Using NanoSIMS coupled with microfluidics to visualize the early stages of coral infection by Vibrio coralliilyticus.

BACKGROUND: Global warming has triggered an increase in the prevalence and severity of coral disease, yet little is known about coral/pathogen interactions in the early stages of infection. The point of entry of the pathogen and the route that they take once inside the polyp is currently unknown, as is the coral's capacity to respond to infection. To address these questions, we developed a novel method that combines stable isotope labelling and microfluidics with transmission electron microscopy (TEM) and nanoscale secondary ion mass spectrometry (NanoSIMS), to monitor the infection process between Pocillopora damicornis and Vibrio coralliilyticus under elevated temperature. RESULTS: Three coral fragments were inoculated with 15N-labeled V. coralliilyticus and then fixed at 2.5, 6 and 22 h post-inoculation (hpi) according to the virulence of the infection. Correlative TEM/NanoSIMS imaging was subsequently used to visualize the penetration and dispersal of V. coralliilyticus and their degradation or secretion products. Most of the V. coralliilyticus cells we observed were located in the oral epidermis of the fragment that experienced the most virulent infection (2.5 hpi). In some cases, these bacteria were enclosed within electron dense host-derived intracellular vesicles. 15N-enriched pathogen-derived breakdown products were visible in all tissue layers of the coral polyp (oral epidermis, oral gastrodermis, aboral gastrodermis), at all time points, although the relative 15N-enrichment depended on the time at which the corals were fixed. Tissues in the mesentery filaments had the highest density of 15N-enriched hotspots, suggesting these tissues act as a "collection and digestion" site for pathogenic bacteria. Closer examination of the sub-cellular structures associated with these 15N-hotspots revealed these to be host phagosomal and secretory cells/vesicles. CONCLUSIONS: This study provides a novel method for tracking bacterial infection dynamics at the levels of the tissue and single cell and takes the first steps towards understanding the complexities of infection at the microscale, which is a crucial step towards understanding how corals will fare under global warming.

Molecular and Clinical Characteristics of Different Toxicity Rates in Anti-CD19 Chimeric Antigen Receptor T Cells: Real-World Experience.

Commercially available anti-CD19 chimeric antigen receptor T cells (CARΤ cells) have offered long-term survival to a constantly expanding patient population. Given that novel toxicities including cytokine release syndrome (CRS) and neurotoxicity (ICANS) have been observed, we aimed to document the safety and toxicity of this treatment in a real-world study. We enrolled 31 adult patients referred to our center for CAR T therapy. Tisagenlecleucel was infused in 12 patients, axicabtagene ciloleucel in 14, and brexucabtagene autoleucel in 5. Cytokine release syndrome was noted in 26 patients while neurotoxicity was observed in 7. Tocilizumab was administered for CRS in 18 patients, along with short-term, low-dose steroid administration in one patient who developed grade III CRS and, subsequently, grade I ICANS. High-dose steroids, along with anakinra and siltuximab, were administered in only two MCL patients. With a median follow-up time of 13.4 months, nine patients were then in CR. The progression-free (PFS) and overall survival (OS) rates were 41.2% and 88.1% at one year, respectively. MCL diagnosis, which coincides with the administration of brexucabtagene autoleucel, was the only factor to be independently associated with poor OS (p < 0.001); meanwhile, increased LDH independently predicted PFS (p = 0.027).In addition, CRP at day 14 was associated with a poor OS (p = 0.001). Therefore, our real-world experience confirmed that commercial CAR T therapy can be administered with minimal toxicity.

How to probe the microscopic onset of irreversibility with ultracold atoms.

The microscopic onset of irreversibility is finally becoming an experimental subject. Recent experiments on microscopic open and even isolated systems have measured statistical properties associated with entropy production, and hysteresis-like phenomena have been seen in cold atom systems with dissipation (i.e. effectively open systems coupled to macroscopic reservoirs). Here we show how experiments on isolated systems of ultracold atoms can show dramatic irreversibility like cooking an egg. In our proposed experiments, a slow forward-and-back parameter sweep will sometimes fail to return the system close to its initial state. This probabilistic hysteresis is due to the same non-adiabatic spreading and ergodic mixing in phase space that explains macroscopic irreversibility, but realized without dynamical chaos; moreover this fundamental mechanism quantitatively determines the probability of return to the initial state as a function of tunable parameters in the proposed experiments. Matching the predicted curve of return probability will be a conclusive experimental demonstration of the microscopic onset of irreversibility.

High-Throughput Sequencing of the T-Cell Receptor Beta Chain Gene Repertoire in Chronic Lymphocytic Leukemia.

