The impact of intermittent hypoxemia on type 1 retinopathy of prematurity in preterm infants.

BACKGROUND: Intermittent hypoxemia (IH) may influence retinopathy of prematurity (ROP) development in preterm infants, however, previous studies had mixed results. This study tests the hypothesis that increased IH is associated with Type 1 ROP; a stage beyond which treatment is indicated. METHODS: IH was quantified by continuously monitoring oxygen saturation (SpO2) using high-resolution pulse oximeters during the first 10 weeks of life. Statistical analyses assessed the relationship and predictive ability of weekly and cumulative IH for Type 1 ROP development. RESULTS: Most analyses showed no association between IH and Type 1 ROP adjusting for gestational age (GA) and birth weight (BW). However, cumulative IH of longer duration during weeks 5-10, 6-10, and 7-10 were significantly associated with Type 1 ROP adjusting for GA and BW, e.g., the adjusted odds ratio of Type 1 ROP was 2.01 (p = 0.03) for every 3.8 seconds increase in IH duration from week 6-10. IH did not provide statistically significant added predictive ability above GA and BW. CONCLUSIONS: For most analyses there was no significant association between IH and Type 1 ROP adjusting for GA and BW. However, infants with longer IH duration during the second month of life had higher risk for Type 1 ROP. IMPACT: The relationship and predictive ability of intermittent hypoxemia (IH) on retinopathy of prematurity (ROP) is controversial. This study shows no significant association between IH events and Type 1 ROP after adjusting for gestational age (GA) and birth weight (BW), except for cumulative IH of longer duration in the second month of life. In this cohort, IH does not provide a statistically significant improvement in ROP prediction over GA and BW. This study is the first to assess the cumulative impact of IH measures on Type 1 ROP. Interventions for reducing IH duration during critical postnatal periods may improve ROP outcomes.

The Impact of Intermittent Hypoxemia on Type 1 Retinopathy of Prematurity in Preterm Infants.

Background: Intermittent hypoxemia (IH) may influence retinopathy of prematurity (ROP) development in preterm infants, however, previous studies had mixed results. This study aims to assess the influence and evaluate the predictive ability of IH measures on Type 1 ROP, a stage beyond which ROP treatment is indicated. Methods: IH was quantified by continuously monitoring oxygen saturation (SpO2) using high-resolution pulse oximeters during the first 10 weeks of life. Statistical analyses assessed the relationship and predictive ability of weekly and cumulative IH variables for Type 1 ROP development. Results: Univariate analyses suggested that IH measures are greater in infants with Type 1 ROP and are predictive of Type 1 ROP development. Multivariable logistic regression analyses revealed that cumulative IH of longer duration during certain postnatal periods are associated with Type 1 ROP development after adjusting for gestational age (GA) or birth weight (BW). Although area under the curve (AUC) analyses revealed added predictivity of cumulative IH variables above GA or BW, these increments in AUC were not statistically significant. Conclusions: The duration of IH events was associated with Type 1 ROP development. Interventions for reducing the duration of IH events may potentially improve ROP outcomes.

Machine learning based classification of mitochondrial morphologies from fluorescence microscopy images of Toxoplasma gondii cysts.

The mitochondrion is intimately linked to energy and overall metabolism and therefore the morphology of mitochondrion can be very informative for inferring the metabolic state of cells. In this study we report an approach for automatic classification of mitochondrial morphologies using supervised machine learning to efficiently classify them from a large number of cells at a time. Fluorescence microscopy images of the chronic encysted form of parasite Toxoplasma gondii were used for this development. Manually classifying these morphologies from the hundreds of parasites within typical tissue cysts is tedious and error prone. In addition, because of inherent biological heterogeneity in morphologies, there can be variability and lack of reproducibility in manual classification. We used image segmentation to detect mitochondrial shapes and used features extracted from them in a multivariate logistic regression model to classify the detected shapes into five morphological classes: Blobs, Tadpoles, Lasso/Donuts, Arcs, and Other. The detected shapes from a subset of images were first used to obtain consensus classification among expert users to obtain a labeled set. The model was trained using the labeled set from five cysts and its performance was tested on the mitochondrial morphologies from ten other cysts that were not used in training. Results showed that the model had an average overall accuracy of 87%. There was high degree of confidence in the classification of Blobs and Arcs (average F scores 0.91 and 0.73) which constituted the majority of morphologies (85%). Although the current development used microscopy images from tissue cysts of Toxoplasma gondii, the approach is adaptable with minor adjustments and can be used to automatically classify morphologies of organelles from a variety of cells.

