Longitudinal characterization of dysfunctional T cell-activation during human acute Ebola infection

Data on immune responses during human Ebola virus disease (EVD) are scanty, due to limitations imposed by biosafety requirements and logistics. A sustained activation of T-cells was recently described but functional studies during the acute phase of human EVD are still missing. Aim of this work was to evaluate the kinetics and functionality of T-cell subsets, as well as the expression of activation, autophagy, apoptosis and exhaustion markers during the acute phase of EVD until recovery. Two EVD patients admitted to the Italian National Institute for Infectious Diseases, Lazzaro Spallanzani, were sampled sequentially from soon after symptom onset until recovery and analyzed by flow cytometry and ELISpot assay. An early and sustained decrease of CD4 T-cells was seen in both patients, with an inversion of the CD4/CD8 ratio that was reverted during the recovery period. In parallel with the CD4 T-cell depletion, a massive T-cell activation occurred and was associated with autophagic/apoptotic phenotype, enhanced expression of the exhaustion marker PD-1 and impaired IFN-gamma production. The immunological impairment was accompanied by EBV reactivation. The association of an early and sustained dysfunctional T-cell activation in parallel to an overall CD4 T-cell decline may represent a previously unknown critical point of Ebola virus (EBOV)-induced immune subversion. The recent observation of late occurrence of EBOV-associated neurological disease highlights the importance to monitor the immuno-competence recovery at discharge as a tool to evaluate the risk of late sequelae associated with resumption of EBOV replication. Further studies are required to define the molecular mechanisms of EVD-driven activation/exhaustion and depletion of T-cells.Ebola virus (EBOV) is one of the most deadly human pathogens, causing a severe hemorrhagic fever syndrome in both humans and non-human primates with fatality rates ranging from 50 to 70%.¹ The recent outbreak of Ebola Virus Diseases (EVD) in West Africa highlights the pathogenic nature of this virus, the high mortality rates and pandemic potential. To date, there have been over 27 700 cases and >11 280 deaths.^{1, 2} Although EVD is usually an acute illness, increasing evidences exist of persistent infections and post infection syndromes,^{3, 4, 5, 6} highlighting the need to identify immune correlates of a protective immune response.Defining human immune responses to EBOV infection, pathogenesis and correlates of protection are important for designing effective therapeutic and vaccination interventions. A decrease in lymphocytes has been observed in studies in mice,⁷ non-human primates⁸ and humans,⁹ and is attributed to apoptotic mechanisms.^{7, 10} Persistent B and T-cell activation has been described in four survivors as long as one month after discharge from the hospital, suggesting recurrent antigenic stimulation.¹¹ While aberrant immune responses have been described after EBOV infection (reviewed in^{12, 13}), and different patterns of inflammatory mediators have been associated with different clinical outcomes,^{9, 10, 11, 14, 15, 16, 17} data on human immune responses to Ebola virus remain scanty, due to difficulties in obtaining sequential samples through the course of illness and to limitations imposed by biosafety requirements for laboratory analyses.We conducted a longitudinal study aimed to characterize the kinetics of T-cell phenotypes, activation/differentiation profile, autophagic/apoptotic markers and functionality in two EVD patients from soon after symptom onset through their hospitalization until recovery.     

Loss of genetic integrity and biological invasions result from stocking and introductions of Barbus barbus: insights from rivers in England

Anthropogenic activities, including the intentional releases of fish for enhancing populations (stocking), are recognized as adversely impacting the adaptive potential of wild populations. Here, the genetic characteristics of European barbel Barbus barbus were investigated using 18 populations in England, where it is indigenous to eastern‐flowing rivers and where stocking has been used to enhance these populations. Invasive populations are also present in western‐flowing rivers following introductions of translocated fish. Two genetic clusters were evident in the indigenous range, centered on catchments in northeast and southeast England. However, stocking activities, including the release of hatchery‐reared fish, have significantly reduced the genetic differentiation across the majority of this range. In addition, in smaller indigenous rivers, populations appeared to mainly comprise fish of hatchery origin. In the nonindigenous range, genetic data largely aligned to historical stocking records, corroborating information that one particular river (Kennet) in southeast England was the original source of most invasive B. barbus in England. It is recommended that these genetic outputs inform management measures to either restore or maintain the original genetic diversity of the indigenous rivers, as this should help ensure populations can maintain their ability to adapt to changing environmental conditions. Where stocking is considered necessary, it is recommended that only broodstock from within the catchment is used.     

Hypothermia during Carotid Endarterectomy: A Safety Study

BackgroundCEA is associated with peri-operative risk of brain ischemia, due both to emboli production caused by manipulation of the plaque and to potentially noxious reduction of cerebral blood flow by carotid clamping. Mild hypothermia (34–35°C) is probably the most effective approach to protect brain from ischemic insult. It is therefore a substantial hypothesis that hypothermia lowers the risk of ischemic brain damage potentially associated with CEA. Purpose of the study is to test whether systemic endovascular cooling to a target of 34.5–35°C, initiated before and maintained during CEA, is feasible and safe.MethodsThe study was carried out in 7 consecutive patients referred to the Vascular Surgery Unit and judged eligible for CEA. Cooling was initiated 60–90 min before CEA, by endovascular approach (Zoll system). The target temperature was maintained during CEA, followed by passive, controlled rewarming (0.4°C/h). The whole procedure was carried out under anesthesia.ResultsAll the patients enrolled had no adverse events. Two patients exhibited a transient bradycardia (heart rate 30 beats/min). There were no significant differences in the clinical status, laboratory and physiological data measured before and after CEA.ConclusionsSystemic cooling to 34.5–35.0°C, initiated before and maintained during carotid clamping, is feasible and safe.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02629653","term_id":"NCT02629653"}}NCT02629653     

Skewed Differentiation of Circulating Vγ9Vδ2 T Lymphocytes in Melanoma and Impact on Clinical Outcome

Objective

The aim of this study was to evaluate over time circulating γδ T lymphocytes in melanoma patients in terms of frequency, effector functions, and relationship with clinical stage and evolution, by comparing preoperative values to those obtained at a mean follow-up of 36 months or in the event of recurrence or disease progression, and to those of healthy controls. Also, we correlated the presence of tumor-infiltrating γδ T lymphocytes with clinical evolution of melanoma.

