Structural insights into loss of function of a pore forming toxin and its role in pneumococcal adaptation to an intracellular lifestyle.

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane ß-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche.

Propagule size and sex ratio influence colonisation dynamics after introduction of a non-native lizard.

The composition of founding populations plays an important role in colonisation dynamics and can influence population growth during early stages of biological invasion. Specifically, founding populations with small propagules (i.e. low number of founders) are vulnerable to the Allee effect and have reduced likelihood of establishment compared to those with large propagules. The founding sex ratio can also impact establishment via its influence on mating success and offspring production. Our goal was to test the effects of propagule size and sex ratio on offspring production and annual population growth following introductions of a non-native lizard species (Anolis sagrei). We manipulated propagule composition on nine small islands, then examined offspring production, population growth and survival rate of founders and their descendants encompassing three generations. By the third reproductive season, per capita offspring production was higher on islands seeded with a relatively large propagule size, but population growth was not associated with propagule size. Propagule sex ratio did not affect offspring production, but populations with a female-biased propagule had positive growth, whereas those with a male-biased propagule had negative growth in the first year. Populations were not affected by propagule sex ratio in subsequent years, possibly due to rapid shifts towards balanced (or slightly female biased) population sex ratios. Overall, we show that different components of population fitness have different responses to propagule size and sex ratio in ways that could affect early stages of biological invasion. Despite these effects, the short life span and high fecundity of A. sagrei likely helped small populations to overcome Allee effects and enabled all populations to successfully establish. Our rare experimental manipulation of propagule size and sex ratio can inform predictions of colonisation dynamics in response to different compositions of founding populations, which is critical in the context of population ecology and invasion dynamics.

Dysfunction of GRAP, encoding the GRB2-related adaptor protein, is linked to sensorineural hearing loss.

We have identified a GRAP variant (c.311A>T; p.Gln104Leu) cosegregating with autosomal recessive nonsyndromic deafness in two unrelated families. GRAP encodes a member of the highly conserved growth factor receptor-bound protein 2 (GRB2)/Sem-5/drk family of proteins, which are involved in Ras signaling; however, the function of the growth factor receptor-bound protein 2 (GRB2)-related adaptor protein (GRAP) in the auditory system is not known. Here, we show that, in mouse, Grap is expressed in the inner ear and the protein localizes to the neuronal fibers innervating cochlear and utricular auditory hair cells. Downstream of receptor kinase (drk), the Drosophila homolog of human GRAP, is expressed in Johnston's organ (JO), the fly hearing organ, and the loss of drk in JO causes scolopidium abnormalities. drk mutant flies present deficits in negative geotaxis behavior, which can be suppressed by human wild-type but not mutant GRAP. Furthermore, drk specifically colocalizes with synapsin at synapses, suggesting a potential role of such adaptor proteins in regulating actin cytoskeleton dynamics in the nervous system. Our findings establish a causative link between GRAP mutation and nonsyndromic deafness and suggest a function of GRAP/drk in hearing.

Reversion of Pneumolysin-Induced Executioner Caspase Activation Redirects Cells to Survival.

Apoptosis is an indispensable mechanism for eliminating infected cells and activation of executioner caspases is considered to be a point of no return. Streptococcus pneumoniae, the most common bacterial pathogen causing community-acquired pneumonia, induces apoptosis via its pore-forming toxin pneumolysin, leading to rapid influxes of mitochondrial calcium [Ca2+]m as well as fragmentation, and loss of motility and membrane potential, which is accompanied by caspase-3/7 activation. Using machine-learning and quantitative live-cell microscopy, we identified a significant number of alveolar epithelial cells surviving such executioner caspase activation after pneumolysin attack. Precise single-cell analysis revealed the [Ca2+]m amplitude and efflux rate as decisive parameters for survival and death, which was verified by pharmacological inhibition of [Ca2+]m efflux shifting the surviving cells towards the dying fraction. Taken together, we identified the regulation of [Ca2+]m as critical for controlling the cellular fate under pneumolysin attack, which might be useful for therapeutic intervention during pneumococcal infection.

Higher severe acute respiratory syndrome coronavirus 2 infection rate in pregnant patients.