Computer Aided Image Processing to Facilitate Determination of Congruence in Manual Classification of Mitochondrial Morphologies in Toxoplasma gondii Tissue Cysts.

Toxoplasma gondii is a parasite that chronically infects about a third of the world's population. During chronic infection, the parasite resides within tissue cysts in the form of poorly understood bradyzoites which can number in the thousands. Our prior work showed that these bradyzoites are metabolically active exhibiting heterogeneous replication potential. The morphological plasticity of the mitochondrion potentially informs about parasite metabolic state. We developed an image processing based program to assist manual classification of mitochondrial morphologies by trained operators to collect data and statistics from the manual classification of shapes. We sought to determine whether certain morphologies were readily classifiable and the congruence among manual classifiers, i.e. the degree to which different operators would place the same objects within the same class. Results from three operators classifying mitochondrial morphologies from 5 tissue cyst images showed that among the four classes, one (Blobs) were the easiest to classify. There was remarkable congruence between 2 of the 3 operators in classifying the objects (96%), while the agreement among all 3 operators was somewhat modest (57%). Such information would be valuable for biologists studying these parasites as well as in development of fully automated methods of morphological classification.

The Effect of Severe Intraventricular Hemorrhage on the Biorhythms of Feeding in Premature Infants.

Background: Suck-swallow rhythmicity and the integration of breathing into infant feeding are developmentally regulated. Neurological injury and breathing abnormalities can both impact feeding in preterm infants. Objective: To determine the effects of neurologic injury independent of effects of disordered breathing on feeding biorhythms in premature infants. Methods: Low-risk preterm infants (LRP), infants with Grade 3-4 Intraventricular Hemorrhage (IVH), those with bronchopulmonary dysplasia (BPD), and those with both BPD and IVH (BPD+IVH) were identified. Forty-seven infants, 32-42 weeks Postmenstrual Age (PMA) were evaluated on one or more occasions (131 studies). Of these, 39 infants (81 studies) were performed at >35 weeks PMA. Coefficient of variation (COV) (=standard deviation of the inter-event (e.g., suck-suck, swallow-breath, etc.) interval divided by the mean of the interval) was used to quantify rhythmic stability. Results: To adjust for PMA, only those infants >35-42 weeks were compared. Suck-suck COV was significantly lower (more rhythmically stable) in the LRP group [COV = 0.274 ± 0.051 (S.D.)] compared to all other groups (BPD = 0.325 ± 0.066; IVH = 0.342 ± 0.072; BPD + IVH = 0.314 ± 0.069; all p < 0.05). Similarly, suck-swallow COV was significantly lower in LRP babies (0.360 ± 0.066) compared to the BPD group (0.475 ± 0.113) and the IVH cohort (0.428 ± 0.075) (p < 0.05). The BPD+IVH group (0.424 ± 0.109), while higher, was not quite statistically significant. Conclusions: Severe IVH negatively impacts suck-suck and suck-swallow rhythms. The independent effect of neurological injury in the form of IVH on feeding rhythms suggests that quantitative analysis of feeding may reflect and predict neurological sequelae.

Response to first dose of inhaled albuterol in mechanically ventilated preterm infants.

BACKGROUND: Bronchodilator responses among preterm infants are heterogeneous. Bedside measurements may identify responders. STUDY DESIGN: Respiratory measurements (Resistance, Compliance, FiO2) and pulse oximetry (SpO2) patterns were downloaded from infants <30 weeks gestational age during the first 2 months of life. Mechanically ventilated infants who received albuterol were included (n = 33). Measurements were compared before and after first albuterol. Secondary analyses assessed subsequent doses. RESULTS: Median gestation and birthweight were 25 3/7 weeks and 730 g, respectively. Mean Resistance decreased post-albuterol (p = 0.007). Sixty-eight percent of infants were responders based on decreased Resistance. Compliance and FiO2 did not significantly differ. Percent time in hypoxemia (SpO2 < 85%) decreased post albuterol (p < 0.02). In responders, Resistance changes diminished with subsequent administration (all p = 0.01). CONCLUSIONS: Ventilator resistance decreased in two-thirds of preterm infants, consistent with studies that utilized formal pulmonary function testing. Albuterol had a variable effect on delivered FiO2; however, hypoxemia may be useful in evaluating albuterol response.

