62 research outputs found
Chlamydia trachomatis responds to heat shock, penicillin induced persistence, and IFN-gamma persistence by altering levels of the extracytoplasmic stress response protease HtrA
Background Chlamydia trachomatis, an obligate intracellular human pathogen, is the most prevalent bacterial sexually transmitted infection worldwide and a leading cause of preventable blindness. HtrA is a virulence and stress response periplasmic serine protease and molecular chaperone found in many bacteria. Recombinant purified C. trachomatis HtrA has been previously shown to have both activities. This investigation examined the physiological role of Chlamydia trachomatis HtrA. Results The Chlamydia trachomatis htrA gene complemented the lethal high temperature phenotype of Escherichia coli htrA- (>42°C). HtrA levels were detected to increase by western blot and immunofluorescence during Chlamydia heat shock experiments. Confocal laser scanning microscopy revealed a likely periplasmic localisation of HtrA. During penicillin induced persistence of Chlamydia trachomatis, HtrA levels (as a ratio of LPS) were initially less than control acute cultures (20 h post infection) but increased to more than acute cultures at 44 h post infection. This was unlike IFN-γ persistence where lower levels of HtrA were observed, suggesting Chlamydia trachomatis IFN-γ persistence does not involve a broad stress response. Conclusion The heterologous heat shock protection for Escherichia coli, and increased HtrA during cell wall disruption via penicillin and heat shock, indicates an important role for HtrA during high protein stress conditions for Chlamydia trachomatis
Biofabrication of customized bone grafts by combination of additive manufacturing and bioreactor knowhow
This study reports on an original concept of additive manufacturing for the fabrication of
tissue engineered constructs (TEC), offering the possibility of concomitantly manufacturing a
customized scaffold and a bioreactor chamber to any size and shape. As a proof of concept
towards the development of anatomically relevant TECs, this concept was utilized for the
design and fabrication of a highly porous sheep tibia scaffold around which a bioreactor
chamber of similar shape was simultaneously built. The morphology of the bioreactor/scaffold
device was investigated by micro-computed tomography and scanning electron microscopy
confirming the porous architecture of the sheep tibiae as opposed to the non-porous nature of
the bioreactor chamber. Additionally, this study demonstrates that both the shape, as well as
the inner architecture of the device can significantly impact the perfusion of fluid within the
scaffold architecture. Indeed, fluid flow modelling revealed that this was of significant
importance for controlling the nutrition flow pattern within the scaffold and the bioreactor
chamber, avoiding the formation of stagnant flow regions detrimental for in vitro tissue
development. The bioreactor/scaffold device was dynamically seeded with human primary
osteoblasts and cultured under bi-directional perfusion for two and six weeks. Primary human
osteoblasts were observed homogenously distributed throughout the scaffold, and were viable
for the six week culture period. This work demonstrates a novel application for additive
manufacturing in the development of scaffolds and bioreactors. Given the intrinsic flexibility
of the additive manufacturing technology platform developed, more complex culture systems
can be fabricated which would contribute to the advances in customized and patient-specific
tissue engineering strategies for a wide range of applications.This work was supported by the NHMRC, the Australian Research Council and Hans Fischer Senior Fellowship, IAS-TUM. Pedro Costa acknowledges the Portuguese Foundation for Science and Technology for his PhD grant (SFRH/BD/62452/2009)
Perspectives of Non-Hospitalised Patients with COVID-19 Self-Isolating for 10 Days at Home: A Qualitative Study in Primary Care in Greece
The aim of this qualitative research, conducted in Spring 2021, was to identify the inconveniences and the psychological and social impact of 10 days of home isolation, required by law, in non-hospitalised COVID-19 patients in Greece and to improve management. Thirty-seven semi-structured telephone interviews were conducted, audio-recorded, and transcribed verbatim. Thematic analysis identified four key emergent themes, i.e., everyday life during self-isolation, psychological issues, social issues, and information and guidance. Food provisioning was of particular concern. Solidarity was expressed to individuals in need. Isolation was not always viable due to space constraints and the necessity to care for sick family members. Fear of transmission to vulnerable groups, hospitalisation, irreversible complications, and death as well as anxiety, insecurity, guilt, and alienation were articulated. COVID-19 disrupted the normal functioning of families and led to revision of interpersonal relationships. Patients avoided re-integration in society due to the transmitter stigma and to limit the risk of infection spread in the community. Over-information promoted fear. Mild illness raised doubts about information validity. Primary care provided monitoring and psychological support. Home isolation caused disruption in various aspects of participants’ life, ranging from logistic problems to dealing with the psychological burden of isolation and illness. Primary care could play a central role in supporting patients
Membrane testosterone binding sites in prostate carcinoma as a potential new marker and therapeutic target: Study in paraffin tissue sections
BACKGROUND: Steroid action is mediated, in addition to classical intracellular receptors, by recently identified membrane sites, that generate rapid non-genomic effects. We have recently identified a membrane androgen receptor site on prostate carcinoma cells, mediating testosterone rapid effects on the cytoskeleton and secretion within minutes. METHODS: The aim of this study was to investigate whether membrane androgen receptors are differentially expressed in prostate carcinomas, and their relationship to the tumor grade. We examined the expression of membrane androgen receptors in archival material of 109 prostate carcinomas and 103 benign prostate hyperplasias, using fluorescein-labeled BSA-coupled testosterone. RESULTS: We report that membrane androgen receptors are preferentially expressed in prostate carcinomas, and they correlate to their grade using the Gleason's microscopic grading score system. CONCLUSION: We conclude that membrane androgen receptors may represent an index of tumor aggressiveness and possibly specific targets for new therapeutic regimens
Proceedings of the 24th Paediatric Rheumatology European Society Congress: Part three
From Springer Nature via Jisc Publications Router.Publication status: PublishedHistory: collection 2017-09, epub 2017-09-0
Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries
Abstract
Background
Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres.
Methods
This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries.
Results
In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia.
Conclusion
This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries
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
Expression of potato virus Y cytoplasmic inclusion protein in tobacco results in disorganisation of parenchyma cells, distortion of epidermal cells, and induces mitochondrial and chloroplast abnormalities, formation of membrane whorls and atypical
Infection of plant cells by potyviruses induces the formation of cytoplasmic inclusions ranging in size from 200 to 1000 nm. To determine if the ability to form these ordered, insoluble structures is intrinsic to the potyviral cytoplasmic inclusion protein, we have expressed the cytoplasmic inclusion protein from Potato virus Y in tobacco under the control of the chrysanthemum ribulose-1,5-bisphosphate carboxylase small subunit promoter, a highly active, green tissue promoter. No cytoplasmic inclusions were observed in the leaves of transgenic tobacco using transmission electron microscopy, despite being able to clearly visualize these inclusions in Potato virus Y infected tobacco leaves under the same conditions. However, we did observe a wide range of tissue and sub-cellular abnormalities associated with the expression of the Potato virus Y cytoplasmic inclusion protein. These changes included the disruption of normal cell morphology and organization in leaves, mitochondrial and chloroplast internal reorganization, and the formation of atypical lipid accumulations. Despite these significant structural changes, however, transgenic tobacco plants were viable and the results are discussed in the context of potyviral cytoplasmic inclusion protein function
In vitro characterisation of koala Chlamydia pneumoniae: Morphology, inclusion development and doubling time
Chlamydia pneumoniae is a common human and animal pathogen associated with upper and lower respiratory tract infections. Of the animal C. pneumoniae isolates, the koala nasal isolate (LPCoLN) is by far the best genetically characterised. This current study was designed to characterise the morphology and developmental events for the LPCoLN isolate, and our results showed several striking in vitro growth differences when compared to the human isolate, AR39. The LPCoLN inclusion size and morphology was distinct from AR39, and a much faster doubling time (3.4–4.9 h versus 5.9–8.7 h doubling time) was observed when grown in HEp-2 cell monolayers. Confocal and electron microscopy of LPCoLN confirmed large (9–30 μm in diameter) inclusions, that were heterogeneously shaped, compared to the small (5–9 μm in diameter), uniformly shaped inclusions of AR39. The morphology of the LPCoLN elementary body was round, and had a narrow or nonexistent periplasmic space, compared to the 'pear-shaped' morphology of AR39 EBs. While both isolates showed evidence of inclusion fusion, the level of fusion was much higher for LPCoLN (100%) compared to AR39 (30–40%). Our findings have provided new insights and identified key differences in the in vitro doubling time, size and morphology of an animal C. pneumoniae isolate
Multiphasic construct studied in an ectopic osteochondral defect model
Free to read at publisher In vivo osteochondral defect models predominantly consist of small animals, such as rabbits. Although they have an advantage of low cost and manageability, their joints are smaller and more easily healed compared with larger animals or humans. We hypothesized that osteochondral cores from large animals can be implanted subcutaneously in rats to create an ectopic osteochondral defect model for routine and high-throughput screening of multiphasic scaffold designs and/or tissue-engineered constructs (TECs). Bovine osteochondral plugs with 4 mm diameter osteochondral defect were fitted with novel multiphasic osteochondral grafts composed of chondrocyte-seeded alginate gels and osteoblast-seeded polycaprolactone scaffolds, prior to being implanted in rats subcutaneously with bone morphogenic protein-7. After 12 weeks of in vivo implantation, histological and micro-computed tomography analyses demonstrated that TECs are susceptible to mineralization. Additionally, there was limited bone formation in the scaffold. These results suggest that the current model requires optimization to facilitate robust bone regeneration and vascular infiltration into the defect site. Taken together, this study provides a proof-of-concept for a high-throughput osteochondral defect model. With further optimization, the presented hybrid in vivo model may address the growing need for a cost-effective way to screen osteochondral repair strategies before moving to large animal preclinical trials
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