BACKGROUND: During the early months of the coronavirus disease 2019 pandemic, risks associated with severe acute respiratory syndrome coronavirus 2 in pregnancy were uncertain. Pregnant patients can serve as a model for the success of clinical and public health responses during public health emergencies as they are typically in frequent contact with the medical system. Population-based estimates of severe acute respiratory syndrome coronavirus 2 infections in pregnancy are unknown because of incomplete ascertainment of pregnancy status or inclusion of only single centers or hospitalized cases. Whether pregnant women were protected by the public health response or through their interactions with obstetrical providers in the early months of pandemic is not clearly understood. OBJECTIVE: This study aimed to estimate the severe acute respiratory syndrome coronavirus 2 infection rate in pregnancy and to examine the disparities by race and ethnicity and English language proficiency in Washington State. STUDY DESIGN: Pregnant patients with a polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 infection diagnosed between March 1, 2020, and June 30, 2020 were identified within 35 hospitals and clinics, capturing 61% of annual deliveries in Washington State. Infection rates in pregnancy were estimated overall and by Washington State Accountable Community of Health region and cross-sectionally compared with severe acute respiratory syndrome coronavirus 2 infection rates in similarly aged adults in Washington State. Race and ethnicity and language used for medical care of pregnant patients were compared with recent data from Washington State. RESULTS: A total of 240 pregnant patients with severe acute respiratory syndrome coronavirus 2 infections were identified during the study period with 70.7% from minority racial and ethnic groups. The principal findings in our study were as follows: (1) the severe acute respiratory syndrome coronavirus 2 infection rate was 13.9 per 1000 deliveries in pregnant patients (95% confidence interval, 8.3-23.2) compared with 7.3 per 1000 (95% confidence interval, 7.2-7.4) in adults aged 20 to 39 years in Washington State (rate ratio, 1.7; 95% confidence interval, 1.3-2.3); (2) the severe acute respiratory syndrome coronavirus 2 infection rate reduced to 11.3 per 1000 deliveries (95% confidence interval, 6.3-20.3) when excluding 45 cases of severe acute respiratory syndrome coronavirus disease 2 detected through asymptomatic screening (rate ratio, 1.3; 95% confidence interval, 0.96-1.9); (3) the proportion of pregnant patients in non-White racial and ethnic groups with severe acute respiratory syndrome coronavirus disease 2 infection was 2- to 4-fold higher than the race and ethnicity distribution of women in Washington State who delivered live births in 2018; and (4) the proportion of pregnant patients with severe acute respiratory syndrome coronavirus 2 infection receiving medical care in a non-English language was higher than estimates of pregnant patients receiving care with limited English proficiency in Washington State (30.4% vs 7.6%). CONCLUSION: The severe acute respiratory syndrome coronavirus 2 infection rate in pregnant people was 70% higher than similarly aged adults in Washington State, which could not be completely explained by universal screening at delivery. Pregnant patients from nearly all racial and ethnic minority groups and patients receiving medical care in a non-English language were overrepresented. Pregnant women were not protected from severe acute respiratory syndrome coronavirus 2 infection in the early months of the pandemic. Moreover, the greatest burden of infections occurred in nearly all racial and ethnic minority groups. These data coupled with a broader recognition that pregnancy is a risk factor for severe illness and maternal mortality strongly suggested that pregnant people should be broadly prioritized for coronavirus disease 2019 vaccine allocation in the United States similar to some states.

Disease severity, pregnancy outcomes, and maternal deaths among pregnant patients with severe acute respiratory syndrome coronavirus 2 infection in Washington State.

BACKGROUND: Evidence is accumulating that coronavirus disease 2019 increases the risk of hospitalization and mechanical ventilation in pregnant patients and for preterm delivery. However, the impact on maternal mortality and whether morbidity is differentially affected by disease severity at delivery and trimester of infection are unknown. OBJECTIVE: This study aimed to describe disease severity and outcomes of severe acute respiratory syndrome coronavirus 2 infections in pregnancy across the Washington State, including pregnancy complications and outcomes, hospitalization, and case fatality. STUDY DESIGN: Pregnant patients with a polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 infection between March 1, 2020, and June 30, 2020, were identified in a multicenter retrospective cohort study from 35 sites in Washington State. Sites captured 61% of annual state deliveries. Case-fatality rates in pregnancy were compared with coronavirus disease 2019 fatality rates in similarly aged adults in Washington State using rate ratios and rate differences. Maternal and neonatal outcomes were compared by trimester of infection and disease severity at the time of delivery. RESULTS: The principal study findings were as follows: (1) among 240 pregnant patients in Washington State with severe acute respiratory syndrome coronavirus 2 infections, 1 in 11 developed severe or critical disease, 1 in 10 were hospitalized for coronavirus disease 2019, and 1 in 80 died; (2) the coronavirus disease 2019-associated hospitalization rate was 3.5-fold higher than in similarly aged adults in Washington State (10.0% vs 2.8%; rate ratio, 3.5; 95% confidence interval, 2.3-5.3); (3) pregnant patients hospitalized for a respiratory concern were more likely to have a comorbidity or underlying conditions including asthma, hypertension, type 2 diabetes mellitus, autoimmune disease, and class III obesity; (4) 3 maternal deaths (1.3%) were attributed to coronavirus disease 2019 for a maternal mortality rate of 1250 of 100,000 pregnancies (95% confidence interval, 257-3653); (5) the coronavirus disease 2019 case fatality in pregnancy was a significant 13.6-fold (95% confidence interval, 2.7-43.6) higher in pregnant patients than in similarly aged individuals in Washington State with an absolute difference in mortality rate of 1.2% (95% confidence interval, -0.3 to 2.6); and (6) preterm birth was significantly higher among women with severe or critical coronavirus disease 2019 at delivery than for women who had recovered from coronavirus disease 2019 (45.4% severe or critical coronavirus disease 2019 vs 5.2% mild coronavirus disease 2019; P<.001). CONCLUSION: Coronavirus disease 2019 hospitalization and case-fatality rates in pregnant patients were significantly higher than in similarly aged adults in Washington State. These data indicate that pregnant patients are at risk of severe or critical disease and mortality compared to nonpregnant adults, and also at risk for preterm birth.

