Epstein-Barr Virus and immune status imprint the immunogenomics of non-Hodgkin lymphomas occurring in immune-suppressed environments.

Non-Hodgkin lymphomas (NHL) commonly occur in immune-deficient (ID) patients, both HIV-infected and transplanted, and are often EBV-driven with cerebral localization, raising the question of tumor immunogenicity, a critical issue for treatment responses. We investigated the immunogenomics of 68 lymphoproliferative disorders from 51 ID (34 posttransplant, 17 HIV+) and 17 immunocompetent patients. Overall, 72% were Large B Cells Lymphoma (LBCL) and 25% were primary central-nervous-system lymphoma (PCNSL) while 40% were EBV-positive. Tumor whole-exome and RNA sequencing, along with a bioinformatics pipeline allowed analysis of tumor mutational burden (TMB), tumor landscape and microenvironment (TME) and prediction of tumor neoepitopes. Both TMB (2.2 vs 3.4/Mb, p=0.001) and neoepitopes numbers (40 vs 200, p=0.00019) were lower in EBVpositive than in EBV-negative NHL, regardless of the immune status. In contrast both EBV and the immune status influenced the tumor mutational profile, with HNRNPF and STAT3 mutations exclusively observed in EBV-positive and ID NHL, respectively. Peripheral blood T-cell responses against tumor neoepitopes were detected in all EBV-negative cases but in only half EBV-positive ones, including responses against IgH-derived MHC-class-II restricted neoepitopes. The TME analysis showed higher CD8 T cell infiltrates in EBVpositive vs EBV-negative NHL, together with a more tolerogenic profile composed of Tregs, type-M2 macrophages and an increased expression of negative immune-regulators. Our results highlight that the immunogenomics of NHL in patients with immunodeficiency primarily relies on the tumor EBV status, while T cell recognition of tumor- and IgH-specific neoepitopes is conserved in EBV-negative patients, offering potential opportunities for future T cell-based immune therapies.

Comprehensive analysis of early T cell responses to acute Zika Virus infection during the first epidemic in Bahia, Brazil.

BACKGROUND: In most cases, Zika virus (ZIKV) causes a self-limited acute illness in adults, characterized by mild clinical symptoms that resolve within a few days. Immune responses, both innate and adaptive, play a central role in controlling and eliminating virus-infected cells during the early stages of infection. AIM: To test the hypothesis that circulating T cells exhibit phenotypic and functional activation characteristics during the viremic phase of ZIKV infection. METHODS: A comprehensive analysis using mass cytometry was performed on peripheral blood mononuclear cells obtained from patients with acute ZIKV infection (as confirmed by RT-PCR) and compared with that from healthy donors (HD). The frequency of IFN-γ-producing T cells in response to peptide pools covering immunogenic regions of structural and nonstructural ZIKV proteins was quantified using an ELISpot assay. RESULTS: Circulating CD4+ and CD8+ T lymphocytes from ZIKV-infected patients expressed higher levels of IFN-γ and pSTAT-5, as well as cell surface markers associated with proliferation (Ki-67), activation ((HLA-DR, CD38) or exhaustion (PD1 and CTLA-4), compared to those from HD. Activation of CD4+ and CD8+ memory T cell subsets, including Transitional Memory T Cells (TTM), Effector Memory T cells (TEM), and Effector Memory T cells Re-expressing CD45RA (TEMRA), was prominent among CD4+ T cell subset of ZIKV-infected patients and was associated with increased levels of IFN-γ, pSTAT-5, Ki-67, CTLA-4, and PD1, as compared to HD. Additionally, approximately 30% of ZIKV-infected patients exhibited a T cell response primarily directed against the ZIKV NS5 protein. CONCLUSION: Circulating T lymphocytes spontaneously produce IFN-γ and express elevated levels of pSTAT-5 during the early phase of ZIKV infection whereas recognition of ZIKV antigen results in the generation of virus-specific IFN-γ-producing T cells.

End-to-end quality assurance for stereotactic radiotherapy with Fricke-Xylenol orange-Gelatin gel dosimeter and dual-wavelength cone-beam optical CT readout.

