The enigma of IgE+ B-cell memory in human subjects

Our understanding of the origin and fate of the IgE-switched B cell has been markedly improved by studies in mouse models. The immediate precursor of the IgE-switched B cell is either a relatively naive nonswitched B cell or a mature IgG-switched B cell. These 2 routes are referred to as the direct and indirect pathways, respectively. IgE responses derived from each pathway differ significantly, largely reflecting the difference in time spent in a germinal center and thus time for clonal expansion, somatic hypermutation, affinity maturation, and acquisition of a memory phenotype. The clinical and therapeutic implications for IgE responses in human subjects are still a matter of debate, largely because the immunization procedures used in the animal models are significantly different from classical atopic sensitization to allergens from pollen and mites. On the basis of the limited information available, it seems likely that these atopic IgE responses are characterized by a relatively low IgG/IgE ratio, low B-cell memory, and modest affinity maturation, which fits well with the direct switching pathway. It is still unresolved how the IgE response evolves to cover a wide epitope repertoire involving many epitopes per allergen, as well as many different allergens from a single allergen source. © 2013 American Academy of Allergy, Asthma & Immunology.

Neoadjuvant crizotinib in advanced inflammatory myofibroblastic tumour with ALK gene rearrangement

Background Inflammatory myofibroblastic tumours (IMTs) are rare sarcomas that were first described in the lung. They are composed of myofibroblastic mesenchymal spindle cells accompanied by an inflammatory infiltrate of plasma cells. Complete resection is the treatment of choice. There is currently no standard treatment for inoperable or recurrent disease. Expression of ALK protein triggered by ALK gene rearrangement at chromosome 2p23 has been found in 36%-60% of IMTs. Case report We report a rapid early response to crizotinib as neoadjuvant therapy, enabling surgical excision of a large ALK-translocated IMT, which resulted in complete disease clearance. To the best of our knowledge, this is the first case in the literature of a patient with IMT in whom crizotinib was used successfully in the neoadjuvant or curative setting.

The effect of microscopic texture on the direct plasma surface passivation of Si solar cells

Textured silicon surfaces are widely used in manufacturing of solar cells due to increasing the light absorption probability and also the antireflection properties. However, these Si surfaces have a high density of surface defects that need to be passivated. In this study, the effect of the microscopic surface texture on the plasma surface passivation of solar cells is investigated. The movement of 105 H+ ions in the texture-modified plasma sheath is studied by Monte Carlo numerical simulation. The hydrogen ions are driven by the combined electric field of the plasma sheath and the textured surface. The ion dynamics is simulated, and the relative ion distribution over the textured substrate is presented. This distribution can be used to interpret the quality of the Si dangling bonds saturation and consequently, the direct plasma surface passivation.

Plasma-induced death of HepG2 cancer cells : intracellular effects of reactive species

Reports show that cold atmospheric-pressure plasmas can induce death of cancer cells in several minutes. However, very little is presently known about the mechanism of the plasma-induced death of cancer cells. In this paper, an atmospheric-pressure plasma plume is used to treat HepG2 cells. The experimental results show that the plasma can effectively control the intracellular concentrations of ROS, NO and lipid peroxide. It is shown that these concentrations are directly related to the mechanism of the HepG2 death, which involves several stages. First, the plasma generates NO species, which increases the NO concentration in the extracellular medium. Second, the intracellular NO concentration is increased due to the NO diffusion from the medium. Third, an increase in the intracellular NO concentration leads to the increase of the intracellular ROS concentration. Fourth, the increased oxidative stress results in more effective lipid peroxidation and consequently, cell injury. The combined action of NO, ROS and lipid peroxide species eventually results in the HepG2 cell death. The mechanism of death of human hepatocellular carcinoma cells (HepG2) induced by atmospheric-pressure room-temperature plasma, related to the plasma-controlled intracellular concentrations of reactive oxygen species (ROS), nitric oxide (NO) and lipid peroxide is revealed. Only 34.75 s are required to reduce the number of the viable HepG2 cells by 50%.