Filmless quality assurance of a Leksell Gamma Knife® Icon™.

PURPOSE: The annual quality assurance (QA) of Leksell Gamma Knife® (LGK) systems are typically performed using films. Film is a good candidate for small field dosimetry due to its high spatial resolution and availability. However, there are multiple challenges with using film; film does not provide real-time measurement and requires batch-specific calibration. Our findings show that active detector-based QA can simplify the procedure and save time without loss of accuracy. METHODS: Annual QA tests for a LGK Icon™ system were performed using both film-based and filmless techniques. Output calibration, relative output factors (ROF), radiation profiles, sector uniformity/source counting, and verification of the unit center point (UCP) and radiation focal point (RFP) coincidence tests were performed. Radiochromic films, two ionization chambers, and a synthetic diamond detector were used for the measurements. Results were compared and verified with the treatment planning system (TPS). RESULTS: The measured dose rate of the LGK Icon was within 0.4% of the TPS value set at the time of commissioning using an ionization chamber. ROF for the 8 and 4-mm collimators were found to be 0.3% and 1.8% different from TPS values using the MicroDiamond detector and 2.6% and 1.9% different for film, respectively. Excellent agreement was found between TPS and measured dose profiles using the MicroDiamond detector which was within 1%/1 mm vs 2%/1 mm for film. Sector uniformity was found to be within 1% for all eight sectors measured using an ionization chamber. Verification of UCP and RFP coincidence using the MicroDiamond detector and pinprick film test was within 0.3 mm at isocenter for both. CONCLUSION: The annual QA of a LGK Icon was successfully performed by employing filmless techniques. Comparable results were obtained using radiochromic films. Utilizing active detectors instead of films simplifies the QA process and saves time without loss of accuracy.

Development-specific transcriptomic profiling suggests new mechanisms for anoxic survival in the ventricle of overwintering turtles.

Oxygen deprivation swiftly damages tissues in most animals, yet some species show remarkable abilities to tolerate little or even no oxygen. Painted turtles exhibit a development-dependent tolerance that allows adults to survive anoxia approximately four times longer than hatchlings: adults survive ∼170 days and hatchlings survive ∼40 days at 3°C. We hypothesized that this difference is related to development-dependent differences in ventricular gene expression. Using a comparative ontogenetic approach, we examined whole transcriptomic changes before, during and 5 days after a 20-day bout of anoxic submergence at 3°C. Ontogeny accounted for more gene expression differences than treatment (anoxia or recovery): 1175 versus 237 genes, respectively. Of the 237 differences, 93 could confer protection against anoxia and reperfusion injury, 68 could be injurious and 20 may be constitutively protective. Most striking during anoxia was the main expression pattern of all 76 annotated ribosomal protein (R-protein) mRNAs, which decreased in anoxia-tolerant adults, but increased in anoxia-sensitive hatchlings, suggesting adult-specific regulation of translational suppression. These genes, along with 60 others that decreased their levels in adults and either increased or remained unchanged in hatchlings, implicate antagonistic pleiotropy as a mechanism to resolve the long-standing question about why hatchling painted turtles overwinter in terrestrial nests, rather than emerge and overwinter in water during their first year. In summary, developmental differences in the transcriptome of the turtle ventricle revealed potentially protective mechanisms that contribute to extraordinary adult-specific anoxia tolerance, and provide a unique perspective on differences between the anoxia-induced molecular responses of anoxia-tolerant and anoxia-sensitive phenotypes within a species.

