Diverse Requirements for Src-Family Tyrosine Kinases Distinguish Chlamydial Species

Chlamydiae are well known for their species specificity and tissue tropism, and yet the individual species and strains show remarkable genomic synteny and share an intracellular developmental cycle unique in the microbial world. Only a relatively few chlamydial genes have been linked to specific disease or tissue tropism. Here we show that chlamydial species associated with human infections, Chlamydia trachomatis and C. pneumoniae, exhibit unique requirements for Src-family kinases throughout their developmental cycle. Utilization of Src-family kinases by C. trachomatis includes tyrosine phosphorylation of the secreted effector Tarp during the entry process, a functional role in microtubule-dependent trafficking to the microtubule organizing center, and a requirement for Src-family kinases for successful initiation of development. Nonhuman chlamydial species C. caviae and C. muridarum show none of these requirements and, instead, appear to be growth restricted by the activities of Src-family kinases. Depletion of Src-family kinases triggers a more rapid development of C. caviae with up to an 800% increase in infectious progeny production. Collectively, the results suggest that human chlamydial species have evolved requirements for tyrosine phosphorylation by Src-family kinases that are not seen in other chlamydial species. The requirement for Src-family kinases thus represents a fundamental distinction between chlamydial species that would not be readily apparent in genomic comparisons and may provide insights into chlamydial disease association and species specificity

Neutrophil Extracellular Traps Go Viral

Neutrophils are the most numerous immune cells. Their importance as the first line of defense against bacterial and fungal pathogens is well described. In contrast, the role of neutrophils in controlling viral infections is less clear. Bacterial and fungal pathogens can stimulate neutrophils extracellular traps (NETs) in a process called NETosis. Although NETosis has previously been described as a special form of programmed cell death, there are forms of NET production that do not end with the demise of neutrophils. As an end result of NETosis, genomic DNA complexed with microbicidal proteins is expelled from neutrophils. These structures can kill pathogens or at least prevent their local spread within host tissue. On the other hand, disproportionate NET formation can cause local or systemic damage. Only recently, it was recognized that viruses can also induce NETosis. In this review, we discuss the mechanisms by which NETs are produced in the context of viral infection and how this may contribute to both antiviral immunity and immunopathology. Finally, we shed light on viral immune evasion mechanisms targeting NETs

Heme Mediated STAT3 Activation in Severe Malaria

The mortality of severe malaria [cerebral malaria (CM), severe malaria anemia (SMA), acute lung injury (ALI) and acute respiratory distress syndrome (ARDS)] remains high despite the availability associated with adequate treatments. Recent studies in our laboratory and others have revealed a hitherto unknown correlation between chemokine CXCL10/CXCR3, Heme/HO-1 and STAT3 and cerebral malaria severity and mortality. Although Heme/HO-1 and CXCL10/CXCR3 interactions are directly involved in the pathogenesis of CM and fatal disease, the mechanism dictating how Heme/HO-1 and CXCL10/CXCR3 are expressed and regulated under these conditions is still unknown. We therefore tested the hypothesis that these factors share common signaling pathways and may be mutually regulated.We first clarified the roles of Heme/HO-1, CXCL10/CXCR3 and STAT3 in CM pathogenesis utilizing a well established experimental cerebral malaria mouse (ECM, P. berghei ANKA) model. Then, we further determined the mechanisms how STAT3 regulates HO-1 and CXCL10 as well as mutual regulation among them in CRL-2581, a murine endothelial cell line.The results demonstrate that (1) STAT3 is activated by P. berghei ANKA (PBA) infection in vivo and Heme in vitro. (2) Heme up-regulates HO-1 and CXCL10 production through STAT3 pathway, and regulates CXCL10 at the transcriptional level in vitro. (3) HO-1 transcription is positively regulated by CXCL10. (4) HO-1 regulates STAT3 signaling.Our data indicate that Heme/HO-1, CXCL10/CXCR3 and STAT3 molecules as well as related signaling pathways play very important roles in the pathogenesis of severe malaria. We conclude that these factors are mutually regulated and provide new opportunities to develop potential novel therapeutic targets that could be used to supplement traditional prophylactics and treatments for malaria and improve clinical outcomes while reducing malaria mortality. Our ultimate goal is to develop novel therapies targeting Heme or CXCL10-related biological signaling molecules associated with development of fatal malaria