High-throughput, next-generation sequencing (NGS) offers a unique opportunity for in-depth characterization of adaptive immune receptor repertoires. Nevertheless, limitations and pitfalls exist in every step of both the experimental and the analytical procedure, leading to discrepancies in the literature and incomprehensive and/or altogether misleading results. Thus, standardization of protocols in NGS immunogenetics is urgently needed.Here, we describe the experimental protocol that we developed for T-cell receptor beta chain (TRB) gene repertoire analysis in chronic lymphocytic leukemia, aiming to provide a reproducible and biologically meaningful output. Although optimized for TRBV-TRBD-TRBJ gene rearrangements, this protocol may be customized for other adaptive immune receptor sequences, as well.

Vibrio coralliilyticus infection triggers a behavioural response and perturbs nutritional exchange and tissue integrity in a symbiotic coral.

Under homoeostatic conditions, the relationship between the coral Pocillopora damicornis and Vibrio coralliilyticus is commensal. An increase in temperature, or in the abundance of V. coralliilyticus, can turn this association pathogenic, causing tissue lysis, expulsion of the corals' symbiotic algae (genus Symbiodinium), and eventually coral death. Using a combination of microfluidics, fluorescence microscopy, stable isotopes, electron microscopy and NanoSIMS isotopic imaging, we provide insights into the onset and progression of V. coralliilyticus infection in the daytime and at night, at the tissue and (sub-)cellular level. The objective of our study was to connect the macro-scale behavioural response of the coral to the micro-scale nutritional interactions that occur between the host and its symbiont. In the daytime, polyps enhanced their mucus production, and actively spewed pathogens. Vibrio infection primarily resulted in the formation of tissue lesions in the coenosarc. NanoSIMS analysis revealed infection reduced 13C-assimilation in Symbiodinium, but increased 13C-assimilation in the host. In the night incubations, no mucus spewing was observed, and a mucus film was formed on the coral surface. Vibrio inoculation and infection at night showed reduced 13C-turnover in Symbiodinium, but did not impact host 13C-turnover. Our results show that both the nutritional interactions that occur between the two symbiotic partners and the behavioural response of the host organism play key roles in determining the progression and severity of host-pathogen interactions. More generally, our approach provides a new means of studying interactions (ranging from behavioural to metabolic scales) between partners involved in complex holobiont systems, under both homoeostatic and pathogenic conditions.

[Circumcision and HIV]. / Circoncision et VIH.

The idea that circumcision decreases the risk of sexual transmission of HIV was first proposed in the 1980s, at the time of the worldwide emergence of HIV infection. Many descriptive studies have subsequently been conducted to confirm this effect. Over the last two years, three experimental studies have provided scientific proof of the protective effect of circumcision, evaluated to be about 60%. These studies were recently validated by the WHO. The underlying mechanism of this protective effect remains unclear, but appears to be related more to the number of CD4+ lymphocytes on the mucosal surface of the prepuce in uncircumcised men than to keratinisation of the glans in circumcised men. Paradoxically, the practical implications are unclear, as large-scale prophylactic circumcision, depending on the country, would raise problems of acceptability, material feasibility and even efficacy if the population, considering itself to be protected, abandons conventional safe sex precautions which remain essential.

Neurological deficit is predicted by S100B in children after cardiac surgery.

INTRODUCTION: Children undergoing cardiac surgery may suffer from brain injuries after surgery and develop neurological deficit. Early diagnosis of brain injury after surgery would enable early therapeutic interventions. The aim of the study is to test whether S100B can serve as a biomarker for brain injury after cardiac surgery. METHODS: Seventy-five patients were enrolled in the study. Serum S100B was collected at the beginning of the surgery, and 6, 12, 24 h after surgery. S100B z-scores were calculated based on norms for age. Neurological evolutions were done before surgery and at discharge by the Pediatric Stroke Outcome Measure (PSOM). New neurological deficit (NND) was defined as a 1 point increase on the PSOM scale. RESULTS: Twenty patients had an NND after cardiac surgery. Medical background was similar between the groups with and without NND. S100B z-scores were significantly higher in the NND group at all time points after surgery. Using a cut-off of 3 z-score at 6 h after surgery, the positive predictive value was 79% and the negative predictive value was 90%. CONCLUSIONS: S100B is a potent early biomarker for brain injury after cardiac surgery. Hopefully, S100B could be used to prevent progression of brain injuries after cardiac surgery.

Programmed cell death of the dinoflagellate Peridinium gatunense is mediated by CO(2) limitation and oxidative stress.