Adaptive seasonal shift towards investment in fewer, larger offspring: Evidence from field and laboratory studies.

Seasonal changes in reproduction have been described for many taxa. As reproductive seasons progress, females often shift from greater energetic investment in many small offspring towards investing less total energy into fewer, better provisioned (i.e. larger) offspring. The underlying causes of this pattern have not been assessed in many systems. Two primary hypotheses have been proposed to explain these patterns. The first is an adaptive hypothesis from life-history theory: early offspring have a survival advantage over those produced later. Accordingly, selection favours females that invest in offspring quantity early in the season and offspring quality later. The second hypothesis suggests these patterns are not intrinsic but result from passive responses to seasonal changes in the environment experienced by reproducing females (i.e. maternal environment). To disentangle the causes underlying this pattern, which has been reported in brown anole lizards (Anolis sagrei), we performed complementary field and laboratory studies. The laboratory study carefully controlled maternal environments and quantified reproductive patterns throughout the reproductive season for each female. The field study measured similar metrics from free ranging lizards across an entire reproductive season. In the laboratory, females increased relative effort per offspring as the reproductive season progressed; smaller eggs were laid earlier, larger eggs were laid later. Moreover, we observed significant among-individual variation in seasonal changes in reproduction, which is necessary for traits to evolve via natural selection. Because these patterns consistently emerge under controlled laboratory conditions, they likely represent an intrinsic and potentially adaptive adjustment of reproductive effort as predicted by life-history theory. The field study revealed similar trends, further suggesting that intrinsic patterns observed in the laboratory are strong enough to persist despite the environmental variability that characterizes natural habitats. The observed patterns are indicative of an adaptive seasonal shift in parental investment in response to a deteriorating offspring environment: allocating greater resources to late-produced offspring likely enhances maternal fitness.

Alveolar Macrophage Apoptosis-associated Bacterial Killing Helps Prevent Murine Pneumonia.

Rationale: Antimicrobial resistance challenges therapy of pneumonia. Enhancing macrophage microbicidal responses would combat this problem but is limited by our understanding of how alveolar macrophages (AMs) kill bacteria. Objectives: To define the role and mechanism of AM apoptosis-associated bacterial killing in the lung. Methods: We generated a unique CD68.hMcl-1 transgenic mouse with macrophage-specific overexpression of the human antiapoptotic Mcl-1 protein, a factor upregulated in AMs from patients at increased risk of community-acquired pneumonia, to address the requirement for apoptosis-associated killing. Measurements and Main Results: Wild-type and transgenic macrophages demonstrated comparable ingestion and initial phagolysosomal killing of bacteria. Continued ingestion (for ≥12 h) overwhelmed initial killing, and a second, late-phase microbicidal response killed viable bacteria in wild-type macrophages, but this response was blunted in CD68.hMcl-1 transgenic macrophages. The late phase of bacterial killing required both caspase-induced generation of mitochondrial reactive oxygen species and nitric oxide, the peak generation of which coincided with the late phase of killing. The CD68.hMcl-1 transgene prevented mitochondrial reactive oxygen species but not nitric oxide generation. Apoptosis-associated killing enhanced pulmonary clearance of Streptococcus pneumoniae and Haemophilus influenzae in wild-type mice but not CD68.hMcl-1 transgenic mice. Bacterial clearance was enhanced in vivo in CD68.hMcl-1 transgenic mice by reconstitution of apoptosis with BH3 mimetics or clodronate-encapsulated liposomes. Apoptosis-associated killing was not activated during Staphylococcus aureus lung infection. Conclusions: Mcl-1 upregulation prevents macrophage apoptosis-associated killing and establishes that apoptosis-associated killing is required to allow AMs to clear ingested bacteria. Engagement of macrophage apoptosis should be investigated as a novel, host-based antimicrobial strategy.

Evaluation of a new secondary dose calculation software for Gamma Knife radiosurgery.

