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.

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.

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.

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.

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.

Fiberoptic bronchoscopy and bronchoalveolar lavage for the evaluation of pulmonary disease in children with primary immunodeficiency and cancer.

BACKGROUND: Patients with childhood cancer or primary immunodeficiencies (PID) are at high risk for developing pulmonary infections and non-infectious complications. The broad differential diagnoses and the critical condition of these patients often drive physicians to start broad-spectrum antibiotic therapy before a definite diagnostic procedure is performed. A definite diagnosis may be achieved in these situations by fiberoptic bronchoscopy (FOB) and bronchoalveolar lavage (BAL). PATIENTS AND METHODS: The records of 58 PIDs and cancer (immunocompromised group) pediatric patients who underwent 62 fiberoptic bronchoscopies between 2000 and 2004 were retrospectively reviewed and compared to 158 non-cancer patients who underwent 182 fiberoptic bronchoscopies during the same period. RESULTS: The overall diagnostic rate achieved by macroscopic inspection of purulent secretions or hemorrhage, abnormal cell count, and infectious agent isolation in the immunocompromised patients was 84%. A definite organism was recovered in 53.2% of the patients. Probable infection defined as purulent secretions or abnormal cell count without infectious agent isolation was diagnosed in another 21% of the patients. The rate of complications was 30.6%. In the control group, the overall diagnostic rate was 76.9% (n.s) and an infectious agent was demonstrated in 12.1% (P < 0.001). Probable infection was diagnosed in 24.2% (n.s) while the rate of complications was lower (15%) (P < 0.01). CONCLUSIONS: Rapid and accurate diagnoses were achieved in most procedures performed on immunocompromised patients. Although the rate of complications was higher in the immunocompromised group, they were usually very mild with no mortality. Based on these results, broncoalveolar lavage should be considered as an initial diagnostic tool in pediatric immunocompromised patients with pulmonary complications.

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.

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.

Acute respiratory distress syndrome in children: a 10 year experience.

BACKGROUND: Acute respiratory distress syndrome is a well-recognized condition resulting in high permeability pulmonary edema associated with a high morbidity. OBJECTIVES: To examine a 10 year experience of predisposing factors, describe the clinical course, and assess predictors of mortality in children with this syndrome. METHODS: The medical records of all admissions to the pediatric intensive care unit over a 10 year period were evaluated to identify children with ARDS. Patients were considered to have ARDS if they met all of the following criteria: acute onset of diffuse bilateral pulmonary infiltrates of non-cardiac origin and severe hypoxemia defined by < 200 partial pressure of oxygen during > or = 6 cm H2O positive end-expiratory pressure for a minimum of 24 hours. The medical records were reviewed for demographic, clinical, and physiologic information including PaO2/forced expiratory O2, alveolar-arterial O2 difference, and ventilation index. RESULTS: We identified 39 children with the adult respiratory distress syndrome. Mean age was 7.4 years (range 50 days to 16 years) and the male:female ratio was 24:15. Predisposing insults included sepsis, pneumonias, malignancy, major trauma, shock, aspiration, near drowning, burns, and envenomation. The mortality rate was 61.5%. Predictors of death included the PaO2/FIO2, ventilation index and A-aDO2 on the second day after diagnosis. Nonsurvivors had significantly lower PaO2/FIO2 (116 +/- 12 vs. 175 +/- 8.3, P < 0.001), and higher A-aDO2 (368 +/- 28.9 vs. 228.0 +/- 15.5, P < 0.001) and ventilation index (43.3 +/- 2.9 vs. 53.1 +/- 18.0, P < 0.001) than survivors. CONCLUSIONS: Local mortality outcome for ARDS is comparable to those in tertiary referral institutions in the United States and Western Europe. The PaO2/FIO2, A-aDO2 and ventilation index are valuable for predicting outcome in ARDS by the second day of conventional therapy. The development of a local risk profile may allow early application of innovative therapies in this population.

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.

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.

Plain radiography for diagnosis of gastrointestinal perforation in immune-compromised children--is it enough?

Gastrointestinal perforation is a surgical emergency in the pediatric patient and any delay in diagnosis might be hazardous. In immunocompromised children, the clinical signs of perforation may be blunted. We describe a child with acute lymphoblastic leukemia (ALL) and a perforated appendix and ileum in whom computerized tomography (CT) revealed extraluminal air that was not initially identified on plain abdominal film. Our case demonstrates the importance of early abdominal CT and ultrasound examination in detecting these potentially lethal complications.

Suppression of somatic embryogenesis in Citrus cell cultures by extracellular proteins.

Nucellar-derived cell cultures of sour orange (Citrus aurantium L.) proliferate as proembryogenic masses. By a change in the carbon source of the medium from sucrose to glycerol they are induced to undergo synchronous embryogenesis forming embryo initials that develop into globular embryos. The proembryogenic masses released glycoproteins to the medium. Exogenous addition of the glycoproteins to cells in glycerol-containing medium modified the course of embryo development in a dose-dependent manner. Addition of 20 µg · ml(-1) of glycoproteins blocked embryogenesis and resulted in an accumulation of embryo initials. When glycoproteins were added to cultures containing advanced globularstage embryos further development was suppressed. The inhibitory component of the glycoproteins was found to be a family of polypeptides with apparent molecular masses of 53-57 kDa. While these proteins normally accumulated only in cultures of proembryogenic masses, they could be induced to accumulate in glycerol-containing medium by the addition of the glycoproteins. Thus, their accumulation was not a direct consequence of the type of growth medium used or the developmental state of the cultures. The results indicate that the 53-to 57 kDa glycoproteins could play a regulatory role in in-vitro embryogenesis in sour orange. The normal progression of embryo development appears to depend, in an obligatory manner, on the absence of these glycosylated extracellular proteins from the medium.

The plastome of Citrus. Physical map, variation among Citrus cultivars and species and comparison with related genera.

A physical plastome map was constructed for Citrus aurantium, and the plastomes of species and cultivars of Citrus and of two Citrus relatives were analysed by Southern blot-hybridisation of labelled total tobacco cpDNA to digests of total Citrus DNA. A resemblance was found between the plastomes of cultivars of C. limon (lemon), C. sinensis (orange), C. aurantium (sour orange), C. paradisii (grapefruit) and C. grandis (pomello). The plastomes of other Citrus types such as mandarin (C. reticulata) and citron (C. medico) differed from each other as well as from the plastomes of the aforementioned group. The plastomes of Poncirus trifoliata and Microcitrus sp. are distinct from each other as well as from the Citrus types.