Prematurity and postnatal alterations in intermittent hypoxaemia.

Intermittent hypoxaemia (IH) events are well described in extremely preterm infants, but the occurrence of IH patterns in more mature preterm infants remains unclear. The objective of this study was to characterise the effect of gestational age on early postnatal patterns of IH in extremely (<28 weeks), very (28-<32 weeks) and moderately (32-<34 weeks) preterm infants. As expected, extremely preterm infants had a significantly higher frequency of IH events of longer durations and greater time with hypoxaemia versus very and moderately preterm infants. In addition, the postnatal decrease in IH duration was comparable in the very and moderately preterm infants. This progression of IH events should assist clinicians and families in managing expectations for resolution of IH events during early postnatal life.

Intermittent Hypoxemia in Preterm Infants: A Potential Proinflammatory Process.

OBJECTIVE: A major consequence of prematurity is intermittent hypoxemia (IH). Data from both adult studies and neonatal animal models suggest that IH is proinflammatory; however, there is limited data in preterm infants. Here, we assess the relationship between IH and systemic inflammation, namely, serum C-reactive protein (CRP) in preterm infants. STUDY DESIGN: Serum CRP was measured at 30 days of life, at the time of peak IH frequency. IH measures (e.g., per cent time in hypoxemia, frequency, duration) were calculated the week prior to CRP collection. Statistical analyses were based on Spearman's correlation. RESULTS: A total of 26 infants were included. Median gestational age and birth weight were 274/7 weeks and 980 g, respectively. There were positive correlations between primary IH measures and CRP levels, especially for events longer than 1-minute duration (r range: 0.56-0.74, all p < 0.01). CONCLUSION: We demonstrate that IH is associated with increased CRP for the first time in preterm infants. Our findings are consistent with studies from adults and neonatal animal models suggesting that IH is a proinflammatory process. KEY POINTS: · IH events are common.. · IH is associated with elevated C-reactive protein.. · Longer IH events (>1 min) are of most significance..

Co-occurrence and phase relationship between alternans of the R wave amplitude (RWAA) and of the T wave (TWA) in ECGs.

Alternans of the T wave in ECG (TWA) has high negative but poor positive predictive value in the prediction of ventricular arrhythmia. Alternans of repolarization duration, i.e. of action potential duration (APD), causes TWA. Prior studies from our group showed that alternans of the maximum rate of depolarization also occurs when APD alternans occurs and the relationship between these two has the potential to affect formation of spatial discord, which may be more arrhythmogenic. Therefore, exploration of the co-occurrence of the alternans of depolarization and repolarization has the potential to improve the prediction. In the present study, we used a mathematical model to show that depolarization alternans appears as alternating amplitude of the R wave in the ECG. We also investigated the link between changes in R wave amplitude and TWA. Results from clinical grade ECGs available in the PhysioNet database show that amplitude of the R wave can change as predicted by our experimental results and the mathematical model. Using TWA as the marker of repolarization alternans and R wave amplitude alternans (RWAA) as the marker of depolarization alternans, we investigated the phase relation between them and observed that, similar to previous results from animals, the phase relation between the two can spontaneously change. That is, alternans of depolarization does co-occur with TWA and the phase relationship between the two is not invariant. These results support further investigation of the use of RWAA as a complementary method to TWA to improve positive predictive value for prediction of ventricular arrhythmia.

Soft Palate Modification Using a Collagen Crosslinking Reagent for Equine Dorsal Displacement of the Soft Palate and Other Upper Airway Breathing Disorders.

