recent evidence suggesting that plasticity is a prominent but incompletely characterized property of lung epithelial cells, and that a focus on understanding this aspect of epithelial cell biology in particular, may be an important window into disease pathobiology and pathogenesis. In addition to their many vital functions in maintaining tissue homeostasis, epithelial cells have emerged as both a central target of disease initiation and an active contributor to disease progression, making a workshop to investigate the role of cell plasticity in lung injury and repair timely. The workshop was organized around four major themes: lung epithelial cell plasticity, signaling control of plasticity, fibroblast plasticity and crosstalk, and translation to human disease. Although this breakdown was recognized to be somewhat artificial, it was felt that this approach would promote cross-fertilization among groups that ordinarily do not communicate and lend itself to the generation of new approaches. The summary reports of individual group discussions below are followed by consensus priorities and recommendations of the workshop participants.

BACKGROUND: Epidemiological studies have established that cardiovascular events account for the greatest number of air pollution-related deaths. However, the underlying structural changes are still unknown.OBJECTIVE: To investigate changes in the ultrastructure of atherosclerotic plaques in Watanabe heritable hyperlipidemic (WHHL) rabbits following the instillation of ambient particulate matter air pollution (particles smaller than 10 mu m in diameter) into the lungs.METHODS: WHHL rabbits (n=8) exposed to 5 mg of ambient particles (Environmental Health Centre - 1993 [EHC-93]; suspended in saline and instilled in the airway) twice per week for four weeks were compared with control WHHL rabbits (n=8) treated with saline alone.RESULTS: All abdominal aortic plaques were examined using light and electron microscopy, which showed the following: increased accumulation of macrophage-derived foam cells immediately below the endothelial plaque surface (P=0.04); increased contact between these foam cells and the dense subendothelial extracellular matrix (P<0.005) with reduction (P<0.0001) and fragmentation (P<0.0001) of this matrix; and emigration of macrophage-derived foam cells from the plaques in exposed rabbits. In addition, immunohistochemistry verified the presence of type IV collagen in the thickened extracellular matrix material subtending the endothelium.CONCLUSIONS: The ultrastructure of atherosclerotic plaques in EHC-93-instilled rabbits differed from the ultrastructure observed in rabbits that did not receive EHC-93. These ultrastructural differences are consistent with greater endothelial instability in the plaques of atherosclerosis-prone rabbits.

BACKGROUND:Although the accepted paradigm is that the proteins stored in eosinophil crystalloid granules are translated from messenger RNA transcribed in the cell nucleus, recent ultrastructural evidence suggests that protein synthesis may also take place within eosinophilic granules.METHODS:We used 2 different methods to detect the presence of DNA and RNA in eosinophil secretory granules. Using bromodeoxyuridine, a thymidine analogue, and bromouridine, a uracil analogue, we labeled the DNA and RNA in eosinophils in vivo in rabbits. Immunoelectron microscopy to localize these molecules was performed on ultrathin sections of blood and bone marrow eosinophils using monoclonal anti-bromodeoxyuridine antibody with IgG as a control. The immunogold grain density was measured in each subcellular compartment within the eosinophils and analyzed using image analysis software. A combination of DNA/CD63 immunofluorescence staining and a fluorescently labeled molecular probe that stains RNA was used to examine the presence of DNA and RNA in the secretory granules of human blood eosinophils.RESULTS:The mean density of bromodeoxyuridine-labeled DNA and bromouridine-labeled RNA immunogold grains in the secretory granules of blood and bone marrow eosinophils were significantly higher (p < 0.0005) than cytoplasmic or background staining. We also demonstrated the existence of DNA and RNA in the CD63-positive secretory granules of human peripheral blood eosinophils by means of immunofluorescent staining and a fluorescently labeled molecular probe.CONCLUSIONS:These results provide evidence that eosinophil granules are the site of DNA and RNA synthesis and suggest the potential for a new role(s) for eosinophil-secretory granules.

Progression of COPD is associated with a measurable increase in small airway wall thickness resulting from a repair and remodeling process that involves fibroblasts of the epithelial mesenchymal trophic unit (EMTU). The present study was designed to examine the organization of fibroblasts within the lamina propria of small airways with respect to their contacts with the epithelium and with each other in persons with COPD. Transmission electron microcopy (TEM) and three-dimensional (3D) reconstructions of serial TEM sections were used to estimate the frequency and determine the nature of the contacts between the epithelium and fibroblasts within the EMTU in small airways from 5 controls (smokers with normal lung function), from 6 persons with mild (GOLD-1) and 5 with moderate (GOLD-2) COPD. In airways from control lungs fibroblasts make frequent contact with cytoplasmic extensions of epithelial cells through apertures in the epithelial basal lamina, but the frequency of these fibroblast-epithelial contacts is reduced in both mild and moderate COPD compared to controls (p < 0.01). The 3D reconstructions showed that the cytoplasmic extensions of lamina propria fibroblasts form a reticulum with fibroblast-fibroblast contacts in an airway from a control subject but this reticulum may be reorganized in airways of COPD patients. Development of COPD is associated with significant disruption of the EMTU due to a reduction of contacts between fibroblasts and the epithelium.

