Update on macrophage clearance of inhaled micro- and nanoparticles

Lung macrophages, that is, the intravascular, interstitial, pleural, and surface macrophages, are part of the mononuclear phagocyte system. They are derived from the hematopoietic stem cell in the bone marrow with the monocytes as their putative precursors. Macrophages residing on the inner surfaces of the lungs and immersed within the lung lining layer, that is, the alveolar and the airway macrophages, are constantly exposed to the environment; it is those cells that are recognized as first line of cellular host defense.

Cellular responses after exposure of lung cell cultures to secondary organic aerosol particles

The scope of this work was to examine in vitro responses of lung cells to secondary organic aerosol (SOA) particles, under realistic ambient air and physiological conditions occurring when particles are inhaled by mammals, using a novel particle deposition chamber. The cell cultures included cell types that are representative for the inner surface of airways and alveoli and are the target cells for inhaled particles. The results demonstrate that an exposure to SOA at ambient-air concentrations of about 10(4) particles/cm(3) for 2 h leads to only moderate cellular responses. There is evidence for (i) cell type specific effects and for (ii) different effects of SOA originating from anthropogenic and biogenic precursors, i.e. 1,3,5-trimethylbenzene (TMB) and alpha-pinene, respectively. There was no indication for cytotoxic effects but for subtle changes in cellular functions that are essential for lung homeostasis. Decreased phagocytic activity was found in human macrophages exposed to SOA from alpha-pinene. Alveolar epithelial wound repair was affected by TMB-SOA exposure, mainly because of altered cell spreading and migration at the edge of the wound. In addition, cellular responses were found to correlate with particle number concentration, as interleukin-8 production was increased in pig explants exposed to TMB-SOA with high particle numbers.

Recent advances into understanding some aspects of the structure and function of mammalian and avian lungs

Recent findings are reported about certain aspects of the structure and function of the mammalian and avian lungs that include (a) the architecture of the air capillaries (ACs) and the blood capillaries (BCs); (b) the pulmonary blood capillary circulatory dynamics; (c) the adaptive molecular, cellular, biochemical, compositional, and developmental characteristics of the surfactant system; (d) the mechanisms of the translocation of fine and ultrafine particles across the airway epithelial barrier; and (e) the particle-cell interactions in the pulmonary airways. In the lung of the Muscovy duck Cairina moschata, at least, the ACs are rotund structures that are interconnected by narrow cylindrical sections, while the BCs comprise segments that are almost as long as they are wide. In contrast to the mammalian pulmonary BCs, which are highly compliant, those of birds practically behave like rigid tubes. Diving pressure has been a very powerful directional selection force that has influenced phenotypic changes in surfactant composition and function in lungs of marine mammals. After nanosized particulates are deposited on the respiratory tract of healthy human subjects, some reach organs such as the brain with potentially serious health implications. Finally, in the mammalian lung, dendritic cells of the pulmonary airways are powerful agents in engulfing deposited particles, and in birds, macrophages and erythrocytes are ardent phagocytizing cellular agents. The morphology of the lung that allows it to perform different functions-including gas exchange, ventilation of the lung by being compliant, defense, and secretion of important pharmacological factors-is reflected in its "compromise design."

Quantitative evaluation of cellular uptake and trafficking of plain and polyethylene glycol-coated gold nanoparticles

This study addresses the cellular uptake and intracellular trafficking of 15-nm gold nanoparticles (NPs), either plain (i.e., stabilized with citrate) or coated with polyethylene glycol (PEG), exposed to human alveolar epithelial cells (A549) at the air-liquid interface for 1, 4, and 24 h. Quantitative analysis by stereology on transmission electron microscopy images reveals a significant, nonrandom intracellular distribution for both NP types. No particles are observed in the nucleus, mitochondria, endoplasmic reticulum, or golgi. The cytosol is not a preferred cellular compartment for both NP types, although significantly more PEG-coated than citrate-stabilized NPs are present there. The preferred particle localizations are vesicles of different sizes (<150, 150-1000, >1000 nm). This is observed for both NP types and indicates a predominant uptake by endocytosis. Subsequent inhibition of caveolin- and clathrin-mediated endocytosis by methyl-beta-cyclodextrin (MbetaCD) results in a significant reduction of intracellular NPs. The inhibition, however, is more pronounced for PEG-coated than citrate-stabilized NPs. The latter are mostly found in larger vesicles; therefore, they are potentially taken up by macropinocytosis, which is not inhibited by MbetaCD. With prolonged exposure times, both NPs are preferentially localized in larger-sized intracellular vesicles such as lysosomes, thus indicating intracellular particle trafficking. This quantitative evaluation reveals that NP surface coatings modulate endocytotic uptake pathways and cellular NP trafficking. Other nonendocytotic entry mechanisms are found to be involved as well, as indicated by localization of a minority of PEG-coated NPs in the cytosol.

