Oscillatory Coupling Between Neural and Cardiac Rhythms.

Oscillations serve a critical role in organizing biological systems. In the brain, oscillatory coupling is a fundamental mechanism of communication. The possibility that neural oscillations interact directly with slower physiological rhythms (e.g., heart rate, respiration) is largely unexplored and may have important implications for psychological functioning. Oscillations in heart rate, an aspect of heart rate variability (HRV), show remarkably robust associations with psychological health. Mather and Thayer proposed coupling between high-frequency HRV (HF-HRV) and neural oscillations as a mechanism that partially accounts for such relationships. We tested this hypothesis by measuring phase-amplitude coupling between HF-HRV and neural oscillations in 37 healthy adults at rest. Robust coupling was detected in all frequency bands. Granger causality analyses indicated stronger heart-to-brain than brain-to-heart effects in all frequency bands except gamma. These findings suggest that cardiac rhythms play a causal role in modulating neural oscillations, which may have important implications for mental health.

Suppressed calcineurin-dependent gene expression identifies lung allograft recipients at increased risk of infection.

Lung transplant immunosuppression regimens generally include the calcineurin inhibitor tacrolimus. We hypothesized that mean residual expression (MRE) of calcineurin-dependent genes assesses rejection and infection risk better than does tacrolimus trough. We prospectively followed 44 lung allograft recipients at 2 to 18 months posttransplant and measured changes in whole blood interleukin-2, interferon-γ, and granulocyte-macrophage colony-stimulating factor gene expression following a tacrolimus dose. Posttransplant duration, immunosuppressive medication levels, and bronchoscopic rejection and infection assessments were compared with MRE by using generalized-estimating equation-adjusted models. Prednisolone effect on MRE was assessed ex vivo in blood samples from nontransplanted controls. Tacrolimus concentration inhibiting 50% of cytokine production (IC50 ) was measured in a pretransplant subset. Results showed that MRE did not change with diagnosis of rejection but that airway infection was associated with a 20% absolute decrease (95% confidence interval 11%-29%). MRE increased with time after transplant but was not associated with tacrolimus trough. Interestingly, MRE correlated inversely with corticosteroid dose in the study cohort and ex vivo. Pretransplant tacrolimus IC50 depended on the cytokine measured and varied between individuals, suggesting a range in baseline responses to tacrolimus. We conclude that MRE identifies infection risk in lung allograft recipients, potentially integrating calcineurin inhibitor and steroid effects on lymphocyte effector function.

Immigrants in immunology: the benefits of lax borders.

The field of immunology has a long history of illuminating fundamental biological processes of critical importance to human health. From an outsider's perspective, the questions are profoundly philosophical and the experimental approaches are elegantly precise. Yet immunology can also appear impenetrable. Here we recount the experience of two graduate students from the fields of ecology and computer science, who have immigrated into immunological terrain attracted by systems-level questions. We argue that such migrations enrich the field of immunology, and that cultural and institutional changes are needed to promote more interdisciplinary explorations.

Spaceflight alters expression of microRNA during T-cell activation.

Altered immune function has been demonstrated in astronauts during spaceflights dating back to Apollo and Skylab; this could be a major barrier to long-term space exploration. We tested the hypothesis that spaceflight causes changes in microRNA (miRNA) expression. Human leukocytes were stimulated with mitogens on board the International Space Station using an onboard normal gravity control. Bioinformatics showed that miR-21 was significantly up-regulated 2-fold during early T-cell activation in normal gravity, and gene expression was suppressed under microgravity. This was confirmed using quantitative real-time PCR (n = 4). This is the first report that spaceflight regulates miRNA expression. Global microarray analysis showed significant (P < 0.05) suppression of 85 genes under microgravity conditions compared to normal gravity samples. EGR3, FASLG, BTG2, SPRY2, and TAGAP are biologically confirmed targets and are co-up-regulated with miR-21. These genes share common promoter regions with pre-mir-21; as the miR-21 matures and accumulates, it most likely will inhibit translation of its target genes and limit the immune response. These data suggest that gravity regulates T-cell activation not only by transcription promotion but also by blocking translation via noncoding RNA mechanisms. Moreover, this study suggests that T-cell activation itself may induce a sequence of gene expressions that is self-limited by miR-21.