Room-temperature, atmospheric plasma needle reduces adenovirus gene expression in HEK 293A host cells

Room-temperature, atmospheric-pressure plasma needle treatment is used to effectively minimize the adenovirus (AdV) infectivity as quantified by the dramatic reduction of its gene expression in HEK 293A primary human embryonic kidney cells studied by green fluorescent protein imaging. The AdV titer is reduced by two orders of magnitude within only 8 min of the plasma exposure. This effect is due to longer lifetimes and higher interaction efficacy of the plasma-generated reactive species in confined space exposed to the plasma rather than thermal effects commonly utilized in pathogen inactivation. This generic approach is promising for the next-generation anti-viral treatments and imunotherapies.

Highly efficient silicon nanoarray solar cells by a single-step plasma-based process

Highly efficient solar cells (conversion efficiency 11.9%, fill factor 70%) based on the vertically aligned single-crystalline nanostructures are fabricated without any pre-fabricated p-n junctions in a very simple, single-step process of Si nanoarray formation by etching p-type Si(100) wafers in low-temperature environment-friendly plasmas of argon and hydrogen mixtures.

Dielectric barrier discharge plasma in Ar/O2 promoting apoptosis behavior in A549 cancer cells

The Ar/O2plasma needle in the induction of A549 cancer cells apoptosis process is studied by means of real-time observation. The entire process of programmed cell death is observed. The typical morphological changes of A549 apoptosis are detected by 4′, 6-diamidino-2-phenylindole staining, for example, chromatin condensation and nuclear fragmentation. Cell viability is determined and quantified by neutral red uptake assay, and the survival rate of A549 from Ar/O2plasmas is presented. Further spectral analysis indicates the reactive species, including O and OH play crucial roles in the cell inactivation.

Plasma ATP concentration and venous oxygen content in the forearm during dynamic handgrip exercise

Background: It has been proposed that adenosine triphosphate (ATP) released from red blood cells (RBCs) may contribute to the tight coupling between blood flow and oxygen demand in contracting skeletal muscle. To determine whether ATP may contribute to the vasodilatory response to exercise in the forearm, we measured arterialised and venous plasma ATP concentration and venous oxygen content in 10 healthy young males at rest, and at 30 and 180 seconds during dynamic handgrip exercise at 45% of maximum voluntary contraction (MVC). Results: Venous plasma ATP concentration was elevated above rest after 30 seconds of exercise (P < 0.05), and remained at this higher level 180 seconds into exercise (P < 0.05 versus rest). The increase in ATP was mirrored by a decrease in venous oxygen content. While there was no significant relationship between ATP concentration and venous oxygen content at 30 seconds of exercise, they were moderately and inversely correlated at 180 seconds of exercise (r = -0.651, P = 0.021). Arterial ATP concentration remained unchanged throughout exercise, resulting in an increase in the venous-arterial ATP difference. Conclusions: Collectively these results indicate that ATP in the plasma originated from the muscle microcirculation, and are consistent with the notion that deoxygenation of the blood perfusing the muscle acts as a stimulus for ATP release. That ATP concentration was elevated just 30 seconds after the onset of exercise also suggests that ATP may be a contributing factor to the blood flow response in the transition from rest to steady state exercise.

Pathogenicity of Plesiomonas shigelloides : interactions with eukaryotic host cells in vitro