Cellular immune responses to HCV core increase and HCV RNA levels decrease during successful antiretroviral therapy

Background Hepatitis C virus (HCV) infection is a major cause of morbidity in HIV infected individuals. Coinfection with HIV is associated with diminished HCV-specific immune responses and higher HCV RNA levels. Aims To investigate whether long-term combination antiretroviral therapy (cART) restores HCV-specific T cell responses and improves the control of HCV replication. Methods T cell responses were evaluated longitudinally in 80 HIV/HCV coinfected individuals by ex vivo interferon-γ-ELISpot responses to HCV core peptides, that predominantly stimulate CD4+ T cells. HCV RNA levels were assessed by real-time PCR in 114 individuals. Results The proportion of individuals with detectable T cell responses to HCV core peptides was 19% before starting cART, 24% in the first year on cART and increased significantly to 45% and 49% after 33 and 70 months on cART (p=0.001). HCV-specific immune responses increased in individuals with chronic (+31%) and spontaneously cleared HCV infection (+30%). Median HCV RNA levels before starting cART were 6.5 log10 IU/ml. During long-term cART, median HCV-RNA levels slightly decreased compared to pre-cART levels (−0.3 log10 IU/ml, p=0.02). Conclusions Successful cART is associated with increasing cellular immune responses to HCV core peptides and with a slight long-term decrease in HCV RNA levels. These findings are in line with the favourable clinical effects of cART on the natural history of hepatitis C and with the current recommendation to start cART earlier in HCV/HIV coinfected individuals.

Posttraumatic stress disorder and soluble cellular adhesion molecules at rest and in response to a trauma-specific interview in patients after myocardial infarction

Posttraumatic stress disorder (PTSD) and circulating cellular adhesion molecules (CAMs) predict cardiovascular risk. We hypothesized a positive relationship between PTSD caused by myocardial infarction (MI) and soluble CAMs. We enrolled 22 post-MI patients with interviewer-rated PTSD and 22 post-MI patients with no PTSD. At 32±6months after index MI, all patients were re-scheduled to undergo the Clinician-Administered PTSD Scale (CAPS) interview and had blood collected to assess soluble CAMs at rest and after the CAPS interview. Relative to patients with no PTSD, those with PTSD had significantly higher levels of soluble vascular cellular adhesion molecule (sVCAM)-1 and intercellular adhesion molecule (sICAM)-1 at rest and, controlling for resting CAM levels, significantly higher sVCAM-1 and sICAM-1 after the interview. Greater severity of PTSD predicted significantly higher resting levels of sVCAM-1 and soluble P-selectin in patients with PTSD. At follow-up, patients with persistent PTSD (n=15) and those who had remitted (n=7) did not significantly differ in CAM levels at rest and after the interview; however, both these groups had significantly higher sVCAM-1 and sICAM-1 at rest and also after the interview compared to patients with no PTSD. Elevated levels of circulating CAMs might help explain the psychophysiologic link of PTSD with cardiovascular risk.

Structure and function of CinD (YtjD) of Lactococcus lactis, a copper-induced nitroreductase involved in defense against oxidative stress

In Lactococcus lactis IL1403, 14 genes are under the control of the copper-inducible CopR repressor. This so-called CopR regulon encompasses the CopR regulator, two putative CPx-type copper ATPases, a copper chaperone, and 10 additional genes of unknown function. We addressed here the function of one of these genes, ytjD, which we renamed cinD (copper-induced nitroreductase). Copper, cadmium, and silver induced cinD in vivo, as shown by real-time quantitative PCR. A knockout mutant of cinD was more sensitive to oxidative stress exerted by 4-nitroquinoline-N-oxide and copper. Purified CinD is a flavoprotein and reduced 2,6-dichlorophenolindophenol and 4-nitroquinoline-N-oxide with k(cat) values of 27 and 11 s(-1), respectively, using NADH as a reductant. CinD also exhibited significant catalase activity in vitro. The X-ray structure of CinD was resolved at 1.35 A and resembles those of other nitroreductases. CinD is thus a nitroreductase which can protect L. lactis against oxidative stress that could be exerted by nitroaromatic compounds and copper.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

Cleavage of IgG1 and IgG3 by gingipain K from Porphyromonas gingivalis may compromise host defense in progressive periodontitis