PURPOSE: The end-to-end (E2E) quality assurance (QA) test is a unique tool for validating the treatment chain undergone by patients in external radiotherapy. It should be conducted in three dimensions (3D) to get accurate results. This study aims to implement these tests with Fricke-Xylenol orange-Gelatin (FXG) gel dosimeter and a newly developed dual-wavelength reading method on the Vista16™ optical Computed Tomography (CT) scanner (ModusQA) for three treatment techniques in stereotactic radiotherapy, on Novalis (Varian) and CyberKnife (Accuray) linear accelerators. METHODS: The tests were performed in head phantoms. Gel measurements were compared with planned dose distributions and measured by film and ion chamber measurements by plotting isodose curves and dose profiles, and by conducting a 3D local gamma-index analysis (2%/2mm criteria). RESULTS: Gamma passing rates were higher than 95 %. Point dose differences between treatment planning and gel and ion chamber measurements at the isocenter were < 2.3 % for both treatments delivered on the Novalis accelerator, while this difference was higher than 4 % for the treatment delivered on the CyberKnife, highlighting a small overdosing of the tumor volume. A good agreement was observed between gel and film dose profiles. CONCLUSIONS: This study presents the successful implementation of 3D E2E QA tests for stereotactic radiotherapy with FXG gel dosimetry and a dual-wavelength reading method on an optical CT scanner. This dosimetric method provides 3D absolute dose distributions in the 0.25 - 10 Gy dose range with a high spatial resolution and a dose uncertainty of around 2 % (k=1).

Identification of natural killer markers associated with fatal outcome in COVID-19 patients.

Introduction: Increasing evidence has shown that coronavirus disease 19 (COVID-19) severity is driven by a dysregulated immunological response. Previous studies have demonstrated that natural killer (NK) cell dysfunction underpins severe illness in COVID-19 patients, but have lacked an in-depth analysis of NK cell markers as a driver of death in the most critically ill patients. Methods: We enrolled 50 non-vaccinated hospitalized patients infected with the initial virus or the alpha variant of SARS-CoV-2 with moderate or severe illness, to evaluate phenotypic and functional features of NK cells. Results: Here, we show that, consistent with previous studies, evolution NK cells from COVID-19 patients are more activated, with the decreased activation of natural cytotoxicity receptors and impaired cytotoxicity and IFN-γ production, in association with disease regardless of the SARS-CoV-2 strain. Fatality was observed in 6 of 17 patients with severe disease; NK cells from all of these patients displayed a peculiar phenotype of an activated memory-like phenotype associated with massive TNF-α production. Discussion: These data suggest that fatal COVID-19 infection is driven by an uncoordinated inflammatory response in part mediated by a specific subset of activated NK cells.

Preliminary dosimetric characterization of EDBreast gel.

Purpose. EDBreast gel is an alternative Fricke gel dosimeter, read by Magnetic Resonance Imaging, in which sucrose is added to lower diffusion effects. This paper aims at determining the dosimetric characteristics of this dosimeter.Methods. The characterization has been performed in high energy photon beams. The dose-response of the gel has been evaluated as well as its detection limit, its fading effects, the reproducibility of its response and its stability over time. Its energy and dose-rate dependence has been investigated, and the overall dose uncertainty budget established. Once characterized, the dosimetry method has been applied to a simple reference irradiation case in a 6 MV photon beam, with the measurement of the lateral dose profile of a 2 × 2 cm2field. The results have been compared with microDiamond measurements.Results. In addition to its low diffusivity, the gel presents a high sensitivity, no dose-rate dependence consideringTPR20-10values ranging from 0.66 to 0.79 and an energy response comparable to ionization chambers. However, its non-linear dose-response induces a high uncertainty on the measured dose (8 % (k=1) at 20 Gy) and reproducibility issues. The profile measurements displayed discrepancies compared to the microDiamond due to diffusion effects. The appropriate spatial resolution was estimated using the diffusion coefficient.Conclusion. EDBreast gel dosimeter presents interesting characteristics for applications in clinics, but the linearity of its dose-response should be improved to lower the uncertainties and to enhance the reproducibility.

Plasticity of natural killer cells in pregnant patients infected with SARS-CoV-2 and their neonates during childbirth.