The mechanical properties of the soft palate can be associated with breathing abnormalities. Dorsal displacement of the soft palate (DDSP) is a naturally occurring equine soft palate disorder caused by displacement of the caudal edge of the soft palate. Snoring and a more serious, sometimes life-threatening, condition called obstructive sleep apnea (OSA) are forms of sleep-related breathing disorders in humans which may involve the soft palate. The goal of this study was to investigate the effect of injecting the protein crosslinker genipin into the soft palate to modify its mechanical properties for the treatment of equine DDSP with potential implications for the treatment of snoring and OSA in humans. Ex vivo experiments consisted of mechanical testing and a wind tunnel study to examine the effect of genipin on the mechanical properties, displacement, and vibration of equine soft palates. A pilot in vivo study was completed using DDSP and control horses to test the safety and effectiveness of injecting a genipin reagent into the soft palate. The wind tunnel testing demonstrated a greater than 50% decrease in transient deformation and a greater than 33% decrease in steady-state vibrations for all doses of genipin tested. Ultimate tensile stress, yield stress, and Young's modulus were higher in the genipin-treated distal soft palate specimens by 52%, 53%, and 63%, respectively. The pilot in vivo study showed a reduction of snoring loudness in all DDSP horses and elimination of DDSP in at least one of three horses. The difficulty of using a 1-meter-long endoscopic injection needle contributed to a consistent overinjection of the equine soft palates, causing excessive stretching (pillowing) and related degradation of the tissue. These ex vivo and in vivo results demonstrated reduced vibration amplitude and flaccidity and increased strength of genipin-treated soft palates, suggesting that genipin crosslinking could become an effective and safe treatment for soft palate related breathing abnormalities.

Effects of Bronchopulmonary Dysplasia on Swallow:Breath Interaction and Phase of Respiration with Swallow During Non-nutritive Suck.

OBJECTIVES: The objective of this study is to describe swallow:breath interaction (SwBr) and phase of respiration incident to swallow (POR) during non-nutritive suck in infants with bronchopulmonary dysplasia and determine if speech-language intervention can modify the characteristics of non-nutritive suck in these infants. METHODS: Logistic regression models were used to describe SwBr and POR in 16 low-risk preterm (LRP) infants and 43 infants with bronchopulmonary dysplasia. Infants with bronchopulmonary dysplasia were randomized to receive individualized intervention from a speech-language pathologist (BPDwithTX) or standard care (BPDnoTX). RESULTS: No significant differences were noted between low-risk infants and either group of BPD infants for the distribution of SwBr types. Infants with bronchopulmonary dysplasia showed minor differences in the progression of POR. Speech-Language intervention did not change the progression of SwBr or POR in infants with bronchopulmonary dysplasia. CONCLUSION: Infants with bronchopulmonary dysplasia can improve the progression of SwBr through practice as effectively as low-risk preterm infants can. The minor differences in POR in infants with bronchopulmonary dysplasia are consistent with dysmature development as seen with other feeding studies of infants with this disease. Speech-Language intervention did not modify the developmental progression of swallow:breath interaction or phase of respiration incident to swallow.

Phase Relation between Depolarization and Repolarization Alternans in ECG.

T-Wave Alternans (TWA) in the electro cardiogram (ECG) has been widely investigated as a potential predictor of ventricular arrhythmia. However, large clinical trials show that TWA has a high negative predictive value (NPV) but poor positive predictive value (PPV). Therefore, there is need for exploration of approaches to improve PPV of TWA. More recent studies suggest that whether alternans is spatially concordant or discordant affects arrhythmic potential. Results of our previous animal and simulation studies show that the phase relation between depolarization and repolarization alternans has an effect on the transition of concordant to discordant alternans. Towards the eventual goal of developing indexes that complement TWA and improve prediction of arrhythmia, the objectives in this study were to verify the existence of R wave amplitude alternans (RWAA, a surrogate of depolarization alternans) and investigate the phase relationship between RWAA and TWA in clinical grade ECGs. Results show that RWAA does occur in ECGs and that the phase relationship between RWAA and TWA can be labile. These results support further investigation of the co-occurrence of these alternans for prediction of arrhythmic events.

Eigen Decomposition of Cardiac Synchronous EEGs for Investigation of Neural Effects of Tempo and Cognition of Songs.

