Genetic subgroups inform on pathobiology in adult and pediatric Burkitt lymphoma.

Burkitt lymphoma (BL) accounts for most pediatric non-Hodgkin lymphomas, being less common but significantly more lethal when diagnosed in adults. Much of the knowledge of the genetics of BL thus far has originated from the study of pediatric BL (pBL), leaving its relationship to adult BL (aBL) and other adult lymphomas not fully explored. We sought to more thoroughly identify the somatic changes that underlie lymphomagenesis in aBL and any molecular features that associate with clinical disparities within and between pBL and aBL. Through comprehensive whole-genome sequencing of 230 BL and 295 diffuse large B-cell lymphoma (DLBCL) tumors, we identified additional significantly mutated genes, including more genetic features that associate with tumor Epstein-Barr virus status, and unraveled new distinct subgroupings within BL and DLBCL with 3 predominantly comprising BLs: DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Each BL subgroup is characterized by combinations of common driver and noncoding mutations caused by aberrant somatic hypermutation. The largest subgroups of BL cases, IC-BL and DGG-BL, are further characterized by distinct biological and gene expression differences. IC-BL and DGG-BL and their prototypical genetic features (ID3 and TP53) had significant associations with patient outcomes that were different among aBL and pBL cohorts. These findings highlight shared pathogenesis between aBL and pBL, and establish genetic subtypes within BL that serve to delineate tumors with distinct molecular features, providing a new framework for epidemiologic, diagnostic, and therapeutic strategies.

Genome-wide discovery of somatic coding and noncoding mutations in pediatric endemic and sporadic Burkitt lymphoma.

Although generally curable with intensive chemotherapy in resource-rich settings, Burkitt lymphoma (BL) remains a deadly disease in older patients and in sub-Saharan Africa. Epstein-Barr virus (EBV) positivity is a feature in more than 90% of cases in malaria-endemic regions, and up to 30% elsewhere. However, the molecular features of BL have not been comprehensively evaluated when taking into account tumor EBV status or geographic origin. Through an integrative analysis of whole-genome and transcriptome data, we show a striking genome-wide increase in aberrant somatic hypermutation in EBV-positive tumors, supporting a link between EBV and activation-induced cytidine deaminase (AICDA) activity. In addition to identifying novel candidate BL genes such as SIN3A, USP7, and CHD8, we demonstrate that EBV-positive tumors had significantly fewer driver mutations, especially among genes with roles in apoptosis. We also found immunoglobulin variable region genes that were disproportionally used to encode clonal B-cell receptors (BCRs) in the tumors. These include IGHV4-34, known to produce autoreactive antibodies, and IGKV3-20, a feature described in other B-cell malignancies but not yet in BL. Our results suggest that tumor EBV status defines a specific BL phenotype irrespective of geographic origin, with particular molecular properties and distinct pathogenic mechanisms. The novel mutation patterns identified here imply rational use of DNA-damaging chemotherapy in some patients with BL and targeted agents such as the CDK4/6 inhibitor palbociclib in others, whereas the importance of BCR signaling in BL strengthens the potential benefit of inhibitors for PI3K, Syk, and Src family kinases among these patients.

Stepping behaviour contributes little to balance control against continuous mediolateral trunk perturbations.

Human bipedal gait is exceptionally stable, but the underlying strategies to maintain stability are unclear, especially in the frontal plane. Our study investigated balance strategies of healthy adults subjected to continuous mediolateral oscillations at the trunk during walking. We used a backpack with a passive inverted pendulum to create perturbations that were fixed, in-phase or out-of-phase with subjects' trunk. We evaluated subjects' corrective strategies and whether they yielded equivalent stability, measured by the margin of stability and the local divergence exponent. The margin of stability measure quantified adjustments in step behaviour relative to the centre of mass, and the local divergence exponent measure characterized the chaotic behaviour of the system throughout the entire trial. Among the conditions, there was no significant difference in the step width. We found a higher margin of stability for the out-of-phase condition and the lowest local divergence exponent for the in-phase condition and the highest for the fixed condition. These results indicate that the in-phase condition was more stable with respect to fluctuations throughout gait cycles, and the out-of-phase condition was more stable in terms of foot placement relative to centre of mass. To maintain equivalent or greater gait stability, subjects elected to reduce the motion of their centre of mass rather than alter step width. The reduction in centre of mass motion without a reduction in step width suggests direct control of the centre of mass to maintain stability was preferred over adjusting stepping behaviour.