Ancient remains preserved in glaciers present a unique opportunity for us to advance our knowledge of human origins, diversity, and health, a central focus of anthropological studies. Cellular components of hard and soft tissue from frozen human remains dated between 1670 to 1850 cal AD recovered from a glacier in Canada were studied. Despite the expected ice crystal damage in some samples, regions of recognizable structure and ultrastructure were observed. We found that the state of preservation was tissue specific and that in some tissues the organelles were better preserved than in others. Skeletal, connective, nervous, and epithelial tissues were recognizable in some of the samples. DNA had been previously extracted from these remains and this study illustrates that the ability to successfully extract DNA may correlate with good preservation of histology.

Introduction: Granzyme B (GrB) is a cytotoxic serine protease that acts in chronic inflammation and disease through immune cell-mediated apoptosis and putatively through the degradation of extracel...

Liver mitochondrial toxicity is a concern, particularly in HIV/hepatitis C virus (HCV) coinfection. Liver biopsies from HIV/HCV co-infected patients, 14 ON-highly active antiretroviral therapy (HAART) and nine OFF-HAART, were assessed by electron microscopy quantitative morphometric analyses. Hepatocytes tended to be larger ON-HAART than OFF-HAART (P = 0.05), but mitochondrial volume, cristae density, lipid volume, mitochondrial DNA and RNA levels were similar. We found no evidence of increased mitochondrial toxicity in individuals currently on HAART, suggesting that concomitant HAART should not delay HCV therapy.

The rates of reaction of muonium atoms with solutes, ionic and organic, were studied in solvents of wildly differing polarities (water, methanol, and hexane) and their rate constants were compared, where possible. In these reactions — which are those of a highly reactive atom, an isotope of hydrogen — it transpires that the reaction rates are higher in solvents in which the solute is more soluble and muonium diffuses faster. This study leads to various kinetic-solvent-effect ratios and to the observation of the reaction of muonium with free radicals being among the fastest reactions recorded so far between two neutral species in solution.Key words: muonium atoms, kinetic isotope effects, solvent-dependent rates, non-aqueous solvents, muon spin rotation technique.

Neutrophil emigration in the lung differs substantially from that in systemic vascular beds where extravasation occurs primarily through postcapillary venules. Migration into the alveolus occurs directly from alveolar capillaries and appears to progress through a sequence of steps uniquely influenced by the cellular anatomy and organization of the alveolar wall. The cascade of adhesive and stimulatory events so critical to the extravasation of neutrophils from postcapillary venules in many tissues is not evident in this setting. Compelling evidence exists for unique cascades of biophysical, adhesive, stimulatory, and guidance factors that arrest neutrophils in the alveolar capillary bed and direct their movement through the endothelium, interstitial space, and alveolar epithelium. A prominent path accessible to the neutrophil appears to be determined by the structural interactions of endothelial cells, interstitial fibroblasts, as well as type I and type II alveolar epithelial cells.

Atheromatous diseases are lipid and cell-rich vascular disorders that include coronary artery disease (CAD), transplant vascular disease (TVD), and restenosis. Considering the inflammatory nature of these diseases, cytotoxic immune mechanisms such as the FasL and granzyme/perforin pathways most likely play important roles in the development and remodeling of many lesions. Furthermore, although the contributions of immune responses to each disease vary, the correspondent localization of certain mediators and effectors suggests that they may contribute to a spectrum of atheromatous diseases. In this review, the contribution of immune cell-mediated cell death in the onset and pathogenesis of CAD and TVD is examined.

The mechanism of formation of muonium atoms from positive muons was studied here through measurements of the yield of diamagnetic muon states in dipolar aprotic solvents and for scavenger solutions in hexane and methanol. The results are compared with published data on common solvents covering a full range of the physicochemical properties of liquids that affect an ionic formation mechanism, namely their static dielectric constants, electron mobilities, and radiolysis yields of electrons. It is concluded that muonium is not formed by a thermal charge-neutralization reaction in these chemically-active media, though that mechanism does contribute to muonium formation in inert media like liquefied noble gases. It is clear that muonium materializes on a much shorter timescale than the recently proposed "delayed" mechanism (microseconds) and the earlier "spur" model (nanoseconds). In contrast, the data referring to all these liquids are consistent with the intra-track "hot" model. This is the only Mu-formation model proposed so far in which the immediate precursors of Mu (Mu(hot)) are neither scavengable nor ionic.Key words: muonium atoms, formation mechanism, hot model, spur model, delayed-muonium-formation model, diamagnetic yields.

Studies of the yields of muonium atoms and diamagnetic-species in hexane in the presence of added scavengers have shown, unequivocally, that muonium is not formed by the “delayed” (microsecond) mechanism proposed recently. Instead, the lifetimes evaluated show that if the combination reaction of μ++e−→Mu contributes to muonium formation in “chemically active” liquids, then it is complete within <10−10 s. In fact the results are consistent only with the “hot” model, in which the initial distribution of muons between muonium atoms and diamagnetic molecules is determined at the epithermal level, as the last step of the muon’s charge-exchangecycles and thermalization.