There is growing evidence of palliative effects of listening to songs on neural and cardiovascular function. It is also known that listening to songs can entrain cardiac variability. These results suggest that the neural changes in response to listening to songs in turn affect cardiac rhythm. How these effects come about is less clearly known. Therefore, investigation of the changes in neural rhythms that are synchronous with cardiac rhythm is likely to shed further light on the mechanisms via which songs produce these effects. Towards this aim, we conducted eigen decomposition of cardiac-synchronized EEGs to investigate the effects of tempo and cognition by auditory stimuli (listening to songs). For evaluating the effects of tempo, songs of slow and fast tempo were used, and for cognition, each subjects' favorite song was used. ECG and six EEGs (F3, F4, T3, T4, P3, P4) were recorded as subjects listened to songs. For cardiac synchronization, R waves from the ECG were localized and the EEGs during 300-millisecond segments ending at each R wave were extracted. Eigen decomposition of the covariance matrix of these EEG segments was performed. Results from 14 subject showed that, compared with other locations, P3 appears to have the ability to discriminate between songs. All songs lowered the second and the third largest eigenvalues compared to control, among these, the slow tempo song induced more significant decreases in T3, T4 and P3. During the slow song, 80% of the variance in all six EEGs could be represented with less eigenvalue/vectors while during the favorite song this number was larger. These results show that eigen decomposition of cardiac synchronized EEGs has the potential to investigate effects of music on neural and cardiovascular systems.

Baroreflex Sensitivity During Listening to Music Computed from Time Domain Sequences and Frequency Domain Transfer Function.

Listening to music has been known to affect autonomic function of cardiovascular regulation. Baroreflex is a feedback control loop that uses rate changes of the heart in order to regulate beat by beat changes in blood pressure (BP). In this study, we used two approaches to compute measures of sensitivity of the baroreflex (BRS), a time domain sequence approach and frequency domain transfer functions. Subjects listened to slow and fast tempo songs during the study. Electrocardiogram (ECG) and non-invasive continuous BP were recorded in 14 subjects (7 males and females). From these signals, either beat by beat or equi-sampled in time RR intervals and systolic BP (SBP) were computed. BRS was then estimated using RR and SBP. Our results show that the sequence method consistently provided higher values of BRS than the transfer function method (up to two fold). The two measures were reasonably well correlated $( \mathrm {R}>0.84)$ during control and the slow song, but not during the fast song. The BRS was lower $( \sim 20$%) than control when listening to fast songs $( \mathrm {p}<0.005)$. These results show the effects of listening to songs on BRS changes, but also show that the two methods to estimate BRS, although reasonably correlated, do not always provide similar estimates of BRS.

Blood transfusions in preterm infants: changes on perfusion index and intermittent hypoxemia.

BACKGROUND: Red blood cell (RBC) transfusion decreases intermittent hypoxemia (IH) events beyond the first week of life. This benefit may be related to improved perfusion to the respiratory control network. Perfusion index (PI) is a perfusion measure provided by the pulse oximeter. We hypothesized that the benefit in IH after RBC transfusion is associated with an increase in PI. In addition, we assessed the value of PI and clinical measures in predicting the effect of RBC transfusion on IH. STUDY DESIGN AND METHODS: We prospectively enrolled infants less than 30 weeks' gestation age. PI and oxygen saturation (SpO2 ) were monitored with high-resolution pulse oximeters 24 hours before and after RBC transfusion. Data were analyzed at three postnatal periods: Epoch 1, first week of life (1 to 7 days of life); Epoch 2, 2 to 4 weeks of life (8 to 28 days of life); and Epoch 3, 4 to 8 weeks of life. RESULTS: A total of 118 transfusions were analyzed. IH measures significantly decreased after transfusion in Epochs 2 and 3. PI significantly increased after transfusion, but it did not correlate with the decrease in IH measures. Mechanical ventilation, fraction of inspired oxygen (FiO2 ), and IH measures influenced the effects on oxygenation. CONCLUSIONS: RBC transfusion improved IH after the first week of life. The benefit in IH did not correlate with PI increase after transfusion. Pretransfusion respiratory support and IH measures predicted the effect of transfusion on oxygenation.

Prenatal Opioid Exposure and Intermittent Hypoxemia in Preterm Infants: A Retrospective Assessment.