Spaceflight and simulated microgravity cause a significant reduction of key gene expression in early T-cell activation.

Healthy immune function depends on precise regulation of lymphocyte activation. During the National Aeronautics and Space Administration (NASA) Apollo and Shuttle eras, multiple spaceflight studies showed depressed lymphocyte activity under microgravity (µg) conditions. Scientists on the ground use two models of simulated µg (sµg): 1) the rotating wall vessel (RWV) and 2) the random positioning machine (RPM), to study the effects of altered gravity on cell function before advancing research to the true µg when spaceflight opportunities become available on the International Space Station (ISS). The objective of this study is to compare the effects of true µg and sµg on the expression of key early T-cell activation genes in mouse splenocytes from spaceflight and ground animals. For the first time, we compared all three conditions of microgravity spaceflight, RPM, and RWV during immune gene activation of Il2, Il2rα, Ifnγ, and Tagap; moreover, we confirm two new early T-cell activation genes, Iigp1 and Slamf1. Gene expression for all samples was analyzed using quantitative real-time PCR (qRT-PCR). Our results demonstrate significantly increased gene expression in activated ground samples with suppression of mouse immune function in spaceflight, RPM, and RWV samples. These findings indicate that sµg models provide an excellent test bed for scientists to develop baseline studies and augment true µg in spaceflight experiments. Ultimately, sµg and spaceflight studies in lymphocytes may provide insight into novel regulatory pathways, benefiting both future astronauts and those here on earth suffering from immune disorders.

Cutting edge: NKG2D-dependent cytotoxicity is controlled by ligand distribution in the target cell membrane.

Although the importance of membrane microdomains in receptor-mediated activation of lymphocytes has been established, much less is known about the role of receptor ligand distribution on APC and target cells. Detergent-resistant membrane domains, into which GPI-linked proteins partition, are enriched in cholesterol and glycosphingolipids. ULBP1 is a GPI-linked ligand for natural cytotoxicity receptor NKG2D. To investigate how ULBP1 distribution on target cells affects NKG2D-dependent NK cell activation, we fused the extracellular domain of ULBP1 to the transmembrane domain of CD45. Introduction of this transmembrane domain eliminated the association of ULBP1 with the detergent-resistant membrane fraction and caused a significant reduction of cytotoxicity and degranulation by NK cells. Clustering and lateral diffusion of ULBP1 was not affected by changes in the membrane anchor. These results show that the partitioning of receptor ligands in discrete membrane domains of target cells is an important determinant of NK cell activation.

The Mammalian Family of Katanin Microtubule-Severing Enzymes.

The katanin family of microtubule-severing enzymes is critical for cytoskeletal rearrangements that affect key cellular processes like division, migration, signaling, and homeostasis. In humans, aberrant expression, or dysfunction of the katanins, is linked to developmental, proliferative, and neurodegenerative disorders. Here, we review current knowledge on the mammalian family of katanins, including an overview of evolutionary conservation, functional domain organization, and the mechanisms that regulate katanin activity. We assess the function of katanins in dividing and non-dividing cells and how their dysregulation promotes impaired ciliary signaling and defects in developmental programs (corticogenesis, gametogenesis, and neurodevelopment) and contributes to neurodegeneration and cancer. We conclude with perspectives on future katanin research that will advance our understanding of this exciting and dynamic class of disease-associated enzymes.

Mesenchymal Stem Cell Regulation of Macrophage Phagocytosis; Quantitation and Imaging.

