A model of cerebral aspergillosis in non-immunosuppressed nursing rats

Central nervous system aspergillosis is an often fatal complication of invasive Aspergillus infection. Relevant disease models are needed to study the pathophysiology of cerebral aspergillosis and to develop novel therapeutic approaches. This study presents a model of central nervous system aspergillosis that mimics important aspects of human disease. Eleven-day-old non-immunosuppressed male Wistar rats were infected by an intracisternal injection of 10 mul of a conidial suspension of Aspergillus fumigatus. An inoculum of 7.18 log(10) colony-forming units (CFU) consistently produced cerebral infection and resulted in death of all animals (n = 25) within 3-10 days. Median survival time was 3 days. Histomorphologically, all animals developed intracerebral abscesses (2-26 per brain) containing abundant fungal hyphae and neutrophils. Fungal culture of cortical homogenates yielded maximal growth on day 3 after infection (5.4 log(10) CFU/g, n = 15) that declined over time. Galactomannan concentrations in cortical homogenates, assessed as an index for hyphal burden, peaked on days 3-5. Fungal infection spread to peripheral organs in 83% of animals. Fungal burden in lung, liver, spleen and kidney was two orders of magnitude lower than in the brain. The successful establishment of a model of cerebral aspergillosis in a non-immunosuppressed host provides the opportunity to investigate mechanisms of disease and to develop novel treatment regimens for this commonly fatal infection.

Role of beta1-, beta2-, and beta3-adrenoceptors in contractile hypersensitivity in a model of small bowel transplantation

BACKGROUND: Chronic extrinsic denervation induced by small bowel transplantation (SBT) results in adrenergic hypersensitivity in rat ileum. This study evaluated the role of neuronal and/or muscular beta1-, beta2-, and beta3-adrenoceptor (AR) mechanisms on contractility. METHODS: Ileal longitudinal muscle strips from Lewis rats (n = 6 rats per group, 8 strips per rat): naive controls (NC), 4 months after sham operation (SC) or after syngeneic orthotopic SBT were studied in vitro. Spontaneous contractile activity and dose responses (10(-8)-10(-4) mol) to isoprenaline (IP), a nonspecific beta-AR agonist were studied with or without selective antagonists (10(-5) mol), for beta1- (atenolol), beta2- (ICI 118551), or beta3- (SR 59230A) AR subtypes in the presence or absence of tetrodotoxin (TTX; 10(-6) mol; nerve blocker). RESULTS: pEC50 (neg log of EC50, which is the concentration where 50% of inhibition was observed) of IP was 7.2 +/- 0.2 (mean value +/- SEM) in SBT vs 6.3 +/- 0.1 in SC and 6.3 +/- 0.2 in NC (both P < .05 vs SBT), reflecting adrenergic hypersensitivity. Beta1- and beta2-AR blockade induced a TTX-sensitive right shift of the curve only in SBT and normalized pEC50 values from 7.2 +/- 0.2 to 6.4 +/- 0.1 and 7.2 +/- 0.2 to 6.6 +/- 0.1, respectively (P < .05). Beta3-AR blockade shifted the curve independent of the presence of TTX to the right in all groups (all P < .05). CONCLUSIONS: In rat ileum, adrenergic inhibition of contractility was dependent on muscular beta3-AR pathways, whereas posttransplant hypersensitivity was due to upregulated neuronal beta1- and beta2-AR mechanisms that were inactive before SBT.

Essential role of choline for pneumococcal virulence in an experimental model of meningitis

Objectives: The goal of the present study was to elucidate the contribution of the newly recognized virulence factor choline to the pathogenesis of Streptococcus pneumoniae in an animal model of meningitis. Results: The choline containing strain D39Cho(-) and its isogenic choline-free derivative D39Cho(-)licA64 -each expressing the capsule polysaccharide 2 - were introduced intracisternally at an inoculum size of 10(3) CFU into 11 days old Wistar rats. During the first 8 h post infection both strains multiplied and stimulated a similar immune response that involved expression of high levels of proinflammatory cytokines, the matrix metalloproteinase 9 (MMP-9), IL-10, and the influx of white blood cells into the CSF. Virtually identical immune response was also elicited by intracisternal inoculation of 10(7) CFU equivalents of either choline-containing or choline-free cell walls. At sampling times past 8 h strain D39Cho(-) continued to replicate accompanied by an intense inflammatory response and strong granulocytic pleiocytosis. Animals infected with D39Cho(-) died within 20 h and histopathology revealed brain damage in the cerebral cortex and hippocampus. In contrast, the initial immune response generated by the choline-free strain D39Cho(-)licA64 began to decline after the first 8 h accompanied by elimination of the bacteria from the CSF in parallel with a strong WBC response peaking at 8 h after infection. All animals survived and there was no evidence for brain damage. Conclusion: Choline in the cell wall is essential for pneumococci to remain highly virulent and survive within the host and establish pneumococcal meningitis.