Results

Mean frequencies of circulating γδ T cells before and after melanoma removal were very similar and comparable to healthy subjects, but patients who progressed to stage III or IV showed a significantly decreased frequency of circulating Vγ9Vδ2 T cells. The distribution of Vγ9Vδ2 memory and effector subsets was similar in healthy subjects and melanoma patients at diagnosis, but circulating γδ T cells of patients after melanoma removal had a skewed terminally-differentiated effector memory phenotype. Highly suggestive of progressive differentiation toward a cytotoxic phenotype, Vγ9Vδ2T cells from patients at follow up had increased cytotoxic potential and limited cytokine production capability, while the opposite pattern was detected in Vγ9Vδ2T cells from patients before melanoma removal.

Conclusions

Follow-up data also showed that tumor infiltrating γδ T cells were significantly associated with lower mortality and relapse rates, suggesting that they may serve as a prognostic biomarker, for human melanoma.     

Genomic and physiological analysis reveals versatile metabolic capacity of deep-sea <Emphasis Type="Italic">Photobacterium phosphoreum</Emphasis> ANT-2200

Bacteria of the genus Photobacterium thrive worldwide in oceans and show substantial eco-physiological diversity including free-living, symbiotic and piezophilic life styles. Genomic characteristics underlying this variability across species are poorly understood. Here we carried out genomic and physiological analysis of Photobacterium phosphoreum strain ANT-2200, the first deep-sea luminous bacterium of which the genome has been sequenced. Using optical mapping we updated the genomic data and reassembled it into two chromosomes and a large plasmid. Genomic analysis revealed a versatile energy metabolic potential and physiological analysis confirmed its growth capacity by deriving energy from fermentation of glucose or maltose, by respiration with formate as electron donor and trimethlyamine N-oxide (TMAO), nitrate or fumarate as electron acceptors, or by chemo-organo-heterotrophic growth in rich media. Despite that it was isolated at a site with saturated dissolved oxygen, the ANT-2200 strain possesses four gene clusters coding for typical anaerobic enzymes, the TMAO reductases. Elevated hydrostatic pressure enhances the TMAO reductase activity, mainly due to the increase of isoenzyme TorA1. The high copy number of the TMAO reductase isoenzymes and pressure-enhanced activity might imply a strategy developed by bacteria to adapt to deep-sea habitats where the instant TMAO availability may increase with depth.     

Specific protein homeostatic functions of small heat‐shock proteins increase lifespan

During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat‐shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress‐denatured substrates and/or to prevent aggregation of disease‐associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70‐dependent refolding of stress‐denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70‐independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo.     

Pyrazolo[1,5-a]quinazoline scaffold as 5-deaza analogue of pyrazolo[5,1-c][1,2,4]benzotriazine system: synthesis of new derivatives,biological activity on GABAA receptor subtype and molecular dynamic study

To investigate the binding affinity of GABA_A receptor subtype, new pyrazolo [1,5-a]quinazolines were designed, synthesized, and in vitro evaluated. These compounds, 5-deaza analogues of pyrazolo[5,1-c][1,2,4]benzotriazine derivatives which were already studied in our research group, permit us to evaluate the relevance of the nitrogen or the oxygen atom at 5-position of the tricyclic scaffold. Molecular dynamic study was done on a set of the new and known ligands to rationalize and to explain the lack of affinity on the 4- or 5-substituted new derivative. In fact, from biological results, it can be found that the only 5-unsubstituted new derivative, compound 15, has receptor recognition (K_i?=?834.7?nM).     

Deletion of PsbM in tobacco alters the QB site properties and the electron flow within photosystem II

Photosystem II, the oxygen-evolving complex of photosynthetic organisms, includes an intriguingly large number of low molecular weight polypeptides, including PsbM. Here we describe the first knock-out of psbM using a transplastomic, reverse genetics approach in a higher plant. Homoplastomic Delta psbM plants exhibit photoautotrophic growth. Biochemical, biophysical, and immunological analyses demonstrate that PsbM is not required for biogenesis of higher order photosystem II complexes. However, photosystem II is highly light-sensitive, and its activity is significantly decreased in Delta psbM, whereas kinetics of plastid protein synthesis, reassembly of photosystem II, and recovery of its activity are comparable with the wild type. Unlike wild type, phosphorylation of the reaction center proteins D1 and D2 is severely reduced, whereas the redox-controlled phosphorylation of photosystem II light-harvesting complex is reversely regulated in Delta psbM plants because of accumulation of reduced plastoquinone in the dark and a limited photosystem II-mediated electron transport in the light. Charge recombination in Delta psbM measured by thermoluminescence oscillations significantly differs from the 2/6 patterns in the wild type. A simulation program of thermoluminescence oscillations indicates a higher Q(B)/Q(-)(B) ratio in dark-adapted mutant thylakoids relative to the wild type. The interaction of the Q(A)/Q(B) sites estimated by shifts in the maximal thermoluminescence emission temperature of the Q band, induced by binding of different herbicides to the Q(B) site, is changed indicating alteration of the activation energy for back electron flow. We conclude that PsbM is primarily involved in the interaction of the redox components important for the electron flow within, outward, and backward to photosystem II.