Current available secondary dose calculation software for Gamma Knife radiosurgery falls short in situations where the target is shallow in depth or when the patient is positioned with a gamma angle other than 90°. In this work, we evaluate a new secondary calculation software which utilizes an innovative method to handle nonstandard gamma angles and image thresholding to render the skull for dose calculation. 800 treatment targets previously treated with our GammaKnife Icon system were imported from our treatment planning system (GammaPlan 11.0.3) and a secondary dose calculation was conducted. The agreement between the new calculations and the TPS were recorded and compared to the original secondary dose calculation agreement with the TPS using a Wilcoxon Signed Rank Test. Further comparisons using a Mann-Whitney test were made for targets treated at a 90° gamma angle against those treated with either a 70 or 110 gamma angle for both the new and commercial secondary dose calculation systems. Correlations between dose deviations from the treatment planning system against average target depth were evaluated using a Kendall's Tau correlation test for both programs. The Wilcoxon Signed Rank Test indicated a significant difference in the agreement between the two secondary calculations and the TPS, with a P-value < 0.0001. With respect to patients treated at nonstandard gamma angles, the new software was largely independent of patient setup, while the commercial software showed a significant dependence (P-value < 0.0001). The new secondary dose calculation software showed a moderate correlation with calculation depth, while the commercial software showed a weak correlation (Tau = -.322 and Tau = -.217 respectively). Overall, the new secondary software has better agreement with the TPS than the commercially available secondary calculation software over a range of diverse treatment geometries.

Pneumolysin and the bacterial capsule of Streptococcus pneumoniae cooperatively inhibit taxis and motility of microglia.

BACKGROUND: Streptococcus pneumoniae is the cause of a highly lethal form of meningitis in humans. Microglial cells in the brain represent the first line of defense against pathogens, and they participate in the inflammatory response. The cholesterol-dependent cytolysin pneumolysin and the bacterial capsule are key pathogenic factors, known to exacerbate the course of pneumococcal meningitis. METHODS: We utilized live imaging and immunostaining of glial cells in dissociated and acute brain slice cultures to study the effect of pneumococcal factors, including the cholesterol-dependent cytolysin pneumolysin and the pneumococcal capsule, on microglial motility and taxis. RESULTS: In brain tissue, primary microglia cells showed an enhanced response towards lysates from bacteria lacking capsules and pneumolysin as they moved rapidly to areas with an abundance of bacterial factors. The presence of bacterial capsules and pneumolysin cumulatively inhibited microglial taxis. In mixed cultures of astrocytes and microglia, the motility of microglia was inhibited by capsular components within minutes after exposure. The reduced motility was partially reversed by mannan, a mannose receptor inhibitor. The effects on microglia were not mediated by astrocytes because pure microglial cells responded to various pneumococcal lysates similarly with distinct cell shape changes as seen in mixed cultures. CONCLUSIONS: Our data indicate that microglia possess the capacity for a very agile response towards bacterial pathogens, but key pathogenic factors, such as pneumococcal capsules and pneumolysin, inhibited this response shortly after a bacterial challenge. Furthermore, we demonstrate for the first time that the bacterial capsule affects cellular behaviors such as motility and taxis.

Do Covariances Between Maternal Behavior and Embryonic Physiology Drive Sex-Ratio Evolution Under Environmental Sex Determination?

Fisherian sex-ratio theory predicts sexual species should have a balanced primary sex ratio. However, organisms with environmental sex determination (ESD) are particularly vulnerable to experiencing skewed sex ratios when environmental conditions vary. Theoretical work has modeled sex-ratio dynamics for animals with ESD with regard to 2 traits predicted to be responsive to sex-ratio selection: 1) maternal oviposition behavior and 2) sensitivity of embryonic sex determination to environmental conditions, and much research has since focused on how these traits influence offspring sex ratios. However, relatively few studies have provided estimates of univariate quantitative genetic parameters for these 2 traits, and the existence of phenotypic or genetic covariances among these traits has not been assessed. Here, we leverage studies on 3 species of reptiles (2 turtle species and a lizard) with temperature-dependent sex determination (TSD) to assess phenotypic covariances between measures of maternal oviposition behavior and thermal sensitivity of the sex-determining pathway. These studies quantified maternal behaviors that relate to nest temperature and sex ratio of offspring incubated under controlled conditions. A positive covariance between these traits would enhance the efficiency of sex-ratio selection when primary sex ratio is unbalanced. However, we detected no such covariance between measures of these categories of traits in the 3 study species. These results suggest that maternal oviposition behavior and thermal sensitivity of sex determination in embryos might evolve independently. Such information is critical to understand how animals with TSD will respond to rapidly changing environments that induce sex-ratio selection.

Prognostic and Pathogenic Role of Angiopoietin-1 and -2 in Pneumonia.