Degradation of immunoglobulins is an effective strategy of bacteria to evade the immune system. We have tested whether human IgG is a substrate for gingipain K of Porphyromonas gingivalis and found that the enzyme can hydrolyze subclass 1 and 3 of human IgG. The heavy chain of IgG(1) was cleaved at a single site within the hinge region, generating Fab and Fc fragments. IgG(3) was also cleaved within the heavy chain, but at several sites around the CH2 region. Investigation of the enzyme kinetics of IgG proteolysis by gingipain K, using FPLC- and isothermal titration calorimetry-based assays followed by Hill plots, revealed non-Michaelis-Menten kinetics involving a mechanism of positive cooperativity. In ex vivo studies, it was shown that gingipain K retained its IgG hydrolyzing activity in human plasma despite the high content of natural protease inhibitors; that IgG(1) cleavage products were detected in gingival crevicular fluid samples from patients with severe periodontitis; and that gingipain K treatment of serum samples from patients with high antibody titers against P. gingivalis significantly hindered opsonin-dependent phagocytosis of clinical isolates of P. gingivalis by neutrophils. Altogether, these findings underline a biological function of gingipain K as an IgG protease of pathophysiological importance.

Coupling biomechanics to a cellular level model: An approach to patient-specific image driven multi-scale and multi-physics tumor simulation

Modeling of tumor growth has been performed according to various approaches addressing different biocomplexity levels and spatiotemporal scales. Mathematical treatments range from partial differential equation based diffusion models to rule-based cellular level simulators, aiming at both improving our quantitative understanding of the underlying biological processes and, in the mid- and long term, constructing reliable multi-scale predictive platforms to support patient-individualized treatment planning and optimization. The aim of this paper is to establish a multi-scale and multi-physics approach to tumor modeling taking into account both the cellular and the macroscopic mechanical level. Therefore, an already developed biomodel of clinical tumor growth and response to treatment is self-consistently coupled with a biomechanical model. Results are presented for the free growth case of the imageable component of an initially point-like glioblastoma multiforme tumor. The composite model leads to significant tumor shape corrections that are achieved through the utilization of environmental pressure information and the application of biomechanical principles. Using the ratio of smallest to largest moment of inertia of the tumor material to quantify the effect of our coupled approach, we have found a tumor shape correction of 20\% by coupling biomechanics to the cellular simulator as compared to a cellular simulation without preferred growth directions. We conclude that the integration of the two models provides additional morphological insight into realistic tumor growth behavior. Therefore, it might be used for the development of an advanced oncosimulator focusing on tumor types for which morphology plays an important role in surgical and/or radio-therapeutic treatment planning.

Plasma membrane repair and cellular damage control: the annexin survival kit

Plasmalemmal injury is a frequent event in the life of a cell. Physical disruption of the plasma membrane is common in cells that operate under conditions of mechanical stress. The permeability barrier can also be breached by chemical means: pathogens gain access to host cells by secreting pore-forming toxins and phospholipases, and the host's own immune system employs pore-forming proteins to eliminate both pathogens and the pathogen-invaded cells. In all cases, the influx of extracellular Ca(2+) is being sensed and interpreted as an "immediate danger" signal. Various Ca(2+)-dependent mechanisms are employed to enable plasma membrane repair. Extensively damaged regions of the plasma membrane can be patched with internal membranes delivered to the cell surface by exocytosis. Nucleated cells are capable of resealing their injured plasmalemma by endocytosis of the permeabilized site. Likewise, the shedding of membrane microparticles is thought to be involved in the physical elimination of pores. Membrane blebbing is a further damage-control mechanism, which is triggered after initial attempts at plasmalemmal resealing have failed. The members of the annexin protein family are ubiquitously expressed and function as intracellular Ca(2+) sensors. Most cells contain multiple annexins, which interact with distinct plasma membrane regions promoting membrane segregation, membrane fusion and--in combination with their individual Ca(2+)-sensitivity--allow spatially confined, graded responses to membrane injury.

The Catholic Doctrine of Transubstantiation: An Exposition and Defense

The purpose of this thesis is to offer both an exposition and defense for the Catholic Church's traditional understanding of eucharistic transubstantiation. I hope to show how a belief in such a doctrine is in no way irrational nor is it indefensible; butinstead, the doctrine of transubstantiation makes sense when it is viewed in light of what Catholic Christians believe about who the human being is, what the human desires, and the special way in which God personally works in human history. The method I am following investigates how the doctrine of transubstantiation coheres with and follows the other beliefs that Catholics hold; that is, beginning with certain presuppositions, there is a certain rational progression to the Catholic understanding of real presence.

Synthesis, binding and cellular uptake properties of oligodeoxynucleotides containing cationic bicyclo-thymidine residues

The synthesis and incorporation into oligodeoxynucleotides of two novel derivatives of bicyclothymidine carrying a cationic diaminopropyl or lysine unit in the C(6′)-β position is described. Compared to unmodified DNA these oligonucleotides show Tm-neutral behavior when paired against complementary DNA and are destabilizing when paired against RNA. Unaided uptake experiments of a decamer containing five lys-bcT units into HeLa and HEK293T cells showed substantial internalization with mostly cytosolic distribution which was not observed in the case of an unmodified control oligonucleotide.