Claudins in lung diseases

Tight junctions are the most apically localized part of the epithelial junctional complex. They regulate the permeability and polarity of cell layers and create compartments in cell membranes. Claudins are structural molecules of tight junctions. There are 27 claudins known, and expression of different claudins is responsible for changes in the electrolyte and solute permeability in cells layers. Studies have shown that claudins and tight junctions also protect multicellular organisms from infections and that some infectious agents may use claudins as targets to invade and weaken the host's defense. In neoplastic diseases, claudin expression may be up- or downregulated. Since their expression is associated with specific tumor types or with specific locations of tumors to a certain degree, they can, in a restricted sense, also be used as tumor markers. However, the regulation of claudin expression is complex involving growth factors and integrins, protein kinases, proto-oncogens and transcription factors. In this review, the significance of claudins is discussed in lung disease and development

CD36 and Fyn kinase mediate malaria-induced lung endothelial barrier dysfunction in mice infected with Plasmodium berghei.

PMC3744507Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT) and CD36-/- mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σalb) compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36-/- mice had near normal fluid conductance but a similar reduction in σalb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS). To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC) from WT and CD36-/- mice were exposed to H2O2. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER) from H2O2 indicating loss of barrier function, CD36-/- MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36-/- endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36-/- lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn-/- mice, which were also protected from H2O2- and malaria-induced lung endothelial leak, respectively. Our results demonstrate that CD36 and Fyn kinase are critical mediators of the increased lung endothelial fluid conductance caused by malaria infection.JH Libraries Open Access Fun

Anti-malarial activity and toxicity assessment of Himatanthus articulatus, a plant used to treat malaria in the Brazilian Amazon

Background: Plasmodium falciparum has become resistant to some of the available drugs. Several plant species are used for the treatment of malaria, such as Himatanthus articulatus in parts of Brazil. The present paper reports the phyto-chemistry, the anti-plasmodial and anti-malarial activity, as well as the toxicity of H. articulatus. Methods: Ethanol and dichloromethane extracts were obtained from the powder of stem barks of H. articulates and later fractionated and analysed. The anti-plasmodial activity was assessed against a chloroquine resistant strain P. falciparum (W2) in vitro, whilst in vivo anti-malarial activity against Plasmodium berghei (ANKA strain) was tested in mice, evaluating the role of oxidative stress (total antioxidant capacity - TEAC; lipid peroxidation – TBARS, and nitrites and nitrates - NN). In addition, cytotoxicity was evaluated using the HepG2 A16 cell-line. The acute oral and sub-chronic toxicity of the ethanol extract were evaluated in both male and female mice. Results: Plumieride was isolated from the ethyl acetate fraction of ethanol extract, Only the dichloromethane extract was active against clone W2. Nevertheless, both extracts reduced parasitaemia in P. berghei-infected mice. Besides, a significant reduction in pulmonary and cerebral levels of NN (nitrites and nitrates) was found, as well as in pulmonary TBARS, indicating a reduced oxidative damage to these organs. The ethanol extract showed low cytotoxicity to HepG2 A16 cells in the concentrations used. No significant changes were observed in the in vivo toxicity studies. Conclusions: The ethanol extract of H. articulatus proved to be promising as anti-malarial medicine and showed low toxicity

The impact of overcrowding on the practice environment for emergency department nurses