Mesenchymal stem cells (MSC) have traditionally been studied for their regenerative properties, but more recently, their immunoregulatory characteristics have been at the forefront. They interact with and regulate immune cell activity. The focus of this study is the MSC regulation of macrophage phagocytic activity. Macrophage (MΦ) phagocytosis is an important part of the innate immune system response to infection, and the mechanisms through which MSC modulate this response are under active investigation. Presented here is a method to study MΦ phagocytosis of non-opsonized zymosan particles conjugated to a pH-sensitive fluorescent molecule while in co-culture with MSC. As phagocytic activity increases and the labeled zymosan particles are enclosed within the acidic environment of the phagolysosome, the fluorescence intensity of the pH-sensitive molecule increases. With the appropriate excitation and emission wavelengths, phagocytic activity is measured using a fluorescent spectrophotometer and kinetic data is presented as changes in relative fluorescent units over a 70 min period. To support this quantitative data, the change in the phagocytic activity is visualized using dynamic imaging. Results using this method demonstrate that when in co-culture, MSC enhance MΦ phagocytosis of non-opsonized zymosan of both naive and IFN-γ treated MΦ. These data add to the current knowledge of MSC regulation of the innate immune system. This method can be applied in future investigations to fully delineate the underlying cellular and molecular mechanisms.

Evaluation of a health information exchange system for microcephaly case-finding - New York City, 2013-2015.

BACKGROUND: Birth defects surveillance in the United States is conducted principally by review of routine but lagged reporting to statewide congenital malformations registries of diagnoses by hospitals or other health care providers, a process that is not designed to rapidly detect changes in prevalence. Health information exchange (HIE) systems are well suited for rapid surveillance, but information is limited about their effectiveness at detecting birth defects. We evaluated HIE data to detect microcephaly diagnosed at birth during January 1, 2013-December 31, 2015 before known introduction of Zika virus in North America. METHODS: Data from an HIE system were queried for microcephaly diagnostic codes on day of birth or during the first two days after birth at three Bronx hospitals for births to New York City resident mothers. Suspected cases identified by HIE data were compared with microcephaly cases that had been identified through direct inquiry of hospital records and confirmed by chart abstraction in a previous study of the same cohort. RESULTS: Of 16,910 live births, 43 suspected microcephaly cases were identified through an HIE system compared to 67 confirmed cases that had been identified as part of the prior study. A total of 39 confirmed cases were found by both studies (sensitivity = 58.21%, 95% CI: 45.52-70.15%; positive predictive value = 90.70%, 95% CI: 77.86-97.41%; negative predictive value = 99.83%, 95% CI: 99.76-99.89% for HIE data). CONCLUSION: Despite limitations, HIE systems could be used for rapid newborn microcephaly surveillance, especially in the many jurisdictions where more labor-intensive approaches are not feasible. Future work is needed to improve electronic medical record documentation quality to improve sensitivity and reduce misclassification.

Tacrolimus trough monitoring guided by mass spectrometry without accounting for assay differences is associated with acute kidney injury in lung transplant recipients.

PURPOSE: Tacrolimus is a nephrotoxic immunosuppressant historically monitored via enzyme-based immunoassay (IA). After 2011, the 2 largest laboratory companies in the United States implemented tacrolimus quantification by liquid chromatography-mass spectrometry (LC-MS); this method excludes metabolites, potentially resulting in lower quantified drug concentrations. We sought to determine if tacrolimus therapeutic drug monitoring via LC-MS, as performed using trough targets originally derived from IA values, influences clinical outcomes. METHODS: In a single-center retrospective cohort study of lung transplant recipients, risks of acute kidney injury, acute renal failure, and new-onset diabetes after transplantation, as well as chronic lung allograft dysfunction-free survival, were compared in 82 subjects monitored by LC-MS and 102 subjects monitored by IA using Cox proportional hazard models adjusted for age, sex, baseline renal function, and race. RESULTS: LC-MS-based monitoring was associated with a greater risk of acute kidney injury (adjusted hazard ratio, 1.65; 95% confidence interval, 1.02-2.67). No statistically significant differences in risks of acute renal failure and new-onset diabetes after transplantation were observed. CONCLUSION: Although LC-MS provides a more accurate representation of the blood concentration of the parent compound tacrolimus exclusive of metabolite, established cut points for tacrolimus dosing may need to be adjusted to account for the increased risk of renal injury.

