Two Distinct Coagulase-Dependent Barriers Protect Staphylococcus aureus from Neutrophils in a Three Dimensional in vitro Infection Model

Staphylococcus aureus is a pyogenic abscess-forming facultative pathogenic microorganism expressing a large set of virulence-associated factors. Among these, secreted proteins with binding capacity to plasma proteins (e.g. fibrinogen binding proteins Eap and Emp) and prothrombin activators such as Coagulase (Coa) and vWbp are involved in abscess formation. By using a three-dimensional collagen gel (3D-CoG) supplemented with fibrinogen (Fib) we studied the growth behavior of S. aureus strain Newman and a set of mutants as well as their interaction with mouse neutrophils by real-time confocal microscopy. In 3D-CoG/Fib, S. aureus forms microcolonies which are surrounded by an inner pseudocapsule and an extended outer dense microcolony-associated meshwork (MAM) containing fibrin. Coa is involved in formation of the pseudocapsule whereas MAM formation depends on vWbp. Moreover, agr-dependent dispersal of late stage microcolonies could be observed. Furthermore, we demonstrate that the pseudocapsule and the MAM act as mechanical barriers against neutrophils attracted to the microcolony. The thrombin inhibitor argatroban is able to prevent formation of both pseudocapsule and MAM and supports access of neutrophils to staphylococci. Taken together, this model can simulate specific stages of S. aureus abscess formation by temporal dissection of bacterial growth and recruitment of immune cells. It can complement established animal infection models in the development of new treatment options

Targeting of the Human Coagulation Factor IX Gene at rDNA Locus of Human Embryonic Stem Cells

BACKGROUND: Genetic modification is a prerequisite to realizing the full potential of human embryonic stem cells (hESCs) in human genetic research and regenerative medicine. Unfortunately, the random integration methods that have been the primary techniques used keep creating problems, and the primary alternative method, gene targeting, has been effective in manipulating mouse embryonic stem cells (mESCs) but poorly in hESCs. METHODOLOGY/PRINCIPAL FINDINGS: Human ribosomal DNA (rDNA) repeats are clustered on the short arm of acrocentric chromosomes. They consist of approximately 400 copies of the 45S pre-RNA (rRNA) gene per haploid. In the present study, we targeted a physiological gene, human coagulation factor IX, into the rDNA locus of hESCs via homologous recombination. The relative gene targeting efficiency (>50%) and homologous recombination frequency (>10(-5)) were more than 10-fold higher than those of loci targeted in previous reports. Meanwhile, the targeted clones retained both a normal karyotype and the main characteristics of ES cells. The transgene was found to be stably and ectopically expressed in targeted hESCs. CONCLUSION/SIGNIFICANCE: This is the first targeting of a human physiological gene at a defined locus on the hESC genome. Our findings indicate that the rDNA locus may serve as an ideal harbor for transgenes in hESCs

Characterization of the model for experimental testicular teratoma in 129/SvJ-mice

An animal model of experimental testicular teratoma has been established to study how a teratoma affects the host testis and how the host testis reacts against the teratoma. 129/SvJ-mice were used as experimental animals. To induce the experimental testicular teratoma, male gonadal ridges from 12-day-old 129/SvJ-mouse fetuses were grafted into the testes of adult mice for 1-12 weeks. The developing tumour was analysed by light and electron microscopy and by immunocytochemical localization of transcription factors SOX9 and c-kit, glial fibrillary acidic protein (GFAP) and type IV collagen. Testicular teratoma was observed in 36 out of 124 testes with implanted fetal gonadal ridges (frequency 29%). One spontaneous testicular teratoma was observed in this material from 70 male mice (1.5%). One week after implantation intracordal clusters of cells were seen in embryonic testicular cords of the graft as the first sign of testicular teratomas. Four weeks after implantation the embryonic testicular cords had totally disappeared from grafts with teratomas, and the tumour tissue had enlarged the testis and invaded the interstitium of the host testis. It consisted of solitary pieces of immature cartilage as well as of glial cells and of primitive neuroepithelium. Six to eight weeks after implantation the tumour tissue had expanded so that the enlarged testis could be detected by macroscopic enlargement of the scrotum. The testicular tissue of the host had practically disappeared, and only solitary disrupted seminiferous tubules of the host were seen surrounding the teratoma. Neuroepithelial structures of some teratomas cultured for 8 weeks had cells with a granular nucleus as a sign of obvious apoptosis. Eleven to 12 weeks after implantation the growth of the teratoma had stopped, and the histology corresponded to that of a mature cystic teratoma. GFAP, SOX9 and type IV collagen were strongly positive in some parts of the tumours cultured for 4 and 8 weeks, while only occasional c-kit-positive areas were observed in tumours cultured for 8 weeks. As conclusions: (1) the metastasizing capacity of the experimental testicular teratoma is very low during 12 weeks, but the behaviour of the tumour in the testicular tissue of the graft is invasive; (2) the growth of experimental testicular teratomas cease 6-8 weeks after implantation of the fetal gonadal ridges with the obvious apoptosis of the immature tissue components; (3) the model of experimental testicular teratoma in the mouse is suitable for studying how the teratoma affects the host testis and how the host testis reacts to teratoma