TGF-beta-exposed plasmacytoid dendritic cells participate in Th17 commitment.

TGF-ß is required for both Foxp3(+) regulatory T cell (Treg) and Th17 commitment. Plasmacytoid DCs (pDC) have been shown to participate to both Treg and Th17 commitment as well. However, few studies have evaluated the direct effect of TGF-ß on pDC, and to our knowledge, no study has assessed the capacity of TGF-ß-exposed pDC to polarize naive CD4(+) T cells. In this paper, we show that TGF-ß-treated pDC favor Th17 but not Treg commitment. This process involves a TGF-ß/Smad signal, because TGF-ß treatment induced Smad2 phosphorylation in pDC and blockade of TGF-ß signaling with the SD208 TGF-ßRI kinase inhibitor abrogated Th17 commitment induced by TGF-ß-treated pDC. Moreover, TGF-ß mRNA synthesis and active TGF-ß release were induced in TGF-ß-treated pDC and anti-TGF-ß Ab blocked Th17 commitment. Unexpectedly, TGF-ß treatment also induced increased IL-6 production by pDC, which serves as the other arm for Th17 commitment driven by TGF-ß-exposed pDC, because elimination of IL-6-mediated signal with either IL-6- or IL-6Rα-specific Abs prevented Th17 commitment. The in vivo pathogenic role of TGF-ß-treated pDC was further confirmed in the Th17-dependent collagen-induced arthritis model in which TGF-ß-treated pDC injection significantly increased arthritis severity and pathogenic Th17 cell accumulation in the draining lymph nodes. Thus, our data reveal a previously unrecognized effect of TGF-ß-rich environment on pDC ability to trigger Th17 commitment. Such findings have implications in the pathogenesis of autoimmune diseases or immune responses against mucosal extracellular pathogens.

Identification of an alternative CD20 transcript variant in B-cell malignancies coding for a novel protein associated to rituximab resistance.

Human CD20 is a B-cell lineage-specific marker expressed by normal and leukemic B cells from the pre-B to the plasma-cell stages and is a target for rituximab (RTX) immunotherapy. A CD20 reverse transcriptase-polymerase chain reaction (PCR) on B-cell lines cDNA yielded a short PCR product (DeltaCD20) corresponding to a spliced mRNA transcript linking the exon 3 and exon 7 ends. We established here that this novel, alternatively spliced CD20 transcript is expressed and detectable at various levels in leukemic B cells, lymphoma B cells, in vivo tonsil- or in vitro CD40L-activated B cells, and Epstein-Barr virus (EBV)-transformed B cells, but not in resting CD19(+)- or CD20(+)-sorted B cells from peripheral blood or bone marrow of healthy donors. The truncated CD20 sequence is within the reading frame, codes a protein of 130 amino acids ( approximately 15-17 kDa) lacking large parts of the 4 transmembrane segments, suggesting that DeltaCD20 is a nonanchored membrane protein. We demonstrated the translation into a DeltaCD20 protein which is associated with the membrane CD20 protein and showed its involvement in RTX resistance. Study of patient samples before and after RTX resistance or escape confirms our in vitro findings.

Evidence for marine microfossils from amber.

Amber usually contains inclusions of terrestrial and rarely limnetic organisms that were embedded in the places were they lived in the amber forests. Therefore, it has been supposed that amber could not have preserved marine organisms. Here, we report the discovery amber-preserved marine microfossils. Diverse marine diatoms as well as radiolarians, sponge spicules, a foraminifer, and a spine of a larval echinoderm were found in Late Albian and Early Cenomanian amber samples of southwestern France. The highly fossiliferous resin samples solidified approximately 100 million years ago on the floor of coastal mixed forests dominated by conifers. The amber forests of southwestern France grew directly along the coast of the Atlantic Ocean and were influenced by the nearby sea: shells and remnants of marine organisms were probably introduced by wind, spray, or high tide from the beach or the sea onto the resin flows.

Load Carriage Device for Studying Medial-Lateral Stability of Walking: Design and Performance Evaluation.

When walking, the trunk not only oscillates in the vertical direction, but also in the medial-lateral direction. We developed a novel backpack that uses the medial-lateral oscillations of the trunk as input motion to drive medial-lateral oscillations of weight carried in a modified backpack. We use a combination of spring and damping elements to control mass motion, resulting in the ability to prescribe a variety of mass oscillation amplitudes and phase angles. We propose the device as a platform that can be used to study medial-lateral stability during walking. In particular, if the body's ability to predict medial-lateral centre-of-mass state is affected by an oscillating external mass. In this paper, we present the design, model, and model evaluation of our novel load carriage device. During testing, our model was able to predict the oscillation dynamics of the carried mass while walking: demonstrating its capability to create a variety of load carriage scenarios for the user.

Generating electricity while walking with a medial-lateral oscillating load carriage device.

Biomechanical energy harvesters generate electricity, from human movement, to power portable electronics. We developed an energy harvesting module to be used in conjunction with a load carriage device that allows carried mass in a backpack to oscillate in the medial-lateral (M-L) direction. The energy harvesting module was designed to tune M-L oscillations of the carried mass to create favourable device-user interaction. We tested seven energy harvesting conditions and compared them to walking with the device when the weight was rigidly fixed to the backpack frame. For each energy harvesting condition, we changed the external load resistance: altering how much electricity was being generated and how much the carried mass would oscillate. We then correlated device behaviour to the biomechanical response of the user. The energy harvesting load carriage system generated electricity with no significant increase in the metabolic power required to walk, when compared to walking with the carried weight rigidly fixed. The device was able to generate up to 0.22 ± 0.03 W of electricity, while walking with 9 kg of carried weight. The device also reduced the interaction forces experienced by the user, in the M-L direction, compared to walking with the device when the mass was rigidly fixed.

Altering Compliance of a Load Carriage Device in the Medial-Lateral Direction Reduces Peak Forces While Walking.

Altering mechanical compliance in load carriage structures has shown to reduce metabolic cost and accelerative forces of carrying weight. Currently, modifications to load carriage structures have been primarily targeted at vertical motion of the carried mass. No study to date has investigated altering load carriage compliance in the medial-lateral direction. We developed a backpack specifically for allowing a carried mass to oscillate in the horizontal direction, giving us the unique opportunity to understand the effects of lateral mass motion on human gait. Previous modelling work has shown that walking economy can be improved through the interaction of a bipedal model with a laterally oscillating walking surface. To test whether a laterally oscillating mass can experimentally improve walking economy, we systematically varied step width above and below the preferred level and compared the effects of carrying an oscillating and fixed mass. Walking with an oscillating mass was found to reduce the accelerative forces of load carriage in both horizontal and vertical directions. However, load eccentricity caused the vertical force component to create a significant bending moment in the frontal plane. Walking with an oscillating mass led to an increase in the metabolic energy expenditure during walking and an increase in positive hip work during stance. The device's ability to reduce forces experienced by the user, due to load carriage, holds promise. However, the requirement of additional metabolic energy to walk with the device requires future study to improve.

Overground vs. treadmill walking on biomechanical energy harvesting: An energetics and EMG study.

A biomechanical energy harvester is a wearable device that generates electricity from human motion. Walking on a treadmill has been used almost exclusively by researchers for studying the energetic effects of biomechanical energy harvesters and wearable robotics. A treadmill provides the advantage of having long duration trials within a stationary motion capture volume. However, no consensus exists on whether the results from treadmill walking accurately represent overground walking. We aim to investigate how a biomechanical energy harvester performs overground compared to on a treadmill by measuring energy expenditure and muscle activity. Participants (n=15) walked both overground and on a treadmill with and without a lower limb-driven biomechanical energy harvester. Energy expenditure was measured using indirect calorimetry and muscle activity was collected with surface electromyograms on seven superficial lower limb muscles. We observed a similar increase in metabolic cost of transport (Δoverground: 0.28±0.24J/kgm, Δtreadmill: 0.30±0.24J/kgm) from normal walking (overground: 2.56±0.33J/kgm, treadmill: 3.39±0.31J/kgm) to harvester walking (overground: 2.83±0.35J/kgm, treadmill: 3.69±0.32J/kgm) in both walking modes (p>0.05). This was accompanied a significant increase in muscle activity of select muscle groups (p<0.05). There was also a significant increase observed during walking on a treadmill compared to overground walking (p<0.05). In conclusion, our results suggest that although the metabolic cost of transport and muscle activation for treadmill walking is higher than that of overground, when studying the effects of a biomechanical energy harvester, treadmill will give similar net increases when compared to a controlled walking condition, such as normal walking, on the same walking surface.

Novel role for STAT3 in transcriptional regulation of NK immune cell targeting receptor MICA on cancer cells.

The role of natural killer group 2, member D receptor (NKG2D)-expressing natural killer (NK) cells in tumor immunosurveillance is now well established. Nevertheless, tumor progression occurs despite tumor immunosurveillance, leading to cancer persistence in immunocompetent hosts. STAT3 plays a pivotal role both in oncogenic functions and in immunosuppression. In this study, we investigated the role of STAT3 in suppressing NK cell-mediated immunosurveillance. Using a colorectal cancer cell line (HT29) that can poorly activate NK, we neutralized STAT3 with pharmacologic inhibitors or siRNA and found that this led to an increase in NK degranulation and IFN-γ production in a TGF-ß1-independent manner. Exposure to NKG2D-neutralizing antibodies partially restored STAT3 activity, suggesting that it prevented NKG2D-mediated NK cell activation. On this basis, we investigated the expression of NKG2D ligands after STAT3 activation in HT29, mesenchymal stem cells, and activated lymphocytes. The NK cell recognition receptor MHC class I chain-related protein A (MICA) was upregulated following STAT3 neutralization, and a direct interaction between STAT3 and the MICA promoter was identified. Because cross-talk between DNA damage repair and NKG2D ligand expression has been shown, we assessed the influence of STAT3 on MICA expression under conditions of genotoxic stress. We found that STAT3 negatively regulated MICA expression after irradiation or heat shock, including in lymphocytes activated by CD3/CD28 ligation. Together, our findings reveal a novel role for STAT3 in NK cell immunosurveillance by modulating the MICA expression in cancer cells.

Neuropilin-2 expression promotes TGF-ß1-mediated epithelial to mesenchymal transition in colorectal cancer cells.

Neuropilins, initially characterized as neuronal receptors, act as co-receptors for cancer related growth factors and were recently involved in several signaling pathways leading to cytoskeletal organization, angiogenesis and cancer progression. Then, we sought to investigate the ability of neuropilin-2 to orchestrate epithelial-mesenchymal transition in colorectal cancer cells. Using specific siRNA to target neuropilin-2 expression, or gene transfer, we first observed that neuropilin-2 expression endows HT29 and Colo320 for xenograft formation. Moreover, neuropilin-2 conferred a fibroblastic-like shape to cancer cells, suggesting an involvement of neuropilin-2 in epithelial-mesenchymal transition. Indeed, the presence of neuropilin-2 in colorectal carcinoma cell lines was correlated with loss of epithelial markers such as cytokeratin-20 and E-cadherin and with acquisition of mesenchymal molecules such as vimentin. Furthermore, we showed by surface plasmon resonance experiments that neuropilin-2 is a receptor for transforming-growth factor-ß1. The expression of neuropilin-2 on colon cancer cell lines was indeed shown to promote transforming-growth factor-ß1 signaling, leading to a constitutive phosphorylation of the Smad2/3 complex. Treatment with specific TGFß-type1 receptor kinase inhibitors restored E-cadherin levels and inhibited in part neuropilin-2-induced vimentin expression, suggesting that neuropilin-2 cooperates with TGFß-type1 receptor to promote epithelial-mesenchymal transition in colorectal cancer cells. Our results suggest a direct role of NRP2 in epithelial-mesenchymal transition and highlight a cross-talk between neuropilin-2 and TGF-ß1 signaling to promote cancer progression. These results suggest that neuropilin-2 fulfills all the criteria of a therapeutic target to disrupt multiple oncogenic functions in solid tumors.

Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) have been identified in humans and mice as a population of immature myeloid cells with the ability to suppress T cell activation. They accumulate in tumor-bearing mice and humans and have been shown to contribute to cancer development. Here, we have isolated tumor-derived exosomes (TDEs) from mouse cell lines and shown that an interaction between TDE-associated Hsp72 and MDSCs determines the suppressive activity of the MDSCs via activation of Stat3. In addition, tumor-derived soluble factors triggered MDSC expansion via activation of Erk. TDE-associated Hsp72 triggered Stat3 activation in MDSCs in a TLR2/MyD88-dependent manner through autocrine production of IL-6. Importantly, decreasing exosome production using dimethyl amiloride enhanced the in vivo antitumor efficacy of the chemotherapeutic drug cyclophosphamide in 3 different mouse tumor models. We also demonstrated that this mechanism is relevant in cancer patients, as TDEs from a human tumor cell line activated human MDSCs and triggered their suppressive function in an Hsp72/TLR2-dependent manner. Further, MDSCs from cancer patients treated with amiloride, a drug used to treat high blood pressure that also inhibits exosome formation, exhibited reduced suppressor functions. Collectively, our findings show in both mice and humans that Hsp72 expressed at the surface of TDEs restrains tumor immune surveillance by promoting MDSC suppressive functions.

Abnormal expression of only the CD34 part of a transgenic CD34/herpes simplex virus-thymidine kinase fusion protein is associated with ganciclovir resistance.

Donor T cell alloreactivity can be efficiently controlled by retrovirus-mediated ex vivo transfer of a "suicide" gene encoding the wild-type herpes simplex virus thymidine kinase (wtHSV-tk) gene, allowing gene-modified cells (GMCs) to be sensitive to ganciclovir (GCV). A limitation to this approach was related to the presence of an inactive form of the wtHSV-tk gene, resulting from alternative splicing. A corrected HSV-tk (cHSV-tk) gene was developed in order to circumvent this problem and was fused to a truncated splice variant of the human CD34 molecule (tCD34) suitable for the selection of retrovirally transduced GMCs. We demonstrate now that, despite this correction, CD34-positive, but GCV-resistant, HUT and primary GMCs can still be generated after transduction with a retroviral vector encoding a tCD34/cHSV-tk fusion protein (FuProtein). Deletions in the HSV-tk part of the transgene account in part for this resistance. However, an additional mechanism involving proteolytic-dependent "breakage" of the FuProtein has been observed: the CD34 part of the FuProtein can be detected by Western blot, separated from its HSV-tk part. Although the HSV-tk protein alone is not detectable in GCV-resistant tCD34/cHSV-tk-transduced HUT cells, it can be detected in 293T cells transduced with another tCD34/HSVTK fusion vector, demonstrating that a posttranslational effect leads to the breakage of the FuProtein. This is to our knowledge the first example of a loss of function of a FuProtein, of which one part is still expressed while the other one, suffering a selection pressure, is no longer detectable.

Role of STAT3 in CD4+CD25+FOXP3+ regulatory lymphocyte generation: implications in graft-versus-host disease and antitumor immunity.

Immunological tolerance is maintained by specialized subsets of T cells including CD4(+)CD25(+)FOXP3(+) regulatory cells (Treg). Previous studies established that Treg thymic differentiation or peripheral conversion depend on CD28 and Lck signaling. Moreover, foxp3 gene transfer in murine CD4(+)CD25(-) T lymphocytes results in the acquisition of suppressive functions. However, molecular pathways leading to FOXP3 expression remain to be described. In this study, we investigated the molecular events driving FOXP3 expression. We demonstrated that CD28 activation in CD4(+)CD25(-) T lymphocytes leads to STAT3 Tyr(705) phosphorylation in an Lck-dependent manner. STAT3 neutralization during naive peripheral CD4(+)CD25(-) T cell conversion into Treg through costimulation with TCR/CD28 and TGF-beta1, decreased FOXP3 expression, prevented the acquisition of suppressive functions and restored the ability of the converted lymphocytes to produce IL-2 and IFN-gamma. Furthermore, we observed that STAT3 ablation using small interfering RNA strategies inhibited FOXP3 expression and suppressive functions among naturally differentiated CD4(+)CD25(+) T lymphocytes, suggesting a direct role of STAT3 in Treg phenotype and function maintenance. CD4(+)CD25(+) T lymphocytes transduced with specific STAT3 small interfering RNA were devoid of suppressive functions and failed to control the occurrence of acute graft-vs-host disease. Finally, STAT3 inhibition in CD4(+) lymphocytes enhanced the anti-tumor immunity conferred by a lymphocyte adoptive transfer. In summary, our findings determine that STAT3 is critical in the molecular pathway required for FOXP3 expression. STAT3 modulation should be taken into account when assessing how regulatory T cells contribute to inflammatory diseases and tumor immunosurveillance.

The key role of vaccine vial monitors in routine and mass immunisation.

The vaccine vial monitor is an excellent monitoring device to assure that only non-heat damaged vaccine is administered to women and children. They help to extend access to immunisation, they increase the efficiency and reduce wastage in the cold-chain and, when visible on all vaccine vials of all types, they will help to streamline distribution systems for immunisation services of the 21st century in all developing countries.

Expression of the myeloid-associated marker CD33 is not an exclusive factor for leukemic plasmacytoid dendritic cells.

A new entity of acute leukemia coexpressing CD4(+)CD56(+) markers without any other lineage-specific markers has been identified recently as arising from lymphoid-related plasmacytoid dendritic cells (pDCs). In our laboratory, cells from a patient with such CD4(+)CD56(+) lineage-negative leukemia were unexpectedly found to also express the myeloid marker CD33. To confirm the diagnosis of pDC leukemia despite the CD33 expression, we demonstrated that the leukemic cells indeed exhibited pDC phenotypic and functional properties. In 7 of 8 other patients with CD4(+)CD56(+) pDC malignancies, we were able to confirm that the tumor cells expressed CD33 although with variable expression levels. CD33 expression was shown by flow cytometry, reverse transcriptase-polymerase chain reaction, and immunoblot analysis. Furthermore, CD33 monoclonal antibody stimulation of purified CD4(+)CD56(+) leukemic cells led to cytokine secretion, thus confirming the presence of a functional CD33 on these leukemic cells. Moreover, we found that circulating pDCs in healthy individuals also weakly express CD33. Overall, our results demonstrate that the expression of CD33 on CD4(+)CD56(+) lineage-negative cells should not exclude the diagnosis of pDC leukemia and underline that pDC-specific markers should be used at diagnosis for CD4(+)CD56(+) malignancies.

Retrovirus-mediated gene transfer in polyclonal T cells results in lower apoptosis and enhanced ex vivo cell expansion of CMV-reactive CD8 T cells as compared with EBV-reactive CD8 T cells.

To modulate alloreactivity after hematopoietic stem cell transplantation, "suicide" gene-modified donor T cells (GMCs) have been administered with an allogeneic T-cell-depleted marrow graft. We previously demonstrated that such GMCs, generated after CD3 activation, retrovirus-mediated transduction, and G418 selection, had an impaired Epstein-Barr virus (EBV) reactivity, likely to result in an altered control of EBV-induced lymphoproliferative disease. To further characterize the antiviral potential of GMCs, we compared the frequencies of cytomegalovirus (CMV)-specific CD8+ T (CMV-T) cells and EBV-specific CD8+ T (EBV-T) cells within GMCs from CMV- and EBV-double seropositive donors. Unlike anti-EBV responses, the anti-CMV responses were not altered by GMC preparation. During the first days of culture, CMV-T cells exhibited a lower level of CD3-induced apoptosis than did EBV-T cells. In addition, the CMV-T cells escaping initial apoptosis subsequently underwent a higher expansion rate than EBV-T cells. The differential early sensitivity to apoptosis could be in relation to the "recent activation" phenotype of EBV-T cells as evidenced by a higher level of CD69 expression. Furthermore, EBV-T cells were found to have a CD45RA-CD27+CCR7- effector memory phenotype, whereas CMV-T cells had a CD45RA+CD27-CCR7- terminal effector phenotype. Such differences could be contributive, because bulk CD8+CD27- cells had a higher expansion than did bulk CD8+CD27+ cells. Overall, ex vivo T-cell culture differentially affects apoptosis, long-term proliferation, and overall survival of CMV-T and EBV-T cells. Such functional differences need to be taken into account when designing cell and/or gene therapy protocols involving ex vivo T-cell manipulation.