INTRODUCTION: Intermittent hypoxemia (IH) is defined as episodic drops in oxygen saturation (SpO2). Preterm infants are at increased risk for IH due to their immature respiratory control/apnea of prematurity. The clinical relevance of IH is a relatively new observation with rising evidence linking IH to neonatal morbidities and long-term impairment. Hence, assessing factors that influence IH in preterm infants is imperative. Given the epidemic of opioid misuse in the USA, there is an urgent need to understand the impact of prenatal opioid exposure on neonatal outcomes. Hence, we wanted to assess the relationship between isolated prenatal opioid exposure and IH in preterm infants. METHODS: In order to accurately calculate IH, SpO2 data were prospectively collected using high-resolution pulse oximeters during the first 8 weeks of life in preterm infants less than 30 weeks gestational age. Data related to prenatal opioid misuse were retrospectively collected from medical charts. Infants with tobacco or poly-drug exposure were excluded. The primary outcome measure is percent time spent with SpO2 below 80% (%time-SpO2 < 80). The secondary outcome measure is the number of severe IH events/week with SpO2 less than 80% (IH-SpO2 < 80). RESULTS: A total of 82 infants with isolated opioid exposure (n = 14) or who were unexposed (n = 68) were included. There were no significant differences in baseline characteristics between opioid exposed and unexposed groups. There was a statistically significant increase of 0.23 (95% CI: 0.03, 0.43, p = 0.03) in mean of the square root of %time-SpO2 < 80. The number of IH-SpO2 < 80 events was higher in the opioid exposed group (mean difference = 2.95, 95% CI: -0.35, 6.25, p-value = 0.08), although statistical significance was not quite attained. CONCLUSION: This study shows that preterm infants prenatally exposed to opioids have increased IH measures compared to unexposed infants. Interestingly, the increased IH in the opioid exposed group persists beyond the immediate postnatal period.

Relationship between perfusion index and patent ductus arteriosus in preterm infants.

BACKGROUND: Perfusion index (PI) is a noninvasive measure of perfusion. ΔPI (difference between pre- and postductal PI) may identify hemodynamically significant PDA. However, studies are limited to brief and intermittent ΔPI sampling. Our objective is to assess the value of continuous high resolution ΔPI monitoring in the diagnosis of PDA. METHODS: Continuous ΔPI monitoring in preterm infants was prospectively performed using two high-resolution pulse oximeters. Perfusion Index measures (ΔPI mean and variability, pre- and postductal PI) were analyzed over a 4-h period prior to echocardiography. A cardiologist blinded to the results evaluated for PDA on echocardiography. Linear mixed regression models were utilized for analyses. RESULTS: We obtained 31 echocardiography observations. Mean ΔPI (-0.23 vs. 0.16; P < 0.05), mean pre-PI (0.86 vs. 1.26; P < 0.05), and ΔPI variability (0.39 vs. 0.61; P = 0.05) were lower in infants with PDA compared to infants without PDA at the time of echocardiography. CONCLUSION: Mean ΔPI, ΔPI variability, and mean pre-PI measured 4 h prior to echocardiography detect PDA in preterm infants. PI is dynamic and should be assessed continuously. Perfusion index is a promising bedside measurement to identify PDA in preterm infants.

Reexamining Chronic Toxoplasma gondii Infection: Surprising Activity for a "Dormant" Parasite.

PURPOSE OF REVIEW: Despite over a third of the world's population being chronically infected with Toxoplasma gondii, little is known about this largely asymptomatic phase of infection. This stage is mediated in vivo by bradyzoites within tissue cysts. The absence of overt symptoms has been attributed to the dormancy of bradyzoites. In this review, we reexamine the conventional view of chronic toxoplasmosis in light of emerging evidence challenging both the nature of dormancy and the consequences of infection in the CNS. RECENT FINDINGS: New and emerging data reveal a previously unrecognized level of physiological and replicative capacity of bradyzoites within tissue cysts. These findings have emerged in the context of a reexamination of the chronic infection in the brain that correlates with changes in neuronal architecture, neurochemistry, and behavior that suggest that the chronic infection is not without consequence. SUMMARY: The emerging data driven by the development of new approaches to study the progression of chronic toxoplasma infection reveals significant physiological and replicative capacity for what has been viewed as a dormant state. The emergence of bradyzoite and tissue cyst biology from what was viewed as a physiological "black box" offers exciting new areas for investigation with direct implications on the approaches to drug development targeting this drug-refractory state. In addition, new insights from studies on the neurobiology on chronic infection reveal a complex and dynamic interplay between the parasite, brain microenvironment, and the immune response that results in the detente that promotes the life-long persistence of the parasite in the host.

Novel Approaches Reveal that Toxoplasma gondii Bradyzoites within Tissue Cysts Are Dynamic and Replicating Entities In Vivo.