Evaluation of the Role of Access Providers Discussion of Dutch Pirate Bay Case Law and Introducing Principles on Directness, Effectiveness, Costs, Relevance, and Time

Internet service providers (ISPs) play a pivotal role in contemporary society because they provide access to the Internet. The primary task of ISPs – to blindly transfer information across the network – has recently come under pressure, as has their status as neutral third parties. Both the public and the private sector have started to require ISPs to interfere with the content placed and transferred on the Internet as well as access to it for a variety of purposes, including the fight against cybercrime, digital piracy, child pornography, etc. This expanding list necessitates a critical assessment of the role of ISPs. This paper analyses the role of the access provider. Particular attention is paid to Dutch case law, in which access providers were forced to block The Pirate Bay. After analysing the position of ISPs, we will define principles that can guide the decisions of ISPs whether to take action after a request to block access based on directness, effectiveness, costs, relevance and time.

A reduced model clarifies the role of feedback loops and time delays in the Drosophila circadian oscillator.

Although several detailed models of molecular processes essential for circadian oscillations have been developed, their complexity makes intuitive understanding of the oscillation mechanism difficult. The goal of the present study was to reduce a previously developed, detailed model to a minimal representation of the transcriptional regulation essential for circadian rhythmicity in Drosophila. The reduced model contains only two differential equations, each with time delays. A negative feedback loop is included, in which PER protein represses per transcription by binding the dCLOCK transcription factor. A positive feedback loop is also included, in which dCLOCK indirectly enhances its own formation. The model simulated circadian oscillations, light entrainment, and a phase-response curve with qualitative similarities to experiment. Time delays were found to be essential for simulation of circadian oscillations with this model. To examine the robustness of the simplified model to fluctuations in molecule numbers, a stochastic variant was constructed. Robust circadian oscillations and entrainment to light pulses were simulated with fewer than 80 molecules of each gene product present on average. Circadian oscillations persisted when the positive feedback loop was removed. Moreover, elimination of positive feedback did not decrease the robustness of oscillations to stochastic fluctuations or to variations in parameter values. Such reduced models can aid understanding of the oscillation mechanisms in Drosophila and in other organisms in which feedback regulation of transcription may play an important role.

ROLE OF VITAMIN-D3 AND RETINOIC ACID IN A HUMAN THP-1 MACROPHAGE MODEL OF MYCOBACTERIUM TUBERCULOSIS INFECTION

Mycobacterium tuberculosis (Mtb) replicates within the human macrophages and we investigated the activating effects of retinoic acid (RA) and vitamin D3 (VD) on macrophages in relation to the viability of Mtb. A combination of these vitamins (RAVD) enhanced the receptors on THP-1 macrophage (Mannose receptor and DC-SIGN) that increased mycobacterial uptake but inhibited thesubsequent intracellular growth of Mtb by inducing reactive oxygen species (ROS) and autophagy. RAVD also enhanced antigen presenting and homing receptors in THPs that suggested an activated phenotype for THPs following RAVD treatment. RAVD mediated activation was also associated with a marked phenotypic change in Mtb infected THPs that fused with adjacent cells to formmultinucleate giant cells (MNGCs). Typically MNGCs occurred over 30 days of in vitro culture and contained non-replicating persisting Mtb for as long as 60 days in culture. We propose that the RAVD mediated inhibition of replicating Mtb leading to persistence of non-replicating Mtb within THPs may provide a novel human macrophage model simulating formation of MNGCs in humanlungs.

Characterization of the ebp(fm) pilus-encoding operon of Enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection.