The COVID-19 pandemic has occurred due to infection caused by the SARS-CoV-2 coronavirus, which impacts gestation and pregnancy. In SARS-CoV-2 infection, only very rare cases of vertical transmission have been reported, suggesting that fetal immune imprinting due to a maternal infection is probably a result of changes in maternal immunity. Natural killer (NK) cells are the leading maternal immune cells that act as a natural defense system to fight infections. They also play a pivotal role in the establishment and maintenance of pregnancy. While peripheral NK cells display specific features in patients infected with SARS-CoV-2 in the general population, information remains elusive in pregnant mothers and neonates. In the present study, we analyzed the characteristics of NK cells isolated from both neonatal umbilical cord blood and maternal peripheral blood close to the time of delivery. Phenotype and functions were compared in 18 healthy pregnant women and 34 COVID-19 patients during pregnancy within an ongoing infection (PCR+; N = 15) or after recovery (IgG+PCR-; N = 19). The frequency of NK cells from infected women and their neonates was correlated with the production of inflammatory cytokines in the serum. The expression of NKG2A and NKp30, as well as degranulation of NK cells in pregnant women with ongoing infection, were both negatively correlated to estradiol level. Furthermore, NK cells from the neonates born to infected women were significantly decreased and also correlated to estradiol level. This study highlights the relationship between NK cells, inflammation, and estradiol in patients with ongoing infection, providing new insights into the impact of maternal SARS-CoV-2 infection on the neonate.

Memory CD4+ T-Cell Lymphocytic Angiopathy in Fatal Forms of COVID-19 Pulmonary Infection.

The immunopathological pulmonary mechanisms leading to Coronavirus Disease (COVID-19)-related death in adults remain poorly understood. Bronchoalveolar lavage (BAL) and peripheral blood sampling were performed in 74 steroid and non-steroid-treated intensive care unit (ICU) patients (23-75 years; 44 survivors). Peripheral effector SARS-CoV-2-specific T cells were detected in 34/58 cases, mainly directed against the S1 portion of the spike protein. The BAL lymphocytosis consisted of T cells, while the mean CD4/CD8 ratio was 1.80 in non-steroid- treated patients and 1.14 in steroid-treated patients. Moreover, strong BAL SARS-CoV-2 specific T-cell responses were detected in 4/4 surviving and 3/3 non-surviving patients. Serum IFN-γ and IL-6 levels were decreased in steroid-treated patients when compared to non-steroid treated patients. In the lung samples from 3 (1 non-ICU and 2 ICU) additional deceased cases, a lymphocytic memory CD4 T-cell angiopathy colocalizing with SARS-CoV-2 was also observed. Taken together, these data show that disease severity occurs despite strong antiviral CD4 T cell-specific responses migrating to the lung, which could suggest a pathogenic role for perivascular memory CD4 T cells upon fatal COVID-19 pneumonia.

Impact of Anti PD-1 Immunotherapy on HIV Reservoir and Anti-Viral Immune Responses in People Living with HIV and Cancer.

The role of immune checkpoints (ICPs) in both anti-HIV T cell exhaustion and HIV reservoir persistence, has suggested that an HIV cure therapeutic strategy could involve ICP blockade. We studied the impact of anti-PD-1 therapy on HIV reservoirs and anti-viral immune responses in people living with HIV and treated for cancer. At several timepoints, we monitored CD4 cell counts, plasma HIV-RNA, cell associated (CA) HIV-DNA, EBV, CMV, HBV, HCV, and HHV-8 viral loads, activation markers, ICP expression and virus-specific T cells. Thirty-two patients were included, with median follow-up of 5 months. The CA HIV-DNA tended to decrease before cycle 2 (p = 0.049). Six patients exhibited a ≥0.5 log10 HIV-DNA decrease at least once. Among those, HIV-DNA became undetectable for 10 months in one patient. Overall, no significant increase in HIV-specific immunity was observed. In contrast, we detected an early increase in CTLA-4 + CD4+ T cells in all patients (p = 0.004) and a greater increase in CTLA-4+ and TIM-3 + CD8+ T cells in patients without HIV-DNA reduction compared to the others (p ≤ 0.03). Our results suggest that ICP replacement compensatory mechanisms might limit the impact of anti-PD-1 monotherapy on HIV reservoirs, and pave the way for combination ICP blockade in HIV cure strategies.

Fricke-Xylenol orange-Gelatin gel characterization with dual wavelength cone-beam optical CT scanner for applications in stereotactic and dynamic radiotherapy.

PURPOSE: This study is about the development of a new dual wavelength reading method of Fricke-Xylenol orange-Gelatin (FXG) gel dosimeters on the Vista16™ optical Computed Tomography (CT) scanner to perform 3D dose distribution measurements in stereotactic and dynamic radiotherapy treatments. METHODS: The dosimetric characteristics of an optimized FXG gel composition and its optical CT readout have been evaluated. A dual wavelength reading method has been developed on the CT scanner at wavelengths 590 nm and 633 nm. Small-field dose profile measurements with FXG gel and microDiamond (PTW) detectors were compared by γ-index analysis (0.5%/0.5 mm) to validate this method. RESULTS: This reading method exhibits linear calibration curves in the 0-4 Gy and 2-10 Gy dose ranges at 590 nm and 633 nm respectively. The absorbed dose values below 4 Gy, measured at 590 nm, and those above 4 Gy, measured at 633 nm, are combined to plot a complete profile. A γ passing rate of 93.4% was achieved. CONCLUSIONS: The new reading method of FXG gel dosimeters has been implemented on the Vista16™ scanner to span absorbed doses representative of stereotactic and dynamic radiotherapy treatments and enable 3D measurements in tumor volumes and surrounding healthy tissues. Small-field profile measurements validated this reading method as FXG gel dosimeters and microDiamond detectors were in very close agreement. This dosimetric method is a promising candidate for 3D quality assurance end-to-end tests in stereotactic and dynamic radiotherapy.

Iron-loaded transferrin potentiates erythropoietin effects on erythroblast proliferation and survival: a novel role through transferrin receptors.

Red blood cell production, or erythropoiesis, is a proliferative process that requires tight regulation. Erythropoietin (Epo) is a glycoprotein cytokine that plays a major role in erythropoiesis by triggering erythroid progenitors/precursors of varying sensitivity. The concentration of Epo in bone marrow is hypothesized to be suboptimal, and the survival of erythroid cells has been suggested to depend on Epo sensitivity. However, the key factors that control Epo sensitivity remain unknown. Two types of transferrin receptors (TfRs), TfR1 and TfR2, are known to play a role in iron uptake in erythroid cells. Here, we hypothesized that TfRs may additionally modulate Epo sensitivity during erythropoiesis by modulating Epo receptor (EpoR) signaling. Using an Epo-sensitive UT-7 (UT7/Epo) erythroid cell and human erythroid progenitor cell models, we report that iron-loaded transferrin, that is, holo-transferrin (holo-Tf), synergizes with suboptimal Epo levels to improve erythroid cell survival, proliferation, and differentiation. This is accomplished via the major signaling pathways of erythropoiesis, which include signal transducer and activator of transcription 5 (STAT5), mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and phosphoinositide-3-kinase (PI3K)/AKT. Furthermore, we found that this cooperation is improved by, but does not require, the internalization of TfR1. Interestingly, we observed that loss of TfR2 stabilizes EpoR levels and abolishes the beneficial effects of holo-Tf. Overall, these data reveal novel signaling properties of TfRs, which involve the regulation of erythropoiesis through EpoR signaling.

2D multislice MP2RAGE sequence for fast T1 mapping at 7 T: Application to mouse imaging and MR thermometry.

PURPOSE: To develop a 2D radial multislice MP2RAGE sequence for fast and reliable T1 mapping at 7 T in mice and for MR thermometry. METHODS: The 2D-MP2RAGE sequence was performed with the following parameters: TI1 -TI2 -MP2RAGETR = 1000-3000-9000 ms. The multiple dead times within the sequence were used for interleaved multislice acquisition, enabling one to acquire six slices in 9 seconds. The excitation pulse shape, inversion selectivity, and interslice gap were optimized. In vitro comparison with the inversion-recovery sequence was performed. The T1 variations with temperature were measured on tubes with T1 ranging from 800 ms to 2000 ms. The sequence was used to acquire T1 maps continuously during 30 minutes on the brain and abdomen of healthy mice. RESULTS: A three-lobe cardinal sine excitation pulse, combined with an inversion slice thickness and an interslice gap of respectively 150% and 50% of the imaging slice thickness, led to a SD and bias of the T1 measurements below 1% and 2%, respectively. A linear dependence of T1 with temperature was measured between 10°C and 60°C. In vivo, less than 1% variation was measured between successive T1 maps in the mouse brain. In the abdomen, no obvious in-plane motion artifacts were observed but respiratory motion in the slice dimension led to 6% T1 underestimation. CONCLUSION: The multislice MP2RAGE sequence could be used for fast whole-body T1 mapping and MR thermometry. Its reconstruction method would enable on-the-fly reconstruction.

A variant erythroferrone disrupts iron homeostasis in SF3B1-mutated myelodysplastic syndrome.

Myelodysplastic syndromes (MDS) with ring sideroblasts are hematopoietic stem cell disorders with erythroid dysplasia and mutations in the SF3B1 splicing factor gene. Patients with MDS with SF3B1 mutations often accumulate excessive tissue iron, even in the absence of transfusions, but the mechanisms that are responsible for their parenchymal iron overload are unknown. Body iron content, tissue distribution, and the supply of iron for erythropoiesis are controlled by the hormone hepcidin, which is regulated by erythroblasts through secretion of the erythroid hormone erythroferrone (ERFE). Here, we identified an alternative ERFE transcript in patients with MDS with the SF3B1 mutation. Induction of this ERFE transcript in primary SF3B1-mutated bone marrow erythroblasts generated a variant protein that maintained the capacity to suppress hepcidin transcription. Plasma concentrations of ERFE were higher in patients with MDS with an SF3B1 gene mutation than in patients with SF3B1 wild-type MDS. Thus, hepcidin suppression by a variant ERFE is likely responsible for the increased iron loading in patients with SF3B1-mutated MDS, suggesting that ERFE could be targeted to prevent iron-mediated toxicity. The expression of the variant ERFE transcript that was restricted to SF3B1-mutated erythroblasts decreased in lenalidomide-responsive anemic patients, identifying variant ERFE as a specific biomarker of clonal erythropoiesis.

The fraction of CD117/c-KIT-expressing erythroid precursors predicts ESA response in low-risk myelodysplastic syndromes.

BACKGROUND: Compelling evidence has emerged for the relevance of flow cytometry (FC) in the diagnostic work-up of myelodysplastic syndromes (MDS) but due to technical issues, the erythroid lineage has been under investigated, specifically in the therapeutic context. METHODS: Using the "no red cell lysis" method developed to set up the RED-score, we specifically quantified the fraction of CD117/c-KIT-expressing erythroid precursors in a cohort of 144 MDS patients and studied the correlation with response to erythropoiesis-stimulating agents (ESA) in a sub cohort of 63 low-risk MDS patients. RESULTS: We confirmed the previously reported increase in CD117/c-KIT-expressing erythroid precursors in a subset of MDS patients and demonstrated a strong association between a cut off of CD117/c-KIT-expressing erythroid precursors ≥3% and ESA response (P = 0.001), independent of red blood cell requirement. From our observations, we hypothesized that a decrease in CD117/c-KIT-expressing erythroid precursors could be a mechanism of ESA failure. Moreover, the fraction of CD117/c-KIT-expressing erythroid precursors was correlated with progression-free survival in low-risk MDS patients (P = 0.018). In vitro, we demonstrated in an EPO dependent cell line that CD117/c-KIT expression is necessary for cell survival under EPO stimulation. CONCLUSIONS: The quantification of the CD117/c-KIT-expressing erythroid precursors could be proposed as a new theranostic and prognostic marker in MDS treated by ESA. Future studies will be required to determine whether modulating CD117/c-KIT expression and signaling could be used to improve anemia in MDS. © 2019 International Clinical Cytometry Society.

Dyserythropoiesis evaluated by the RED score and hepcidin:ferritin ratio predicts response to erythropoietin in lower-risk myelodysplastic syndromes.

Erythropoiesis-stimulating agents are generally the first line of treatment of anemia in patients with lower-risk myelodysplastic syndrome. We prospectively investigated the predictive value of somatic mutations, and biomarkers of ineffective erythropoiesis including the flow cytometry RED score, serum growth-differentiation factor-15, and hepcidin levels. Inclusion criteria were no prior treatment with erythropoiesis-stimulating agents, low- or intermediate-1-risk myelodysplastic syndrome according to the International Prognostic Scoring System, and a hemoglobin level <10 g/dL. Patients could be red blood cell transfusion-dependent or not and were given epoetin zeta 40 000 IU/week. Serum erythropoietin level, iron parameters, hepcidin, flow cytometry Ogata and RED scores, and growth-differentiation factor-15 levels were determined at baseline, and molecular analysis by next-generation sequencing was also conducted. Erythroid response (defined according to the International Working Group 2006 criteria) was assessed at week 12. Seventy patients, with a median age of 78 years, were included in the study. There were 22 patients with refractory cytopenia with multilineage dysplasia, 19 with refractory cytopenia with unilineage dysplasia, 14 with refractory anemia with ring sideroblasts, four with refractory anemia with excess blasts-1, six with chronic myelomonocytic leukemia, two with del5q-and three with unclassifiable myelodysplastic syndrome. According to the revised International Prognostic Scoring System, 13 had very low risk, 47 had low risk, nine intermediate risk and one had high-risk disease. Twenty patients were transfusion dependent. Forty-eight percent had an erythroid response and the median duration of the response was 26 months. At baseline, non-responders had significantly higher RED scores and lower hepcidin:ferritin ratios. In multivariate analysis, only a RED score >4 (P=0.05) and a hepcidin:ferritin ratio <9 (P=0.02) were statistically significantly associated with worse erythroid response. The median response duration was shorter in patients with growth-differentiation factor-15 >2000 pg/mL and a hepcidin:ferritin ratio <9 (P=0.0008 and P=0.01, respectively). In multivariate analysis, both variables were associated with shorter response duration. Erythroid response to epoetin zeta was similar to that obtained with other erythropoiesis-stimulating agents and was correlated with higher baseline hepcidin:ferritin ratio and lower RED score. ClinicalTrials.gov registration: NCT 03598582.

Architectural and functional heterogeneity of hematopoietic stem/progenitor cells in non-del(5q) myelodysplastic syndromes.

Myelodysplastic syndromes (MDSs) are hematopoietic stem cell disorders in which recurrent mutations define clonal hematopoiesis. The origin of the phenotypic diversity of non-del(5q) MDS remains unclear. Here, we investigated the clonal architecture of the CD34+CD38- hematopoietic stem/progenitor cell (HSPC) compartment and interrogated dominant clones for MDS-initiating cells. We found that clones mainly accumulate mutations in a linear succession with retention of a dominant subclone. The clone detected in the long-term culture-initiating cell compartment that reconstitutes short-term human hematopoiesis in xenotransplantation models is usually the dominant clone, which gives rise to the myeloid and to a lesser extent to the lymphoid lineage. The pattern of mutations may differ between common myeloid progenitors (CMPs), granulomonocytic progenitors (GMPs), and megakaryocytic-erythroid progenitors (MEPs). Rare STAG2 mutations can amplify at the level of GMPs, from which it may drive the transformation to acute myeloid leukemia. We report that major truncating BCOR gene mutation affecting HSPC and CMP was beneath the threshold of detection in GMP or MEP. Consistently, BCOR knock-down (KD) in normal CD34+ progenitors modifies their granulocytic and erythroid differentiation. Clonal architecture of the HSPC compartment and mutations selected during differentiation contribute to the phenotypic heterogeneity of MDS. Defining the hierarchy of driver mutations provides insights into the process of transformation and may guide the search for novel therapeutic strategies.

Disruption of the Phosphate Transporter Pit1 in Hepatocytes Improves Glucose Metabolism and Insulin Signaling by Modulating the USP7/IRS1 Interaction.

The liver plays a central role in whole-body lipid and glucose homeostasis. Increasing dietary fat intake results in increased hepatic fat deposition, which is associated with a risk for development of insulin resistance and type 2 diabetes. In this study, we demonstrate a role for the phosphate inorganic transporter 1 (PiT1/SLC20A1) in regulating metabolism. Specific knockout of Pit1 in hepatocytes significantly improved glucose tolerance and insulin sensitivity, enhanced insulin signaling, and decreased hepatic lipogenesis. We identified USP7 as a PiT1 binding partner and demonstrated that Pit1 deletion inhibited USP7/IRS1 dissociation upon insulin stimulation. This prevented IRS1 ubiquitination and its subsequent proteasomal degradation. As a consequence, delayed insulin negative feedback loop and sustained insulin signaling were observed. Moreover, PiT1-deficient mice were protected against high-fat-diet-induced obesity and diabetes. Our findings indicate that PiT1 has potential as a therapeutic target in the context of metabolic syndrome, obesity, and diabetes.