UNLABELLED: Despite their critical role in chronic toxoplasmosis, the biology of Toxoplasma gondii bradyzoites is poorly understood. In an attempt to address this gap, we optimized approaches to purify tissue cysts and analyzed the replicative potential of bradyzoites within these cysts. In order to quantify individual bradyzoites within tissue cysts, we have developed imaging software, BradyCount 1.0, that allows the rapid establishment of bradyzoite burdens within imaged optical sections of purified tissue cysts. While in general larger tissue cysts contain more bradyzoites, their relative "occupancy" was typically lower than that of smaller cysts, resulting in a lower packing density. The packing density permits a direct measure of how bradyzoites develop within cysts, allowing for comparisons across progression of the chronic phase. In order to capture bradyzoite endodyogeny, we exploited the differential intensity of TgIMC3, an inner membrane complex protein that intensely labels newly formed/forming daughters within bradyzoites and decays over time in the absence of further division. To our surprise, we were able to capture not only sporadic and asynchronous division but also synchronous replication of all bradyzoites within mature tissue cysts. Furthermore, the time-dependent decay of TgIMC3 intensity was exploited to gain insights into the temporal patterns of bradyzoite replication in vivo. Despite the fact that bradyzoites are considered replicatively dormant, we find evidence for cyclical, episodic bradyzoite growth within tissue cysts in vivo. These findings directly challenge the prevailing notion of bradyzoites as dormant nonreplicative entities in chronic toxoplasmosis and have implications on our understanding of this enigmatic and clinically important life cycle stage. IMPORTANCE: The protozoan Toxoplasma gondii establishes a lifelong chronic infection mediated by the bradyzoite form of the parasite within tissue cysts. Technical challenges have limited even the most basic studies on bradyzoites and the tissue cysts in vivo. Bradyzoites, which are viewed as dormant, poorly replicating or nonreplicating entities, were found to be surprisingly active, exhibiting not only the capacity for growth but also previously unrecognized patterns of replication that point to their being considerably more dynamic than previously imagined. These newly revealed properties force us to reexamine the most basic questions regarding bradyzoite biology and the progression of the chronic phase of toxoplasmosis. By developing new tools and approaches to study the chronic phase at the level of bradyzoites, we expose new avenues to tackle both drug development and a better understanding of events that may lead to reactivated symptomatic disease.

The cardiomyocyte molecular clock regulates the circadian expression of Kcnh2 and contributes to ventricular repolarization.

BACKGROUND: Sudden cardiac death (SCD) follows a diurnal variation. Data suggest the timing of SCD is influenced by circadian (~24-hour) changes in neurohumoral and cardiomyocyte-specific regulation of the heart's electrical properties. The basic helix-loop-helix transcription factors brain muscle arnt-like1 (BMAL1) and circadian locomotor output control kaput (CLOCK) coordinate the circadian expression of select genes. OBJECTIVE: We sought to test whether Bmal1 expression in cardiomyocytes contributes to K(+) channel expression and diurnal changes in ventricular repolarization. METHODS: We used transgenic mice that allow for the inducible cardiomyocyte-specific deletion of Bmal1 (iCSΔBmal1(-/-)). We used quantitative polymerase chain reaction, voltage clamping, promoter-reporter bioluminescence assays, and electrocardiographic telemetry. RESULTS: Although several K(+) channel gene transcripts were downregulated in iCSΔBmal1(-/-)mouse hearts, only Kcnh2 exhibited a robust circadian pattern of expression that was disrupted in iCSΔBmal1(-/-) hearts. Kcnh2 underlies the rapidly activating delayed-rectifier K(+) current, and the rapidly activating delayed-rectifier K(+) current recorded from iCSΔBmal1(-/-) ventricular cardiomyocytes was ~50% smaller than control ventricular myocytes. Promoter-reporter assays demonstrated that the human Kcnh2 promoter is transactivated by the coexpression of BMAL1 and CLOCK. Electrocardiographic analysis showed that iCSΔBmal1(-/-) mice developed a prolongation in the heart rate-corrected QT interval during the light (resting) phase. This was secondary to an augmented circadian rhythm in the uncorrected QT interval without a corresponding change in the RR interval. CONCLUSION: The molecular clock in the heart regulates the circadian expression of Kcnh2, modifies K(+) channel gene expression, and is important for normal ventricular repolarization. Disruption of the cardiomyocyte circadian clock mechanism likely unmasks diurnal changes in ventricular repolarization that could contribute to an increased risk of cardiac arrhythmias/SCD.