We recently identified 15 genes encoding putative surface proteins with features of MSCRAMMs and/or pili in the Enterococcus faecium TX0016 (DO) genome, including four predicted pilus-encoding gene clusters; we also demonstrated that one of these, ebpABC(fm), is transcribed as an operon, that its putative major pilus subunit, EbpC(fm) (also called pilB), is polymerized into high molecular weight complexes, and that it is enriched among clinical E. faecium isolates. Here, we created a deletion of the ebpABC(fm) operon in an endocarditis-derived E. faecium strain (TX82) and showed, by a combination of whole-cell ELISA, flow cytometry, immunoblot and immunogold electron microscopy, that this deletion abolished EbpC(fm) expression and eliminated EbpC(fm)-containing pili from the cell surface. However, transcription of the downstream sortase, bps(fm), was not affected. Importantly, the ebpABC(fm) deletion resulted in significantly reduced biofilm formation (p < 0.0001) and initial adherence (p < 0.0001) versus the wild-type; both were restored by complementing ebpABC(fm) in trans, which also restored cell surface expression of EbpC(fm) and pilus production. Furthermore, the deletion mutant was significantly attenuated in two independent mixed infection mouse urinary tract experiments, i.e., outnumbered by the wild-type in kidneys (p = 0.0003 and < 0.0001, respectively) and urinary bladders (p = 0.0003 and = 0.002). In conclusion, we have shown that the ebpABC(fm) locus encodes pili on the E. faecium TX82 cell surface and provide the first evidence that pili of this emerging pathogen are important for its ability to form biofilm and to cause infection in an ascending UTI model.

Paleoclimate data-model comparison and the role of climate forcings over the past 1500 years

The past 1500 years provide a valuable opportunity to study the response of the climate system to external forcings. However, the integration of paleoclimate proxies with climate modeling is critical to improving the understanding of climate dynamics. In this paper, a climate system model and proxy records are therefore used to study the role of natural and anthropogenic forcings in driving the global climate. The inverse and forward approaches to paleoclimate data–model comparison are applied, and sources of uncertainty are identified and discussed. In the first of two case studies, the climate model simulations are compared with multiproxy temperature reconstructions. Robust solar and volcanic signals are detected in Southern Hemisphere temperatures, with a possible volcanic signal detected in the Northern Hemisphere. The anthropogenic signal dominates during the industrial period. It is also found that seasonal and geographical biases may cause multiproxy reconstructions to overestimate the magnitude of the long-term preindustrial cooling trend. In the second case study, the model simulations are compared with a coral δ18O record from the central Pacific Ocean. It is found that greenhouse gases, solar irradiance, and volcanic eruptions all influence the mean state of the central Pacific, but there is no evidence that natural or anthropogenic forcings have any systematic impact on El Niño–Southern Oscillation. The proxy climate relationship is found to change over time, challenging the assumption of stationarity that underlies the interpretation of paleoclimate proxies. These case studies demonstrate the value of paleoclimate data–model comparison but also highlight the limitations of current techniques and demonstrate the need to develop alternative approaches.

Decadal Variability in the Northeast Pacific in a Physical-Ecosystem Model: Role of Mixed Layer Depth and Trophic Interactions

A basin-wide interdecadal change in both the physical state and the ecology of the North Pacific occurred near the end of 1976. Here we use a physical-ecosystem model to examine whether changes in the physical environment associated with the 1976-1977 transition influenced the lower trophic levels of the food web and if so by what means. The physical component is an ocean general circulation model, while the biological component contains 10 compartments: two phytoplankton, two zooplankton, two detritus pools, nitrate, ammonium, silicate, and carbon dioxide. The model is forced with observed atmospheric fields during 1960-1999. During spring, there is a similar to 40% reduction in plankton biomass in all four plankton groups during 1977-1988 relative to 1970-1976 in the central Gulf of Alaska (GOA). The epoch difference in plankton appears to be controlled by the mixed layer depth. Enhanced Ekman pumping after 1976 caused the halocline to shoal, and thus the mixed layer depth, which extends to the top of the halocline in late winter, did not penetrate as deep in the central GOA. As a result, more phytoplankton remained in the euphotic zone, and phytoplankton biomass began to increase earlier in the year after the 1976 transition. Zooplankton biomass also increased, but then grazing pressure led to a strong decrease in phytoplankton by April followed by a drop in zooplankton by May: Essentially, the mean seasonal cycle of plankton biomass was shifted earlier in the year. As the seasonal cycle progressed, the difference in plankton concentrations between epochs reversed sign again, leading to slightly greater zooplankton biomass during summer in the later epoch.

Perceived organizational support and intention to stay in host countries among self-initiated expatriates: the role of career satisfaction and networks

Previous literature in SIE (self-initiated expatriation) has been mostly focused on an individual perspective. Studies on SIEs in organizational context are scarce. The current paper sought to examine the effect of perceived organizational support (POS) on SIE employees’ intention to stay in the host country, mediated by career satisfaction. Furthermore, we examined the moderating roles of career-related social networks with host and home country nationals on the effectiveness of POS. Data from 112 SIE employees in Germany were collected and analyzed. Empirical results partially supported our proposed model: there were significant negative indirect effect between POS and intention to stay, when career network size with home country nationals was high. The direct effect between POS and intention to stay was positive. For HR practice, our paper gave insight to understand SIE employees’ needs for support and mobility preferences, which can help organizations to develop more targeted HR development measures and assignment strategies for them.