The phytoplankton assemblage in Lake Kinneret is dominated in spring by a bloom of the dinoflagellate Peridinium gatunense, which terminates sharply in summer [1]. The pH in Peridinium patches rises during the bloom to values higher than pH9 [2] and results in CO(2) limitation. Here we show that depletion of dissolved CO(2) (CO(2(dis))) stimulated formation of reactive oxygen species (ROS) and induced cell death in both natural and cultured Peridinium populations. In contrast, addition of CO(2) prevented ROS formation. Catalase inhibited cell death in culture, implicating hydrogen peroxide (H(2)O(2)) as the active ROS. Cell death was also blocked by a cysteine protease inhibitor, E-64, a treatment which stimulated cyst formation. Intracellular ROS accumulation induced protoplast shrinkage and DNA fragmentation prior to cell death. We propose that CO(2) limitation resulted in the generation of ROS to a level that induced programmed cell death, which resembles apoptosis in animal and plant cells. Our results also indicate that cysteine protease(s) are involved in processes that determine whether a cell is destined to die or to form a cyst.

Prevention of potential errors in resuscitation medications orders by means of a computerised physician order entry in paediatric critical care.

INTRODUCTION: Computerised physician order entry with clinical decision support system (CPOE+CDSS) is an important tool in attempting to reduce medication errors. The objective of this study was to evaluate the impact of a CPOE+CDSS on (1) the frequency of errors in ordering resuscitation (CPR) medications and (2) the time for printing out the order form, in a paediatric critical care department (PCCD). SETTING: An 18-bed PCCD in a tertiary-care children's hospital. DESIGN: Prospective cohort study. MEASURES: Compilation and comparison of number of errors and time to fill in forms before and after implementation of CPOE+CDSS. Time to fill in conventional, simulated and CPOE forms was measured and compared. RESULTS: There were three reported incidents of errors among 13,124 CPR medications orders during the year preceding implementation of CPOE+CDSS. These represent errors that escaped the triple check by three independent staff members. There were no errors after CPOE+CDSS was implemented (100% error reduction for 46,970 orders). Time to completion of drug forms dropped from 14 min 42 s to 2 min 14s (p < 0.001). CONCLUSIONS: CPOE+CDSS completely eliminated errors in filling in the forms and significantly reduced time to completing the form.

Restrictions in the T-cell repertoire of chronic lymphocytic leukemia: high-throughput immunoprofiling supports selection by shared antigenic elements.

Immunoglobulin (IG) gene repertoire restrictions strongly support antigen selection in the pathogenesis of chronic lymphocytic leukemia (CLL). Given the emerging multifarious interactions between CLL and bystander T cells, we sought to determine whether antigen(s) are also selecting T cells in CLL. We performed a large-scale, next-generation sequencing (NGS) study of the T-cell repertoire, focusing on major stereotyped subsets representing CLL subgroups with undisputed antigenic drive, but also included patients carrying non-subset IG rearrangements to seek for T-cell immunogenetic signatures ubiquitous in CLL. Considering the inherent limitations of NGS, we deployed bioinformatics algorithms for qualitative curation of T-cell receptor rearrangements, and included multiple types of controls. Overall, we document the clonal architecture of the T-cell repertoire in CLL. These T-cell clones persist and further expand overtime, and can be shared by different patients, most especially patients belonging to the same stereotyped subset. Notably, these shared clonotypes appear to be disease-specific, as they are found in neither public databases nor healthy controls. Altogether, these findings indicate that antigen drive likely underlies T-cell expansions in CLL and may be acting in a CLL subset-specific context. Whether these are the same antigens interacting with the malignant clone or tumor-derived antigens remains to be elucidated.

Complete population transfer to and from a continuum and the radiative association of cold Na atoms to produce translationally cold Na2 molecules in specific vib-rotational states.

We demonstrate the feasibility of a laser induced complete population transfer to and from a continuum of states. We study the two-photon dissociation of v = 28; J = 1,...,10 sodium dimers. We demonstrate that using just a pair of "counter intuitively" ordered pulses we can dissociate 100% of the molecules in an ensemble. The scheme is shown to be stable with respect to the initial choice of rotational level and to fluctuations in the laser frequency and intensity. We also study the reverse phenomenon of complete population transfer from the continuum. We perform calculations on the radiative association of Na atoms to form the Na2 molecule in specific vib-rotational states. It is shown that two pulses of 20 nsec duration and as little as 6 MW/cm 2 peak power can photo-associate more than 98% of the atoms within a (pulse and velocity determined) relative effective distance, to yield Na2 molecules in the chosen v = 28; J = 10 vib-rotational state. This means that given a density of 10 16 atoms/cm 3 and a temperature of 7K, a 10Hz pulsed laser source of the above parameters can con- vert half of all the Na atoms in the ensemble to v = 28; J = 10 Na2 molecules within 15 seconds of operation.