Diarrhoea is one of the leading causes of morbidity and mortality in populations in developing countries and is a significant health issue throughout the world. Despite the frequency and the severity of the diarrhoeal disease, mechanisms of pathogenesis for many of the causative agents have been poorly characterised. Although implicated in a number of intestinal and extra-intestinal infections in humans, Plesiomonas shigelloides generally has been dismissed as an enteropathogen due to the lack of clearly demonstrated virulence-associated properties such as production of cytotoxins and enterotoxins or invasive abilities. However, evidence from a number of sources has indicated that this species may be the cause of a number of clinical infections. The work described in this thesis seeks to resolve this discrepancy by investigating the pathogenic potential of P. shigelloides using in vitro cell models. The focus of this research centres on how this organism interacts with human host cells in an experimental model. Very little is known about the pathogenic potential of P. shigel/oides and its mechanisms in human infections and disease. However, disease manifestations mimic those of other related microorganisms. Chapter 2 reviews microbial pathogenesis in general, with an emphasis on understanding the mechanisms resulting from infection with bacterial pathogens and the alterations in host cell biology. In addition, this review analyses the pathogenic status of a poorly-defined enteropathogen, P. shigelloides. Key stages of pathogenicity must occur in order for a bacterial pathogen to cause disease. Such stages include bacterial adherence to host tissue, bacterial entry into host tissues (usually required), multiplication within host tissues, evasion of host defence mechanisms and the causation of damage. In this study, these key strategies in infection and disease were sought to help assess the pathogenic potential of P. shigelloides (Chapter 3). Twelve isolates of P. shigelloides, obtained from clinical cases of gastroenteritis, were used to infect monolayers of human intestinal epithelial cells in vitro. Ultrastructural analysis demonstrated that P. shigelloides was able to adhere to the microvilli at the apical surface of the epithelial cells and also to the plasma membranes of both apical and basal surfaces. Furthermore, it was demonstrated that these isolates were able to enter intestinal epithelial cells. Internalised bacteria often were confined within vacuoles surrounded by single or multiple membranes. Observation of bacteria within membranebound vacuoles suggests that uptake of P. shigelloides into intestinal epithelial cells occurs via a process morphologically comparable to phagocytosis. Bacterial cells also were observed free in the host cell cytoplasm, indicating that P. shige/loides is able to escape from the surrounding vacuolar membrane and exist within the cytosol of the host. Plesiomonas shigelloides has not only been implicated in gastrointestinal infections, but also in a range of non-intestinal infections such as cholecystitis, proctitis, septicaemia and meningitis. The mechanisms by which P. shigelloides causes these infections are not understood. Previous research was unable to ascertain the pathogenic potential of P. shigel/oides using cells of non-intestinal origin (HEp-2 cells derived from a human larynx carcinoma and Hela cells derived from a cervical carcinoma). However, with the recent findings (from this study) that P. shigelloides can adhere to and enter intestinal cells, it was hypothesised, that P. shigel/oides would be able to enter Hela and HEp-2 cells. Six clinical isolates of P. shigelloides, which previously have been shown to be invasive to intestinally derived Caco-2 cells (Chapter 3) were used to study interactions with Hela and HEp-2 cells (Chapter 4). These isolates were shown to adhere to and enter both nonintestinal host cell lines. Plesiomonas shigelloides were observed within vacuoles surrounded by single and multiple membranes, as well as free in the host cell cytosol, similar to infection by P. shigelloides of Caco-2 cells. Comparisons of the number of bacteria adhered to and present intracellularly within Hela, HEp-2 and Caco-2 cells revealed a preference of P. shigelloides for Caco-2 cells. This study conclusively showed for the first time that P. shigelloides is able to enter HEp-2 and Hela cells, demonstrating the potential ability to cause an infection and/or disease of extra-intestinal sites in humans. Further high resolution ultrastructural analysis of the mechanisms involved in P. shigelloides adherence to intestinal epithelial cells (Chapter 5) revealed numerous prominent surface features which appeared to be involved in the binding of P. shige/loides to host cells. These surface structures varied in morphology from small bumps across the bacterial cell surface to much longer filaments. Evidence that flagella might play a role in bacterial adherence also was found. The hypothesis that filamentous appendages are morphologically expressed when in contact with host cells also was tested. Observations of bacteria free in the host cell cytosol suggests that P. shigelloides is able to lyse free from the initial vacuolar compartment. The vacuoles containing P. shigel/oides within host cells have not been characterised and the point at which P. shigelloides escapes from the surrounding vacuolar compartment has not been determined. A cytochemical detection assay for acid phosphatase, an enzymatic marker for lysosomes, was used to analyse the co-localisation of bacteria-containing vacuoles and acid phosphatase activity (Chapter 6). Acid phosphatase activity was not detected in these bacteria-containing vacuoles. However, the surface of many intracellular and extracellular bacteria demonstrated high levels of acid phosphatase activity, leading to the proposal of a new virulence factor for P. shigelloides. For many pathogens, the efficiency with which they adhere to and enter host cells is dependant upon the bacterial phase of growth. Such dependency reflects the timing of expression of particular virulence factors important for bacterial pathogenesis. In previous studies (Chapter 3 to Chapter 6), an overnight culture of P. shigelloides was used to investigate a number of interactions, however, it was unknown whether this allowed expression of bacterial factors to permit efficient P. shigelloides attachment and entry into human cells. In this study (Chapter 7), a number of clinical and environmental P. shigelloides isolates were investigated to determine whether adherence and entry into host cells in vitro was more efficient during exponential-phase or stationary-phase bacterial growth. An increase in the number of adherent and intracellular bacteria was demonstrated when bacteria were inoculated into host cell cultures in exponential phase cultures. This was demonstrated clearly for 3 out of 4 isolates examined. In addition, an increase in the morphological expression of filamentous appendages, a suggested virulence factor for P. shigel/oides, was observed for bacteria in exponential growth phase. These observations suggest that virulence determinants for P. shigel/oides may be more efficiently expressed when bacteria are in exponential growth phase. This study demonstrated also, for the first time, that environmental water isolates of P. shigelloides were able to adhere to and enter human intestinal cells in vitro. These isolates were seen to enter Caco-2 host cells through a process comparable to the clinical isolates examined. These findings support the hypothesis of a water transmission route for P. shigelloides infections. The results presented in this thesis contribute significantly to our understanding of the pathogenic mechanisms involved in P. shigelloides infections and disease. Several of the factors involved in P. shigelloides pathogenesis have homologues in other pathogens of the human intestine, namely Vibrio, Aeromonas, Salmonella, Shigella species and diarrhoeaassociated strains of Escherichia coli. This study emphasises the relevance of research into Plesiomonas as a means of furthering our understanding of bacterial virulence in general. As well it provides tantalising clues on normal and pathogenic host cell mechanisms.