High-Throughput Flow Cytometric Method for the Simultaneous Measurement of CAR-T Cell Characterization and Cytotoxicity against Solid Tumor Cell Lines.

High-throughput flow cytometry is an attractive platform for the analysis of adoptive cellular therapies such as chimeric antigen receptor T cell therapy (CAR-T) because it allows for the concurrent measurement of T cell-dependent cellular cytotoxicity (TDCC) and the functional characterization of engineered T cells with respect to percentage of CAR transduction, T cell phenotype, and measurement of T cell function such as activation in a single assay. The use of adherent tumor cell lines can be challenging in these flow-based assays. Here, we present the development of a high-throughput flow-based assay to measure TDCC for a CAR-T construct co-cultured with multiple adherent tumor cell lines. We describe optimal assay conditions (such as adherent cell dissociation techniques to minimize impact on cell viability) that result in robust cytotoxicity assays. In addition, we report on the concurrent use of T cell transduction and activation antibody panels (CD25) that provide further dissection of engineered T cell function. In conclusion, we present the development of a high-throughput flow cytometry method allowing for in vitro interrogation of solid tumor, targeting CAR-T cell-mediated cytotoxicity, CAR transduction, and engineered T cell characterization in a single assay.

Analysis of 30-day mortality for clostridium difficile-associated disease in the ICU setting.

OBJECTIVE: To examine the 30-day mortality rate among patients with Clostridium difficile-associated disease (CDAD) requiring intensive care. DESIGN: A retrospective, single-center, observational, cohort study. SETTING: Barnes-Jewish Hospital, a 1,200-bed, urban, teaching facility. PATIENTS: Adult patients admitted to the ICU identified to have CDAD by enzyme immunoassay. INTERVENTIONS: Retrospective data collection from automated hospital, microbiology, and pharmacy databases. MEASUREMENTS AND MAIN RESULTS: Two hundred seventy-eight patients with CDAD admitted to an ICU were identified over a 2-year period. Two hundred six patients (74.1%) received prior antibiotic therapy. The overall 30-day mortality rate was 36.7% (n = 102). Logistic regression analysis identified septic shock (adjusted odds ratio, 1.96; 95% confidence interval [CI], 1.47 to 2.61; p = 0.018), ward-to-ICU transfer (adjusted odds ratio, 2.12; 95% CI, 1.62 to 2.79; p = 0.006), and increasing APACHE (acute physiology and chronic health evaluation) II scores (1-point increments) [adjusted odds ratio, 1.09; 95% CI, 1.07 to 1.12; p < 0.001] as independent predictors for 30-day mortality. The attributable mortality associated with CDAD was estimated to be 6.1% (95% CI, - 1.7 to 13.9%; p = 0.127). CDAD was associated with an excess ICU length of stay (2.2 days) and hospital length of stay (4.5 days). CONCLUSIONS: We found a high 30-day crude mortality among patients with CDAD in the ICU setting. Although the attributable mortality from CDAD was relatively low, excess length of stay in the ICU and hospital was observed with CDAD. ICUs should routinely employ infection control efforts aimed at minimizing the occurrence of CDAD because of the excess morbidity associated with this nosocomial infection.

Identification of Population Bottlenecks and Colonization Factors during Assembly of Bacterial Communities within the Zebrafish Intestine.