A controlled intervention in reduction of redundant hospital days.

BACKGROUND: Inappropriate use of hospital services, in the form of unjustified hospital stay days (HSD), constitutes a major burden on a health budget. Reduction of unjustified HSD was achieved in a medical ward in a previous intervention study. METHODS: A controlled intervention aimed at reducing unjustified hospital stay was performed on 155 paediatric inpatients and 248 controls, by applying pre-set criteria for hospitalization and comparing to results in previous studies. RESULTS: Unjustified stay was decreased from 32.6% to 14.8% on the study ward, and from 25.7% to 19.3% on the control ward. The children on both wards did not differ significantly in rates of subsequent out of hospital mortality, re-admission, and the subjective evaluation of health by their parents one month following discharge. CONCLUSIONS: This study demonstrates that despite the fact that the per cent of unjustified HSD on a paediatric wars is much lower than on medicine or surgery, a significant reduction in unjustified stay can be achieved by intervention programme.

Gunshot wounds in brains of children: prognostic variables in mortality, course, and outcome.

A retrospective study of 51 children presenting with craniocerebral gunshot lesions was carried out to identify predictors of outcome. The patients ranged in age from 2 months to 17 years, with a mean of 14.5 years. The outcome was good in 20 patients, and seven and four were moderately and severely disabled, respectively. Twenty patients died. Statistical analysis showed prognostic significance of the admission Glasgow Coma Score (GCS), computerized tomographic findings of intraventricular hemorrhage and midline shift, and metabolic abnormalities, including hypokalemia and hyperglycemia. These prognostic factors may have implications regarding counseling of families, utilization of resources, and organ transplantation.

Epinephrine pharmacokinetics and pharmacodynamics following endotracheal administration in dogs: the role of volume of diluent.

OBJECTIVE: to define the optimal volume of dilution for endotracheal(ET) administration of epinephrine (EPI). DESIGN: prospective, randomized, laboratory comparison of four different volumes of dilution of endotracheal epinephrine (1, 2, 5, and 10 ml of normal saline). SETTING: large animal research facility of a university medical center. SUBJECTS AND INTERVENTIONS: epinephrine (0.02 mg/kg) diluted with four different volumes (1, 2, 5, and 10 ml) of normal saline was injected into the ET tube of five anesthetized dogs. Each dog served as its own control and received all four volumes in different sequences at least 1 week apart. Arterial blood samples for plasma epinephrine concentration and blood gases were collected before and 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30 and 60 min after drug administration. Heart rate and arterial blood pressure were continuously monitored with a polygraph recorder. MEASUREMENTS AND MAIN RESULTS: higher volumes of diluent (5 and 10 ml) caused a significant decrease of PaO2, from 147 +/- 8 to 106 +/- 10 torr, compared with the lower volumes of diluent (1 and 2 ml), from 136 +/- 10 to 135 +/- 7 torr (P < 0.05). These effects persisted for over 30 min. Mean plasma epinephrine concentrations significantly increased within 15 s following administration for all the volumes of diluent. Mean plasma epinephrine concentrations, maximal epinephrine concentration (Cmax) and the coefficient of absorption (Ka) were higher in the 5 and 10 ml groups. The time interval to reach maximal concentration (Tmax) was shorter in the 5 and 10 ml groups. Yet these results were not significantly different. Heart rate, systolic and diastolic blood pressures did not differ significantly between the groups throughout the study. CONCLUSIONS: Dilution of endotracheal epinephrine into a 5 ml volume with saline optimizes drug uptake and delivery without adversely affecting oxygenation and ventilation.

Ventilation index and outcome in children with acute respiratory distress syndrome.

The purpose of this investigation was to determine the predictive value of the ventilation index (VI) in children with acute respiratory distress syndrome (ARDS). We performed a 10-year retrospective chart review of children who were admitted to the Pediatric Intensive Care Unit with a diagnosis of ARDS. Acute respiratory distress syndrome was defined as acute onset of diffuse, bilateral pulmonary infiltrates of noncardiac origin, and severe hypoxemia, defined as the ratio of the arterial partial pressure of oxygen to the fraction of inspired oxygen of <200 and a positive end expiratory pressure of 6 cmH2O or greater. Records of daily arterial blood gas results and ventilator settings were reviewed, and the ventilation index (VI=partial pressure of arterial CO2 x peak airway pressure x respiratory rate/1,000) was calculated each time the measurements were made. These values were correlated with outcome (survival or nonsurvival). The VI was not different at the time of diagnosis of ARDS in the patients who lived, compared with those who subsequently died. However, by 3 to 5 days after study entry, the VI of nonsurvivors was significantly higher than for survivors (P < 0.05). The VI for survivors remained between 30 and 35 throughout the study period, whereas the VI of nonsurvivors continued to increase with time. A VI of >65 predicted death with a specificity and positive predictive value of >90% on days 3 through 9. We conclude that the VI provides a reliable prognostic marker in children with ARDS, and its increase above 65 indicates a need for orderly intervention with alternative modalities of care.