The role of microclot formation in an acute subarachnoid hemorrhage model in the rabbit

BACKGROUND Microvascular dysfunction and microthrombi formation are believed to contribute to development of early brain injury (EBI) after aneurysmal subarachnoid hemorrhage (SAH). OBJECTIVE This study aimed to determine (i) extent of microthrombus formation and neuronal apoptosis in the brain parenchyma using a blood shunt SAH model in rabbits; (ii) correlation of structural changes in microvessels with EBI characteristics. METHODS Acute SAH was induced using a rabbit shunt cisterna magna model. Extent of microthrombosis was detected 24 h post-SAH (n = 8) by fibrinogen immunostaining, compared to controls (n = 4). We assessed apoptosis by terminal deoxynucleotidyl transferase nick end labeling (TUNEL) in cortex and hippocampus. RESULTS Our results showed significantly more TUNEL-positive cells (SAH: 115 ± 13; controls: 58 ± 10; P = 0.016) and fibrinogen-positive microthromboemboli (SAH: 9 ± 2; controls: 2 ± 1; P = 0.03) in the hippocampus after aneurysmal SAH. CONCLUSIONS We found clear evidence of early microclot formation in a rabbit model of acute SAH. The extent of microthrombosis did not correlate with early apoptosis or CPP depletion after SAH; however, the total number of TUNEL positive cells in the cortex and the hippocampus significantly correlated with mean CPP reduction during the phase of maximum depletion after SAH induction. Both microthrombosis and neuronal apoptosis may contribute to EBI and subsequent DCI.

Cost-effectiveness of peer role play and standardized patients in undergraduate communication training.

BACKGROUND The few studies directly comparing the methodological approach of peer role play (RP) and standardized patients (SP) for the delivery of communication skills all suggest that both methods are effective. In this study we calculated the costs of both methods (given comparable outcomes) and are the first to generate a differential cost-effectiveness analysis of both methods. METHODS Medical students in their prefinal year were randomly assigned to one of two groups receiving communication training in Pediatrics either with RP (N = 34) or 19 individually trained SP (N = 35). In an OSCE with standardized patients using the Calgary-Cambridge Referenced Observation Guide both groups achieved comparable high scores (results published). In this study, corresponding costs were assessed as man-hours resulting from hours of work of SP and tutors. A cost-effectiveness analysis was performed. RESULTS Cost-effectiveness analysis revealed a major advantage for RP as compared to SP (112 vs. 172 man hours; cost effectiveness ratio .74 vs. .45) at comparable performance levels after training with both methods. CONCLUSIONS While both peer role play and training with standardized patients have their value in medical curricula, RP has a major advantage in terms of cost-effectiveness. This could be taken into account in future decisions.

The Role of Cells in Meniscal Guided Tissue Regeneration: A Proof of Concept Study in a Goat Model.

OBJECTIVE Successful repair of defects in the avascular zone of meniscus remains a challenge in orthopedics. This proof of concept study aimed to investigate a guided tissue regeneration approach for treatment of tears in meniscus avascular zone in a goat model. DESIGN Full-depth longitudinal tear was created in the avascular zone of the meniscus and sutured. In the two treatment groups, porcine collagen membrane was wrapped around the tear without (CM) or with injection of expanded autologous chondrocytes (CM+cells), whereas in the control group the tear remained only sutured. Gait recovery was evaluated during the entire follow-up period. On explantation at 3 and 6 months, macroscopic gross inspection assessed healing of tears, degradation of collagen membrane, potential signs of inflammation, and osteoarthritic changes. Microscopic histology scoring criteria were developed to evaluate healing of tears, the cellular response, and the inflammatory response. RESULTS Gait recovery suggested protective effect of collagen membrane and was supported by macroscopical evaluation where improved tear healing was noted in both treated groups. Histology scoring in CM compared to suture group revealed an increase in tear margins contact, newly formed connective tissue between margins, and cell formations surrounded with new matrix after 3 months yet not maintained after 6 months. In contrast, in the CM+cells group these features were observed after 3 and 6 months. CONCLUSIONS A transient, short-term guided tissue regeneration of avascular meniscal tears occurred upon application of collagen membrane, whereas addition of expanded autologous chondrocytes supported more sustainable longer term tear healing.