UNLABELLED: The zebrafish, Danio rerio, is a powerful model for studying bacterial colonization of the vertebrate intestine, but the genes required by commensal bacteria to colonize the zebrafish gut have not yet been interrogated on a genome-wide level. Here we apply a high-throughput transposon mutagenesis screen to Aeromonas veronii Hm21 and Vibrio sp. strain ZWU0020 during their colonization of the zebrafish intestine alone and in competition with each other, as well as in different colonization orders. We use these transposon-tagged libraries to track bacterial population sizes in different colonization regimes and to identify gene functions required during these processes. We show that intraspecific, but not interspecific, competition with a previously established bacterial population greatly reduces the ability of these two bacterial species to colonize. Further, using a simple binomial sampling model, we show that under conditions of interspecific competition, genes required for colonization cannot be identified because of the population bottleneck experienced by the second colonizer. When bacteria colonize the intestine alone or at the same time as the other species, we find shared suites of functional requirements for colonization by the two species, including a prominent role for chemotaxis and motility, regardless of the presence of another species. IMPORTANCE: Zebrafish larvae, which are amenable to large-scale gnotobiotic studies, comprehensive sampling of their intestinal microbiota, and live imaging, are an excellent model for investigations of vertebrate intestinal colonization dynamics. We sought to develop a mutagenesis and tagging system in order to understand bacterial population dynamics and functional requirements during colonization of the larval zebrafish intestine. We explored changes in bacterial colonization dynamics and functional requirements when bacteria colonize a bacterium-free intestine, one previously colonized by their own species, or one colonized previously or simultaneously with a different species. This work provides a framework for rapid identification of colonization factors important under different colonization conditions. Furthermore, we demonstrate that when colonizing bacterial populations are very small, this approach is not accurate because random sampling of the input pool is sufficient to explain the distribution of inserts recovered from bacteria that colonized the intestines.

Comparison of bivalirudin and argatroban for the management of heparin-induced thrombocytopenia.

STUDY OBJECTIVES: To compare the effectiveness of bivalirudin and argatroban in achieving anticoagulation goals and to compare clinical outcomes assessing the safety and efficacy in patients with known or suspected heparin-induced thrombocytopenia (HIT). DESIGN: Single-center, retrospective analysis. SETTING: Large tertiary care academic medical center. PATIENTS: A total of 138 adults who received either bivalirudin (92 patients) or argatroban (46 patients) for at least 24 hours for known or suspected HIT between January 2007 and July 2008. MEASUREMENTS AND MAIN RESULTS. Data regarding demographics, direct thrombin inhibitor (DTI) dosing and monitoring, and related clinical outcomes were collected; statistical analysis was performed to compare results for patients receiving bivalirudin versus those receiving argatroban. Duration of DTI use ranged from 24-658 hours. At the time of DTI initiation, 108 patients (78%) were in an intensive care unit, with the highest proportion (61/138 [44%]) in the cardiothoracic surgery intensive care unit. The median (interquartile range [IQR]) DTI doses at the time of first reaching therapeutic goal were bivalirudin 0.06 mg/kg/hour (0.04-0.08 mg/kg/hr) and argatroban 1.0 µg/kg/minute (0.5-2.0 µg/kg/min). The median percentage of activated partial thromboplastin time (aPTT) values within therapeutic range while patients were receiving DTI therapy were similar for bivalirudin and argatroban (75% and 70%, respectively, p=0.238). A greater percentage of aPTT values were supratherapeutic with argatroban versus bivalirudin treatment (18% vs 8%, p=0.046). Median time to therapeutic goal was similar for bivalirudin (5.50 hrs [IQR 4-14.5 hrs]) and argatroban (5.75 hrs [IQR 3-17.7 hrs], p=0.499). New thromboembolic events occurred in seven patients (8%) receiving bivalirudin and two (4%) receiving argatroban (p=0.718). Bleeding events occurred at similar rates in both groups (9% for bivalirudin vs 11% for argatroban, p>0.999). CONCLUSIONS: Bivalirudin and argatroban were similar in achieving and maintaining therapeutic anticoagulation goals, clinical outcomes, and safety. This study suggests that bivalirudin represents an alternative in the management of HIT, but prospective studies are needed.