Adapting prognostic respiratory variables of ARDS in children to small-scale community needs.

PURPOSE: The clinical literature on the incidence and subsequent mortality of adult respiratory distress syndrome (ARDS) has come primarily from the experiences of large tertiary referral centers, particularly in Western Europe and North America. Consequently, very little has been published on the incidence, management, and outcome of ARDS in smaller community-based intensive care units. We aimed to delineate early clinical respiratory predictors of death in children with ARDS on the modest scale of a community hospital. MATERIALS AND METHODS: A retrospective chart review of children with ARDS needing conventional mechanical ventilation admitted to our pediatric intensive care unit from 1984 to 1997. The diagnosis of ARDS was based on acute onset of diffuse, bilateral pulmonary infiltrates of noncardiac origin and severe hypoxemia defined by partial pressure of oxygen <200 mm Hg during positive end-expiratory pressure (PEEP) of 6 cm H2O or greater for a minimum of 24 hours. Demographic, clinical, and physiological data including PaO2/ FIO2, A-aDo2, and ventilation index were retrieved. RESULTS: Fifty-six children with ARDS aged 8 +/- 5.5 years (range, 50 days to 21 years) were identified. The mortality rate was 50%. Early predictors of death included the peak inspiratory pressure (PIP), ventilation index, and PEEP on the third day after diagnosis: Nonsurvivors had significantly higher PIP (35.3 +/- 10.5 cm H2O vs 44.4 +/- 10.7 cm H2O, P < .001), PEEP (8 +/- 2.8 cm H2O vs 10.7.0 +/- 3.5 cm H2O, P < .01), and ventilation index (49.14 +/- 20.4 mm Hg x cm H2O/minute vs 61.6 +/- 51.1 mm Hg cm H2O/minute) than survivors. In contrast, PAO2/FIO2 and A-a DO2 were capable of predicting outcome by day 5 and thereafter. CONCLUSIONS: A small-scale mortality outcome for ARDS is comparable to large tertiary referral institutions. The PIP, PEEP, and ventilation index are valuable for predicting outcome in ARDS by the third day of conventional therapy. The development of a local risk profile may assist in decision-making of early application of supportive therapies in this population.

Acute respiratory distress syndrome in children with malignancy--can we predict outcome?

PURPOSE: The purpose of this study was to delineate early respiratory predictors of mortality in children with hemato-oncology malignancy who developed acute respiratory distress syndrome (ARDS). MATERIALS AND METHODS: We conducted a retrospective chart review of children with malignant and ARDS who needed mechanical ventilation and were admitted to a pediatric intensive care unit from January 1987 to January 1997. RESULTS: Seventeen children with ARDS and malignancy aged 10.5 +/- 5.1 years were identified. Six of the 17 children (35.3%) survived. Sepsis syndrome was present in 70.6% of all the children. Peak inspiratory pressure, positive end-expiratory pressure (PEEP), and ventilation index values could distinguish outcome by day 3. A significant relationship between respiratory data and outcome related to efficiency of oxygenation, as determined by PaO(2)/FIO(2) and P(A-a)O(2), was present from day 8 after onset of mechanical ventilation. CONCLUSIONS: Peak inspiratory pressure, PEEP, and ventilation index values could distinguish survivors from nonsurvivors by day 3. This may assist in early application of supportive nonconventional therapies in children with malignancy and ARDS.

Rhabdomyolysis due to hereditary torsion dystonia.

Following an acute dystonic crisis, a 6-year-old boy with hereditary torsion dystonia developed rhabdomyolysis. To our knowledge, hereditary torsion dystonia has never been reported as a cause of rhabdomyolysis. Early diagnosis and treatment of rhabdomyolysis should be considered in children with severe dystonia in order to prevent renal failure.

Laceration of the phrenic artery. A life-threatening complication after repair of pectus excavatum.

We report a case of life-threatening hemothorax three months after surgical repair of pectus excavatum. Angiography revealed the hemorrhage to originate from a laceration of the phrenic artery secondary to dislodgment of the metal strut used for the repair. Awareness of this rare complication in patients after repair of pectus excavatum is required.