RATIONALE: During pneumonia, pathogen-host interaction evokes inflammation and lung barrier dysfunction. Tie2 activation by angiopoietin-1 reduces, whereas Tie2 blockade by angiopoietin-2 increases, inflammation and permeability during sepsis. The role of angiopoietin-1/-2 in pneumonia remains unidentified. OBJECTIVES: To investigate the prognostic and pathogenic impact of angiopoietins in regulating pulmonary vascular barrier function and inflammation in bacterial pneumonia. METHODS: Serum angiopoietin levels were quantified in pneumonia patients of two independent cohorts (n = 148, n = 395). Human postmortem lung tissue, pneumolysin- or angiopoietin-2-stimulated endothelial cells, isolated perfused and ventilated mouse lungs, and mice with pneumococcal pneumonia were investigated. MEASUREMENTS AND MAIN RESULTS: In patients with pneumonia, decreased serum angiopoietin-1 and increased angiopoietin-2 levels were observed as compared with healthy subjects. Higher angiopoietin-2 serum levels were found in patients with community-acquired pneumonia who died within 28 days of diagnosis compared with survivors. Receiver operating characteristic analysis revealed improved prognostic accuracy of CURB-65 for 28-day survival, intensive care treatment, and length of hospital stay if combined with angiopoietin-2 serum levels. In vitro, pneumolysin enhanced endothelial angiopoietin-2 release, angiopoietin-2 increased endothelial permeability, and angiopoietin-1 reduced pneumolysin-evoked endothelial permeability. Ventilated and perfused lungs of mice with angiopoietin-2 knockdown showed reduced permeability on pneumolysin stimulation. Increased pulmonary angiopoietin-2 and reduced angiopoietin-1 mRNA expression were observed in Streptococcus pneumoniae-infected mice. Finally, angiopoietin-1 therapy reduced inflammation and permeability in murine pneumonia. CONCLUSIONS: These data suggest a central role of angiopoietin-1/-2 in pneumonia-evoked inflammation and permeability. Increased angiopoietin-2 serum levels predicted mortality and length of hospital stay, and angiopoietin-1 may provide a therapeutic target for severe pneumonia.

Characterization and validation of an intra-fraction motion management system for masked-based radiosurgery.

PURPOSE: Characterize the intra-fraction motion management (IFMM) system found on the Gamma Knife Icon (GKI), including spatial accuracy, latency, temporal performance, and overall effect on delivered dose. METHODS: A phantom was constructed, consisting of a three-axis translation mount, a remote motorized flipper, and a thermoplastic sphere surrounding a radiation detector. An infrared marker was placed on the translation mount secured to the flipper. The spatial accuracy of the IFMM was measured via the translation mount in all Cartesian planes. The detector was centered at the radiation focal point. A remote signal was used to move the marker out of the IFMM tolerance and pause the beam. A two-channel electrometer was used to record the signals from the detector and the flipper when motion was signaled. These signals determined the latency and temporal performance of the GKI. RESULTS: The spatial accuracy of the IFMM was found to be <0.1 mm. The measured latency was <200 ms. The dose difference with five interruptions was <0.5%. CONCLUSION: This work provides a quantitative characterization of the GKI IFMM system as required by the Nuclear Regulatory Commission. This provides a methodology for GKI users to satisfy these requirements using common laboratory equipment in lieu of a commercial solution.

Sphingosine Kinase 1 Regulates Inflammation and Contributes to Acute Lung Injury in Pneumococcal Pneumonia via the Sphingosine-1-Phosphate Receptor 2.

OBJECTIVES: Severe pneumonia may evoke acute lung injury, and sphingosine-1-phosphate is involved in the regulation of vascular permeability and immune responses. However, the role of sphingosine-1-phosphate and the sphingosine-1-phosphate producing sphingosine kinase 1 in pneumonia remains elusive. We examined the role of the sphingosine-1-phosphate system in regulating pulmonary vascular barrier function in bacterial pneumonia. DESIGN: Controlled, in vitro, ex vivo, and in vivo laboratory study. SUBJECTS: Female wild-type and SphK1-deficient mice, 8-10 weeks old. Human postmortem lung tissue, human blood-derived macrophages, and pulmonary microvascular endothelial cells. INTERVENTIONS: Wild-type and SphK1-deficient mice were infected with Streptococcus pneumoniae. Pulmonary sphingosine-1-phosphate levels, messenger RNA expression, and permeability as well as lung morphology were analyzed. Human blood-derived macrophages and human pulmonary microvascular endothelial cells were infected with S. pneumoniae. Transcellular electrical resistance of human pulmonary microvascular endothelial cell monolayers was examined. Further, permeability of murine isolated perfused lungs was determined following exposition to sphingosine-1-phosphate and pneumolysin. MEASUREMENTS AND MAIN RESULTS: Following S. pneumoniae infection, murine pulmonary sphingosine-1-phosphate levels and sphingosine kinase 1 and sphingosine-1-phosphate receptor 2 expression were increased. Pneumonia-induced lung hyperpermeability was reduced in SphK1 mice compared with wild-type mice. Expression of sphingosine kinase 1 in macrophages recruited to inflamed lung areas in pneumonia was observed in murine and human lungs. S. pneumoniae induced the sphingosine kinase 1/sphingosine-1-phosphate system in blood-derived macrophages and enhanced sphingosine-1-phosphate receptor 2 expression in human pulmonary microvascular endothelial cell in vitro. In isolated mouse lungs, pneumolysin-induced hyperpermeability was dose dependently and synergistically increased by sphingosine-1-phosphate. This sphingosine-1-phosphate-induced increase was reduced by inhibition of sphingosine-1-phosphate receptor 2 or its downstream effector Rho-kinase. CONCLUSIONS: Our data suggest that targeting the sphingosine kinase 1-/sphingosine-1-phosphate-/sphingosine-1-phosphate receptor 2-signaling pathway in the lung may provide a novel therapeutic perspective in pneumococcal pneumonia for prevention of acute lung injury.