Contribution of Coagulases towards Staphylococcus aureus Disease and Protective Immunity

The bacterial pathogen Staphylococcus aureus seeds abscesses in host tissues to replicate at the center of these lesions, protected from host immune cells via a pseudocapsule. Using histochemical staining, we identified prothrombin and fibrin within abscesses and pseudocapsules. S. aureus secretes two clotting factors, coagulase (Coa) and von Willebrand factor binding protein (vWbp). We report here that Coa and vWbp together are required for the formation of abscesses. Coa and vWbp promote the non-proteolytic activation of prothrombin and cleavage of fibrinogen, reactions that are inhibited with specific antibody against each of these molecules. Coa and vWbp specific antibodies confer protection against abscess formation and S. aureus lethal bacteremia, suggesting that coagulases function as protective antigens for a staphylococcal vaccine

Putting prevention into practice: qualitative study of factors that inhibit and promote preventive care by general practitioners, with a focus on elderly patients

<p>Abstract</p> <p>Background</p> <p>General practitioners (GPs) have a key role in providing preventive care, particularly for elderly patients. However, various factors can inhibit or promote the implementation of preventive care. In the present study, we identified and examined factors that inhibit and promote preventive care by German GPs, particularly for elderly patients, and assessed changes in physicians' attitudes toward preventive care throughout their careers.</p> <p>Methods</p> <p>A qualitative, explorative design was used to identify inhibitors and promoters of preventive care in German general medical practice. A total of 32 GPs in Berlin and Hannover were surveyed. Questions about factors that promote or inhibit implementation of preventive care and changes in physicians' perceptions of promoting and inhibiting factors throughout their careers were identified. Episodic interviews, which encouraged the reporting of anecdotes regarding daily knowledge and experiences, were analyzed using ATLAS/ti. Socio-demographic data of GPs and structural information about their offices were collected using short questionnaires. The factors identified as inhibitory or promoting were classified as being related to patients, physicians, or the healthcare system. The changes in GP attitudes toward preventive care throughout their careers were classified as personal transitions or as social and health policy transitions.</p> <p>Results</p> <p>Most of the identified barriers to preventive care were related to patients, such as a lack of motivation for making lifestyle changes and a lack of willingness to pay for preventive interventions. In addition, the healthcare system seemed to inadequately promote preventive care, mainly due to poor reimbursement for preventive care and fragmentation of care. GPs own attitudes and health habits seemed to influence the implementation of preventive care. GPs recognized their own lack of awareness of effective preventive interventions, particularly for elderly patients. GPs were motivated by positive preventive experiences, but often lacked the necessary training to counsel and support their patients.</p> <p>Conclusions</p> <p>German GPs had positive attitudes towards prevention, but the implementation of preventive care was neither systematic nor continuous. Identification and elimination of barriers to preventive care is crucial. Further research is needed to identify effective practice-based approaches to overcome these barriers.</p

The Type I NADH Dehydrogenase of Mycobacterium tuberculosis Counters Phagosomal NOX2 Activity to Inhibit TNF-α-Mediated Host Cell Apoptosis