Overcrowding in the Emergency Department (ED) occurs when there is an imbalance between resource supply and demand. In other words, the ED is challenged to provide quality care within acceptable time frames. Overcrowding literature illustrates that ED overcrowding has become so common that it is now considered normal, which begs the question: are terms such as "overcrowded" still pertinent to the current context of the ED environment in which overcrowding is a normal state? A recent and growing body of Canadian literature describes negative workplace environments in acute care facilities, but there is as yet no literature that examines overcrowding in relation to workplace environments in the ED. This interpretive descriptive study examines the relationship of overcrowding to the practice environment of emergency departments. Data were collected over six months and included in-depth interviews with ten experienced emergency department nurses. These data illustrate the negative impact of overcrowding on the emergency department environment. Emergency department overcrowding has occurred in parallel with fundamental restructuring of the health care system, creating a new era environment. This new environment is characterized by chronic overcrowding and an equally chronic shortage of resources. Within this environment nurses face several new challenges, such as understanding the discourse of overcrowding, reconciling conflicting values, and navigating the cycles of tension that arise from this new situation. Many of the changes to the ED have resulted from changes imposed elsewhere in the system, such as the closure of acute care beds. EDs are consequently in a state of transition as health care providers attempt to put the pieces back together. Within this new era environment many challenges have emerged and data analysis suggests that new era EDs support negative workplace environments. It is crucial that policy makers be well informed when making decisions that impact EDs. In order to be able to influence policy makers it is essential that nurses be proactive and involved in charting the course for the future. It is also necessary for organizations to invest in leadership by creating appropriate positions at appropriate levels so that leaders can be visible and available to create vision and help nurses through the transition to the new era environment. This study argues for the need to create an increased body of research that will inform policy makers about how ED overcrowding negatively affects the practice environment.Applied Science, Faculty ofNursing, School ofGraduat

Regulation of endothelial cell function by p. falciparum

Bibliography: p. 201-244Some pages are in colour.Includes copy of ethics approval and copyright permissions. Original copies with original Partial Copyright Licence.Plasmodiumfalciparum is a protozoan infection of human erythrocytes. Despite optimal therapy, mortality due to multi-organ failure in severe falciparum malaria remains high at 5 to 20%. Correlation of disease severity to multiple clinical markers of endothelial activation suggest a central role for endothelial cells in the pathophysiology of severe malaria. Detailed studies in models of other systemic infections, including bacterial sepsis, have highlighted the central role of endothelial cells in pathogen recognition, barrier function and proinflammatory signaling in determining organ failure and mortality. We hypothesized that in addition to providing points of attachment for sequestering infected red blood cells (IRBC), microvascular endothelial cells can directly recognize parasite products released by adherent IRBC at the time of schizogony through innate receptors. Using clinical P. falciparum isolates we showed that parasite sonicates but not intact IRBC disrupted primary human dermal and lung endothelial cell barrier function in a Src-family kinase-dependent manner. Increased endothelial permeability was characterized by redistribution of junctiona1 proteins Z0-1 and VE-cadherin away from sites of cell-cell contact. The active parasite component appeared to be a merozoite­associated protein. We further examined the ability of P. falciparum sonicates and merozoites to induce proinflammatory signaling and discovered production of broad proinflammatory responses including endothelial chemokines and adhesion molecules. We defined a critical role for Src-family kinase Lyn and downstream p38 MAPK in endothelial proinflammatory responses to P. falciparum sonicate using IL-8 protein production as a functional readout. Further characterization of the active component in P. falciparum sonicates and merozoites revealed parasite histones as an activator of both endothelial permeability and proinflamrnatory protein production. Both activities of histones were found to be dependent on the strong cationic charge of these proteins. Proinflammatory responses to P. falciparum histones were were partially dependent on Toll-like receptor 2 (TLR2). Recombinant human activated protein C (rhAPC) cleaved parasite histones and abrogated the increases in endothelial permeability and IL-8 production. More importantly, levels of both parasite and human histones were markedly elevated in patients with severe malaria as compared to healthy controls, and patients with uncomplicated infection and bacterial sepsis. Together, these findings strongly suggest Src-family kinases and parasite histones as targets for adjunctive therapies in severe falciparum malaria