Evidence of cardiac involvement in the fetal inflammatory response syndrome: disruption of gene networks programming cardiac development in nonhuman primates.

BACKGROUND: Most early preterm births are associated with intraamniotic infection and inflammation, which can lead to systemic inflammation in the fetus. The fetal inflammatory response syndrome describes elevations in the fetal interleukin-6 level, which is a marker for inflammation and fetal organ injury. An understanding of the effects of inflammation on fetal cardiac development may lead to insight into the fetal origins of adult cardiovascular disease. OBJECTIVE: The purpose of this study was to determine whether the fetal inflammatory response syndrome is associated with disruptions in gene networks that program fetal cardiac development. STUDY DESIGN: We obtained fetal cardiac tissue after necropsy from a well-described pregnant nonhuman primate model (pigtail macaque, Macaca nemestrina) of intrauterine infection (n=5) and controls (n=5). Cases with the fetal inflammatory response syndrome (fetal plasma interleukin-6 >11 pg/mL) were induced by either choriodecidual inoculation of a hypervirulent group B streptococcus strain (n=4) or intraamniotic inoculation of Escherichia coli (n=1). RNA and protein were extracted from fetal hearts and profiled by microarray and Luminex (Millipore, Billerica, MA) for cytokine analysis, respectively. Results were validated by quantitative reverse transcriptase polymerase chain reaction. Statistical and bioinformatics analyses included single gene analysis, gene set analysis, Ingenuity Pathway Analysis (Qiagen, Valencia, CA), and Wilcoxon rank sum. RESULTS: Severe fetal inflammation developed in the context of intraamniotic infection and a disseminated bacterial infection in the fetus. Interleukin-6 and -8 in fetal cardiac tissues were elevated significantly in fetal inflammatory response syndrome cases vs controls (P<.05). A total of 609 probe sets were expressed differentially (>1.5-fold change, P<.05) in the fetal heart (analysis of variance). Altered expression of select genes was validated by quantitative reverse transcriptase polymerase chain reaction that included several with known functions in cardiac injury, morphogenesis, angiogenesis, and tissue remodeling (eg, angiotensin I converting enzyme 2, STEAP family member 4, natriuretic peptide A, and secreted frizzled-related protein 4; all P<.05). Multiple gene sets and pathways that are involved in cardiac morphogenesis and vasculogenesis were downregulated significantly by gene set and Ingenuity Pathway Analysis (hallmark transforming growth factor beta signaling, cellular morphogenesis during differentiation, morphology of cardiovascular system; all P<.05). CONCLUSION: Disruption of gene networks for cardiac morphogenesis and vasculogenesis occurred in the preterm fetal heart of nonhuman primates with preterm labor, intraamniotic infection, and severe fetal inflammation. Inflammatory injury to the fetal heart in utero may contribute to the development of heart disease later in life. Development of preterm labor therapeutics must also target fetal inflammation to lessen organ injury and potential long-term effects on cardiac function.

Active release of pneumolysin prepores and pores by mammalian cells undergoing a Streptococcus pneumoniae attack.