The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persisting, intracellular pathogens such as the human pathogen Mycobacterium tuberculosis (Mtb) to inhibit infection-induced apoptosis of macrophages is important for virulence. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase, NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis, but the molecular mechanism of this host pathogen interaction remains elusive. Here we show that the apoptogenic phenotype of MtbΔnuoG was significantly reduced in human macrophages treated with caspase-3 and -8 inhibitors, TNF-α-neutralizing antibodies, and also after infection of murine TNF−/− macrophages. Interestingly, incubation of macrophages with inhibitors of reactive oxygen species (ROS) reduced not only the apoptosis induced by the nuoG mutant, but also its capacity to increase macrophage TNF-α secretion. The MtbΔnuoG phagosomes showed increased ROS levels compared to Mtb phagosomes in primary murine and human alveolar macrophages. The increase in MtbΔnuoG induced ROS and apoptosis was abolished in NOX-2 deficient (gp91−/−) macrophages. These results suggest that Mtb, via a NuoG-dependent mechanism, can neutralize NOX2-derived ROS in order to inhibit TNF-α-mediated host cell apoptosis. Consistently, an Mtb mutant deficient in secreted catalase induced increases in phagosomal ROS and host cell apoptosis, both of which were dependent upon macrophage NOX-2 activity. In conclusion, these results serendipitously reveal a novel connection between NOX2 activity, phagosomal ROS, and TNF-α signaling during infection-induced apoptosis in macrophages. Furthermore, our study reveals a novel function of NOX2 activity in innate immunity beyond the initial respiratory burst, which is the sensing of persistent intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense

Mobile health apps to facilitate self-care: a qualitative study of user experiences

Objective: Consumers are living longer, creating more pressure on the health system and increasing their requirement for self-care of chronic conditions. Despite rapidly-increasing numbers of mobile health applications (‘apps’) for consumers’ self-care, there is a paucity of research into consumer engagement with electronic self-monitoring. This paper presents a qualitative exploration of how health consumers use apps for health monitoring, their perceived benefits from use of health apps, and suggestions for improvement of health apps. Materials and Methods: ‘Health app’ was defined as any commercially-available health or fitness app with capacity for self-monitoring. English-speaking consumers aged 18 years and older using any health app for self-monitoring were recruited for interview from the metropolitan area of Perth, Australia. The semi-structured interview guide comprised questions based on the Technology Acceptance Model, Health Information Technology Acceptance Model, and the Mobile Application Rating Scale, and is the only study to do so. These models also facilitated deductive thematic analysis of interview transcripts. Implicit and explicit responses not aligned to these models were analyzed inductively.Results: Twenty-two consumers (15 female, seven male) participated, 13 of whom were aged 26–35 years. Eighteen participants reported on apps used on iPhones. Apps were used to monitor diabetes, asthma, depression, celiac disease, blood pressure, chronic migraine, pain management, menstrual cycle irregularity, and fitness. Most were used approximately weekly for several minutes per session, and prior to meeting initial milestones, with significantly decreased usage thereafter. Deductive and inductive thematic analysis reduced the data to four dominant themes: engagement in use of the app; technical functionality of the app; ease of use and design features; and management of consumers’ data. Conclusions: The semi-structured interviews provided insight into usage, benefits and challenges of health monitoring using apps. Understanding the range of consumer experiences and expectations can inform design of health apps to encourage persistence in self-monitoring

Preventing Staphylococcus aureus Sepsis through the Inhibition of Its Agglutination in Blood

Staphylococcus aureus infection is a frequent cause of sepsis in humans, a disease associated with high mortality and without specific intervention. When suspended in human or animal plasma, staphylococci are known to agglutinate, however the bacterial factors responsible for agglutination and their possible contribution to disease pathogenesis have not yet been revealed. Using a mouse model for S. aureus sepsis, we report here that staphylococcal agglutination in blood was associated with a lethal outcome of this disease. Three secreted products of staphylococci - coagulase (Coa), von Willebrand factor binding protein (vWbp) and clumping factor (ClfA) – were required for agglutination. Coa and vWbp activate prothrombin to cleave fibrinogen, whereas ClfA allowed staphylococci to associate with the resulting fibrin cables. All three virulence genes promoted the formation of thromboembolic lesions in heart tissues. S. aureus agglutination could be disrupted and the lethal outcome of sepsis could be prevented by combining dabigatran-etexilate treatment, which blocked Coa and vWbp activity, with antibodies specific for ClfA. Together these results suggest that the combined administration of direct thrombin inhibitors and ClfA-antibodies that block S. aureus agglutination with fibrin may be useful for the prevention of staphylococcal sepsis in humans

Isolation, Cloning and Structural Characterisation of Boophilin, a Multifunctional Kunitz-Type Proteinase Inhibitor from the Cattle Tick

Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine α-thrombin·boophilin complex, refined at 2.35 Å resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S1 pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9° and is displaced by 6 Å, while the C-terminal domain rotates almost 6° accompanied by a 3 Å displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P1 residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin·boophilin·trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo