BackgroundRecent evidence suggests that co-clustering of Fas/CD95 death receptor and lipid rafts plays a major role in death receptor-mediated apoptosis.Methodology/Principal FindingsBy a combination of genetic, biochemical, and ultrastructural approaches, we provide here compelling evidence for the involvement of lipid raft aggregates containing recruited Fas/CD95 death receptor, Fas-associated death domain-containing protein (FADD), and procaspase-8 in the induction of apoptosis in human T-cell leukemia Jurkat cells by the antitumor drug edelfosine, the prototype compound of a promising family of synthetic antitumor lipids named as synthetic alkyl-lysophospholipid analogues. Co-immunoprecipitation assays revealed that edelfosine induced the generation of the so-called death-inducing signaling complex (DISC), made up of Fas/CD95, FADD, and procaspase-8, in lipid rafts. Electron microscopy analyses allowed to visualize the formation of raft clusters and their co-localization with DISC components Fas/CD95, FADD, and procaspase-8 following edelfosine treatment of Jurkat cells. Silencing of Fas/CD95 by RNA interference, transfection with a FADD dominant-negative mutant that blocks Fas/CD95 signaling, and specific inhibition of caspase-8 prevented the apoptotic response triggered by edelfosine, hence demonstrating the functional role of DISC in drug-induced apoptosis. By using radioactive labeled edelfosine and a fluorescent analogue, we found that edelfosine accumulated in lipid rafts, forming edelfosine-rich membrane raft clusters in Jurkat leukemic T-cells. Disruption of these membrane raft domains abrogated drug uptake and drug-induced DISC assembly and apoptosis. Thus, edelfosine uptake into lipid rafts was critical for the onset of both co-aggregation of DISC in membrane rafts and subsequent apoptotic cell death.Conclusions/SignificanceThis work shows the involvement of DISC clusters in lipid raft aggregates as a supramolecular and physical entity responsible for the induction of apoptosis in leukemic cells by the antitumor drug edelfosine. Our data set a novel framework and paradigm in leukemia therapy, as well as in death receptor-mediated apoptosis.

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Background Introduction of pneumococcal conjugate vaccines (PCVs) has shown a marked reduction in the disease caused by vaccine serotypes in children providing herd protection to the elderly group. However, the emergence of non-vaccine serotypes is of great concern worldwide. Methods This study includes national laboratory data from invasive pneumococcal disease (IPD) cases affecting pediatric and adult population during 2009–2019. The impact of implementing different vaccine strategies for immunocompetent adults comparing Spanish regions using PCV13 vs regions using PPV23 vaccine was also analyzed for 2017–2019. Results The overall reductions of IPD cases by PCV13 serotypes in children and adults were 88% and 59% respectively during 2009–2019 with a constant increase of serotype 8 in adults since 2015. IPD cases by additional serotypes covered by PPV23 increased from 20% in 2009 to 52% in 2019. In children, serotype 24F was the most frequent in 2019 whereas in adults, serotypes 3 and 8 accounted for 36% of IPD cases. Introduction of PCV13 or PPV23 in the adult calendar of certain Spanish regions reduced up to 25% and 11% respectively the IPD cases by PCV13 serotypes, showing a decrease of serotype 3 when PCV13 was used. Conclusions Use of PCV13 in children has shown a clear impact in pneumococcal epidemiology reducing the burden of IPD in children but also in adults by herd protection although the increase of serotype 8 in adults is worrisome. Vaccination with PCV13 in immunocompetent adults seems to control IPD cases by PCV13 serotypes including serotype 3.

Heat shock protein 90 (Hsp90) is a survival signaling chaperone and a cancer chemotherapeutic target. However, we have found that inhibitors of Hsp90 diminished the apoptotic response induced in leukemic cells by the antitumor alkyl-lysophospholipid analog edelfosine, which acts through lipid raft reorganization. Edelfosine treatment recruited Hsp90, c-Jun N-terminal kinase (JNK) and apoptotic molecules in lipid rafts, but not the JNK regulators apoptosis signal-regulating kinase 1 (ASK1) and Daxx, or the survival signaling molecules extracellular signal-regulated kinase (ERK) and Akt. Following edelfosine treatment, Hsp90 bound to JNK in lipid rafts and Hsp90-JNK clusters were identified at the plasma membrane by immunoelectron microscopy. Hsp90 inhibition reduced JNK protein level in lipid rafts and turned proapoptotic persistent JNK activation into a transient response in edelfosine-treated cells. Decrease in edelfosine-induced JNK activation and apoptosis by Hsp90 inhibition was prevented through proteasome inhibition, suggesting that Hsp90 inhibition diminishes apoptosis by promoting JNK protein degradation. Expression of ASK1 dominant negative mutant did not affect JNK activation and apoptosis following edelfosine treatment. These data indicate that lipid raft-recruited JNK is ASK1-independent and becomes a novel Hsp90 client protein. Our results reveal a new chaperoning role of Hsp90 on JNKmediated apoptosis following its recruitment in lipid rafts.

We describe the validation of an enzyme-linked immunosorbent assay (ELISA) and confirmatory immunoblotting assays based on a recombinant p30 protein (p30r) produced in insect larvae using a baculovirus vector. Such validation included the following: (i) the scaling up and standardization of p30r production and the associated immunoassays, (ii) a broad immunological analysis using a large number of samples (a total of 672) from Spain and different African locations, and (iii) the detection of the ASF virus (ASFV)-antibody responses at different times after experimental infection. Yields of p30r reached up to 15% of the total protein recovered from the infected larvae at 3 days postinfection. Serological analysis of samples collected in Spain revealed that the p30r-based ELISA presented similar sensitivity to and higher specificity than the conventional Office International des Epizooties-approved ASFV ELISA. Moreover, the p30r ELISA was more sensitive than the conventional ELISA test in detecting ASFV-specific antibodies in experimentally infected animals at early times postinfection. Both the recombinant and conventional ELISAs presented variable rates of sensitivity and specificity with African samples, apparently related to their geographical origin. Comparative analyses performed on the sequences, predicted structures, and antigenicities of p30 proteins from different Spanish and African isolates suggested that variability among isolates might correlate with changes in antigenicity, thus affecting detection by the p30r ELISA. Our estimations indicate that more than 40,000 ELISA determinations and 2,000 confirmatory immunoblotting tests can be performed with the p30r protein obtained from a single infected larva, making this a feasible and inexpensive strategy for production of serological tests with application in developing countries.

The complement system is a key component of the host immune response for the recognition and clearance of Streptococcus pneumoniae. In this study, we demonstrate that the amidase LytA, the main pneumococcal autolysin, inhibits complement-mediated immunity independently of effects on pneumolysin by a complex process of impaired complement activation, increased binding of complement regulators, and direct degradation of complement C3. The use of human sera depleted of either C1q or factor B confirmed that LytA prevented activation of both the classical and alternative pathways, whereas pneumolysin inhibited only the classical pathway. LytA prevented binding of C1q and the acute-phase protein C-reactive protein to S. pneumoniae, thereby reducing activation of the classical pathway on the bacterial surface. In addition, LytA increased recruitment of the complement downregulators C4BP and factor H to the pneumococcal cell wall and directly cleaved C3b and iC3b to generate degradation products. As a consequence, C3b deposition and phagocytosis increased in the absence of LytA and were markedly enhanced for the lytA ply double mutant, confirming that a combination of LytA and Ply is essential for the establishment of pneumococcal pneumonia and sepsis in a murine model of infection. These data demonstrate that LytA has pleiotropic effects on complement activation, a finding which, in combination with the effects of pneumolysin on complement to assist with pneumococcal complement evasion, confirms a major role of both proteins for the full virulence of the microorganism during septicemia.

Osteosarcoma is a type of bone tumour characterized by considerable levels of phenotypic heterogeneity, aneuploidy, and a high mutational rate. The life expectancy of osteosarcoma patients has not changed during the last three decades and thus much remains to be learned about the disease biology. Here, we employ a RGB-based single-cell tracking system to study the clonal dynamics occurring in a de novo-induced murine osteosarcoma model. We show that osteosarcoma cells present initial polyclonal dynamics, followed by clonal dominance associated with adaptation to the microenvironment. Interestingly, the dominant clones are composed of subclones with a similar tumour generation potential when they are re-implanted in mice. Moreover, individual spontaneous metastases are clonal or oligoclonal, but they have a different cellular origin than the dominant clones present in primary tumours. In summary, we present evidence that osteosarcomagenesis can follow a neutral evolution model, in which different cancer clones coexist and propagate simultaneously.