EKLF-driven PIT1 expression is critical for mouse erythroid maturation in vivo and in vitro.

The PIT1/SLC20A1 protein, a well-described sodium/phosphate cotransporter and retrovirus receptor, has been identified recently as a modular of proliferation and apoptosis in vitro. The targeted deletion of the PIT1 gene in mice revealed a lethal phenotype due to severe anemia attributed to defects in liver development. However, the presence of immature erythroid cells associated with impaired maturation of the globin switch led us to investigate the role of PIT1 in hematopoietic development. In the present study, specific deletion of PIT1 in the hematopoietic system and fetal liver transplantation experiments demonstrated that anemia was associated with an erythroid cell- autonomous defect. Moreover, anemia was not due to RBC destruction but rather to maturation defects. Because Erythroid Krüppel-like Factor (EKLF)-knockout mice showed similar maturation defects, we investigated the functional link between PIT1 and EKLF. We demonstrated that EKLF increases PIT1 expression during RBC maturation by binding to its promoter in vivo and that shRNA-driven depletion of either PIT1 or EKLF impairs erythroid maturation of G1E cells in vitro, whereas reexpression of PIT1 in EKLF-depleted G1E cells partially restores erythroid maturation. This is the first demonstration of a physiologic involvement of PIT1 in erythroid maturation in vivo.

Identification of a novel transport-independent function of PiT1/SLC20A1 in the regulation of TNF-induced apoptosis.

PiT1/SLC20A1 is a sodium-dependent P(i) transporter expressed by most mammalian cells. Interestingly, PiT1 transcription has been shown to be up-regulated by the tumor necrosis factor α (TNF), and we have now investigated the possible involvement of PiT1 in TNF-induced apoptosis. We show that PiT1-depleted cells are more sensitive to the proapoptotic activity of TNF (i.e. when the antiapoptotic NFκB pathway is inactivated). These observations were made in the human HeLa cancer cell line either transiently or stably depleted in PiT1 by RNA interference and in immortalized mouse embryonic fibroblasts isolated from PiT1 knock-out embryos. Depletion of the closely related family member PiT2 had no effect on TNF-induced apoptosis, showing that this effect was specific to PiT1. The increased sensitivity of PiT1-depleted cells was evident regardless of the presence or absence of extracellular P(i), suggesting that a defect in P(i) uptake was not involved in the observed phenotype. Importantly, we show that the re-expression of a P(i) uptake mutant of PiT1 in PiT1(-/-) mouse embryonic fibroblasts delays apoptosis as efficiently as the WT protein, showing that this function of PiT1 is unrelated to its transport activity. Caspase-8 is more activated in PiT1-depleted cells, and our data reveal that the sustained activation of the MAPK JNK is up-regulated in response to TNF. JNK activity is actually involved in PiT1-depleted cell death because specific JNK inhibitors delay apoptosis.

Efficient virus-induced gene silencing in Arabidopsis using a 'one-step' TYMV-derived vector.

Virus-induced gene silencing (VIGS) is an important tool for the analysis of gene function in plants. This technique exploits recombinant viral vectors harbouring fragments of plant genes in their genome to generate double-stranded RNAs that initiate homology-dependent silencing of the target gene. Several viral VIGS vectors have already been successfully used in reverse-genetics studies of a variety of processes occurring in plants. Here, we show that a viral vector derived from Turnip yellow mosaic virus (TYMV) has the ability to induce VIGS in Arabidopsis thaliana, accession Col-0, provided that it carries an inverted-repeat fragment of the target gene. Robust and reliable gene silencing was observed when plants were inoculated simply by abrasion with intact plasmid DNA harbouring a cDNA copy of the viral genome, thus precluding the need for in vitro transcription, biolistic or agroinoculation procedures. Our results indicate that a 76 bp fragment is sufficient to cause gene silencing in leaves, stems and flowers, and that the TYMV-derived vector also has the ability to target genes expressed in meristematic tissues. The VIGS vector described here may thus represent an ideal tool for improving high-throughput functional genomics in Arabidopsis.