BACKGROUND: Streptococcus pneumoniae is a potent human pathogen. Its pore-forming exotoxin pneumolysin is instrumental for breaching the host's epithelial barrier and for the incapacitation of the immune system. METHODS AND RESULTS: Using a combination of life imaging and cryo-electron microscopy we show that pneumolysin, released by cultured bacteria, is capable of permeabilizing the plasmalemma of host cells. However, such permeabilization does not lead to cell lysis since pneumolysin is actively removed by the host cells. The process of pore elimination starts with the formation of pore-bearing plasmalemmal nanotubes and proceeds by the shedding of pores that are embedded in the membrane of released microvesicles. Pneumolysin prepores are likewise removed. The protein composition of the toxin-induced microvesicles, assessed by mass spectrometry, is suggestive of a Ca(2+)-triggered mechanism encompassing the proteins of the annexin family and members of the endosomal sorting complex required for transport (ESCRT) complex. CONCLUSIONS: S. pneumoniae releases sufficient amounts of pneumolysin to perforate the plasmalemma of host cells, however, the immediate cell lysis, which is frequently reported as a result of treatment with purified and artificially concentrated toxin, appears to be an unlikely event in vivo since the toxin pores are efficiently eliminated by microvesicle shedding. Therefore the dysregulation of cellular homeostasis occurring as a result of transient pore formation/elimination should be held responsible for the damaging toxin action. GENERAL SIGNIFICANCE: We have achieved a comprehensive view of a general plasma membrane repair mechanism after injury by a major bacterial toxin.

Vasculotide reduces pulmonary hyperpermeability in experimental pneumococcal pneumonia.

BACKGROUND: Community-acquired pneumonia (CAP) is a significant cause of morbidity and mortality worldwide. Despite effective antimicrobial therapy, CAP can induce pulmonary endothelial hyperpermeability resulting in life-threatening lung failure due to an exaggerated host-pathogen interaction. Treatment of acute lung injury is mainly supportive because key elements of inflammation-induced barrier disruption remain undetermined. Angiopoietin-1 (Ang-1)-mediated Tie2 activation reduces, and the Ang-1 antagonist Ang-2 increases, inflammation and endothelial permeability in sepsis. Vasculotide (VT) is a polyethylene glycol-clustered Tie2-binding peptide that mimics the actions of Ang-1. The aim of our study was to experimentally test whether VT is capable of diminishing pneumonia-induced lung injury. METHODS: VT binding and phosphorylation of Tie2 were analyzed using tryptophan fluorescence spectroscopy and phospho-Tie-2 enzyme-linked immunosorbent assay. Human and murine lung endothelial cells were investigated by immunofluorescence staining and electric cell-substrate impedance sensing. Pulmonary hyperpermeability was quantified in VT-pretreated, isolated, perfused, and ventilated mouse lungs stimulated with the pneumococcal exotoxin pneumolysin (PLY). Furthermore, Streptococcus pneumoniae-infected mice were therapeutically treated with VT. RESULTS: VT showed dose-dependent binding and phosphorylation of Tie2. Pretreatment with VT protected lung endothelial cell monolayers from PLY-induced disruption. In isolated mouse lungs, VT decreased PLY-induced pulmonary permeability. Likewise, therapeutic treatment with VT of S. pneumoniae-infected mice significantly reduced pneumonia-induced hyperpermeability. However, effects by VT on the pulmonary or systemic inflammatory response were not observed. CONCLUSIONS: VT promoted pulmonary endothelial stability and reduced lung permeability in different models of pneumococcal pneumonia. Thus, VT may provide a novel therapeutic perspective for reduction of permeability in pneumococcal pneumonia-induced lung injury.

Reptile Embryos Lack the Opportunity to Thermoregulate by Moving within the Egg.

Historically, egg-bound reptile embryos were thought to passively thermoconform to the nest environment. However, recent observations of thermal taxis by embryos of multiple reptile species have led to the widely discussed hypothesis that embryos behaviorally thermoregulate. Because temperature affects development, such thermoregulation could allow embryos to control their fate far more than historically assumed. We assessed the opportunity for embryos to behaviorally thermoregulate in nature by examining thermal gradients within natural nests and eggs of the common snapping turtle (Chelydra serpentina; which displays embryonic thermal taxis) and by simulating thermal gradients within nests across a range of nest depths, egg sizes, and soil types. We observed little spatial thermal variation within nests, and thermal gradients were poorly transferred to eggs. Furthermore, thermal gradients sufficiently large and constant for behavioral thermoregulation were not predicted to occur in our simulations. Gradients of biologically relevant magnitude have limited global occurrence and reverse direction twice daily when they do exist, which is substantially faster than embryos can shift position within the egg. Our results imply that reptile embryos will rarely, if ever, have the opportunity to behaviorally thermoregulate by moving within the egg. We suggest that embryonic thermal taxis instead represents a play behavior, which may be adaptive or selectively neutral, and results from the mechanisms for behavioral thermoregulation in free-living stages coming online prior to hatching.