Biomimetic tubular nanofiber mesh and platelet rich plasma-mediated delivery of BMP-7 for large bone defect regeneration.

There is a growing need for successful bone tissue engineering strategies and advanced biomaterials that mimic the structure and function of native tissues carry great promise. Successful bone repair approaches may include an osteoconductive scaffold, osteoinductive growth factors, cells with an osteogenic potential and capacity for graft vascularisation. To increase osteoinductivity of biomaterials, the local combination and delivery of growth factors has been developed. In the present study we investigated the osteogenic effects of calcium phosphate (CaP)-coated nanofiber mesh tube-mediated delivery of BMP-7 from a PRP matrix for the regeneration of critical sized segmental bone defects in a small animal model. Bilateral full-thickness diaphyseal segmental defects were created in twelve male Lewis rats and nanofiber mesh tubes were placed around the defect. Defects received either treatment with a CaP-coated nanofiber mesh tube (n = 6), an un-coated nanofiber mesh tube (n=6) a CaP-coated nanofiber mesh tube with PRP (n=6) or a CaP-coated nanofiber mesh tube in combination with 5 μg BMP-7 and PRP (n = 6). After 12 weeks, bone volume and biomechanical properties were evaluated using radiography, microCT, biomechanical testing and histology. The results demonstrated significantly higher biomechanical properties and bone volume for the BMP group compared to the control groups. These results were supported by the histological evaluations, where BMP group showed the highest rate of bone regeneration within the defect. In conclusion, BMP-7 delivery via PRP enhanced functional bone defect regeneration, and together these data support the use of BMP-7 in the treatment of critical sized defects.

Expression of PTRF in PC3 cells modulates cholesterol dynamics and the actin cytoskeleton impacting secretion pathways

Expression of caveolin-1 is up-regulated in prostate cancer metastasis and is associated with aggressive recurrence of the disease. Intriguingly, caveolin-1 is also secreted from prostate cancer cell lines and has been identified in secreted prostasomes. Caveolin-1 is the major structural component of the plasma membrane invaginations called caveolae. Co-expression of the coat protein Polymerase I and transcript release factor (PTRF) is required for caveolae formation. We recently found that expression of caveolin-1 in the aggressive prostate cancer cell line PC-3 is not accompanied by PTRF, leading to noncaveolar caveolin-1 lipid rafts. Moreover, ectopic expression of PTRF in PC-3 cells sequesters caveolin-1 into caveolae. Here we quantitatively analyzed the effect of PTRF expression on the PC-3 proteome using stable isotope labeling by amino acids in culture and subcellular proteomics. We show that PTRF reduced the secretion of a subset of proteins including secreted proteases, cytokines, and growth regulatory proteins, partly via a reduction in prostasome secretion. To determine the cellular mechanism accounting for the observed reduction in secreted proteins we analyzed total membrane and the detergent-resistant membrane fractions. Our data show that PTRF expression selectively impaired the recruitment of actin cytoskeletal proteins to the detergent-resistant membrane, which correlated with altered cholesterol distribution in PC-3 cells expressing PTRF. Consistent with this, modulating cellular cholesterol altered the actin cytoskeleton and protein secretion in PC-3 cells. Intriguingly, several proteins that function in ER to Golgi trafficking were reduced by PTRF expression. Taken together, these results suggest that the noncaveolar caveolin-1 found in prostate cancer cells generates a lipid raft microenvironment that accentuates secretion pathways, possibly at the step of ER sorting/exit. Importantly, these effects could be modulated by PTRF expression.

Numerical simulation of red blood cells' deformation using SPH method

A numerical simulation method for the Red Blood Cells’ (RBC) deformation is presented in this study. The two-dimensional RBC membrane is modeled by the spring network, where the elastic stretch/compression energy and the bending energy are considered with the constraint of constant RBC surface area. Smoothed Particle Hydrodynamics (SPH) method is used to solve the Navier-Stokes equation coupled with the Plasma-RBC membrane and Cytoplasm- RBC membrane interaction. To verify the method, the motion of a single RBC is simulated in Poiseuille flow and compared with the results reported earlier. Typical motion and deformation mechanism of the RBC is observed.

Cryo-electron tomography of Marburg Virus particles and their morphogenesis within infected cells

Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible "Velcro-like" manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.

Reduction in resting plasma granulysin as a marker of increased training load

Granulysin is a cytolytic granule protein released by natural killer cells and activated cytotoxic T lymphocytes. The influence of exercise training on circulating granulysin concentration is unknown, as is the relationship between granulysin concentration, natural killer cell number and natural killer cell cytotoxicity. We examined changes in plasma granulysin concentration, natural killer cell number and cytotoxicity following acute exercise and different training loads. Fifteen highly trained male cyclists completed a baseline 40-km cycle time trial (TT401) followed by five weeks of normal training and a repeat time trial (TT402). The cyclists then completed four days of high intensity training followed by another time trial (TT403) on day five. Following one final week of normal training cyclists completed another time trial (TT404). Fasting venous blood was collected before and after each time trial to determine granulysin concentration, natural killer cell number and natural killer cell cytotoxicity. Granulysin concentration increased significantly after each time trial (P<0.001). Pre-exercise granulysin concentration for TT403 was significantly lower than pre-exercise concentration for TT401 (-20.3 +/- 7.5%, P<0.026), TT402 (-16.7 +/- 4.3%, P<0.003) and 7T404 (-21 +/- 4.2%, P<0.001). Circulating natural killer cell numbers also increased significantly post-exercise for each time trial (P<0.001), however there was no significant difference across TT40 (P>0.05). Exercise did not significantly alter natural killer cell cytotoxicity on a per cell basis, and there were no significant differences between the four time trials. In conclusion, plasma granulysin concentration increases following moderate duration, strenuous exercise and is decreased in response to a short-term period of intensified training.