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Insect pest infestations are a worldwide problem as a result of human actions. Among them, Aedes aegypti stands out as vector of several diseases. Like other pests, A. aegypti has become resistant to the insecticide used for its control, thus harming the local fauna of non-target insects. Therefore, the aim of this study was to study the physiological selectivity of social wasps exposed to Malathion, as well as to investigate possible resistance to this insecticide. Adults of the swarming species Polybia platycephala and Protopolybia sedula and the independent species Polistes versicolor were exposed to four Malathion concentrations. Overall, Malathion was more selective to P. versicolor and more toxic to P. platycephala, and despite the decrease in the concentration of the product recommended by the manufacturer, which in theory would be sublethal doses, social wasps still exhibited high mortality rates. In comparing the mortalities of species collected in an area where there is no application of the insecticide with an area where there is regular application, the data showed an indication that populations of non-target insects are also becoming resistant to insecticides, especially in urban environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Monocytes are considered crucial actors of inflammation in sickle cell disease (SCD), being responsible for an increased production of proinflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6. Although a role of free heme released by intravascular hemolysis has been suspected, the mechanisms underlying monocyte activation in patients with SCD remain unknown. Using purified human hemoglobin (Hb), we demonstrate herein, that cell-free HbS, unlike HbA or heme, is responsible for a major enhancement in the expression of proinflammatory cytokines by human monocytes. This effect was found mediated by direct interaction with the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex, resulting in the activation of both the nuclear factor-κB (NF-κB) and type I interferon pathways. In Townes SCD mice, injection of HbS, unlike HbA, was responsible for an increased production of proinflammatory cytokines, which was prevented by the TLR4 inhibitor, TAK-242. Our results reveal a novel mechanism of monocyte activation and systemic inflammation in SCD, which opens new promising therapeutic perspectives targeting the HbS-TLR4 interaction., (© 2022 by The American Society of Hematology.)

Targeting immune checkpoints, such as Programmed cell Death 1 (PD1), has improved survival in cancer patients by restoring antitumor immune responses. Most patients, however, relapse or are refractory to immune checkpoint blocking therapies. Neuropilin-1 (NRP1) is a transmembrane glycoprotein required for nervous system and angiogenesis embryonic development, also expressed in immune cells. We hypothesized that NRP1 could be an immune checkpoint co-receptor modulating CD8 + T cells activity in the context of the antitumor immune response. Here, we show that NRP1 is recruited in the cytolytic synapse of PD1 + CD8 + T cells, cooperates and enhances PD-1 activity. In mice, CD8 + T cells specific deletion of Nrp1 improves anti-PD1 antibody antitumor immune responses. Likewise, in human metastatic melanoma, the expression of NRP1 in tumor infiltrating CD8 + T cells predicts poor outcome of patients treated with anti-PD1. NRP1 is a promising target to overcome resistance to anti-PD1 therapies., Competing Interests: The authors declare no competing interests., (© 2022.)

Background: Erythrocytosis is a hematological disorder usually related to hematopoietic stem cell somatic mutations. However, unexplained erythrocytosis remains frequent. In this study, we evaluated the involvement of IgA1, a regulator of erythropoiesis also implicated in IgA nephropathy (IgAN) pathophysiology, in unexplained polycythemia/erythrocytosis (PE) of IgAN patients., Methods: IgAN-PE patients' serum was collected, analyzed and used to study IgA1 effect on proliferation and differentiation of erythroid progenitors. Hematological parameters of transgenic mice for human alpha1 heavy chain were studied. Multicentric observational cohorts of chronic kidney disease (CKD) patients, including both native kidney diseases and renal transplants, were studied to analyze patient hemoglobin levels., Findings: We retrospectively identified 6 patients with IgAN and unexplained PE. In large CKD cohorts, IgAN was associated with PE in 3.5% of patients (p<0.001 compared to other nephropathies). IgAN was an independent factor associated with higher hemoglobin levels (13.1g/dL vs 12.2 g/dL, p=0.01). During post-transplant anemia, anemia recovery was faster in IgAN patients. Elevated polymeric/monomeric IgA1 ratio as well as high Gd-IgA1 rate were observed in circulating IgA1 of the 6 IgAN-PE patients as compared with control or IgAN patients without PE. IgA1 from these patients increased the sensitivity of erythroid progenitors to Epo. In mice, we also observed an elevation of hematocrit in alpha1 knock-in mice compared to wild type controls., Interpretation: These data identify a new etiology of erythrocytosis and demonstrate the role of pIgA1 in human erythropoiesis. This syndrome of IgA-related erythrocytosis should be investigated in case of unexplained erythrocytosis and renal disease., Funding: This work was supported by INSERM (French national institute for health and medical research), Labex GRex and Imagine Institute (Paris, France)., Competing Interests: Declaration of interests No conflict of interest to disclose, (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only currently available curative treatment for sickle cell disease (SCD). However, the effects of HSCT on SCD pathophysiology are poorly elucidated. Here, we assessed red blood cell (RBC) adhesiveness, intensity of hemolysis, vascular tone markers and systemic inflammation, in SCD patients treated with allogeneic HSCT. Thirty-two SCD patients were evaluated before and on long-term follow-up after HSCT. Overall survival was 94% with no severe (grade III-IV) graft- vs -host disease and a 22% rejection rate (graft failure). Hematological parameters, reticulocyte counts, and levels of lactate dehydrogenase (LDH), endothelin-1 and VCAM-1 normalized in SCD patients post-HSCT. Expression of adhesion molecules on reticulocytes and RBC was lower in patients with sustained engraftment. Levels of IL-18, IL-15 and LDH were higher in patients that developed graft failure. Increased levels of plasma pro-inflammatory cytokines, mainly TNF-α, were found in SCD patients long-term after transplantation. SCD patients with sustained engraftment after allo-HSCT showed decreased reticulocyte counts and adhesiveness, diminished hemolysis, and lower levels of vascular tonus markers. Nevertheless, systemic inflammation persists for at least five years after transplantation, indicating that allo-HSCT does not equally affect all aspects of SCD pathophysiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 de Azevedo, Costa, Lima, Maciel, Palma, Darrigo-Júnior, Setanni Grecco, Stracieri, Elias, Pieroni, Guerino-Cunha, Pinto, De Santis, Covas, Hermine, Simões, Oliveira and Malmegrim.)

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., Competing Interests: Conflict of interest disclosure The authors do not have any conflicts of interest to declare in relation to this work., (Copyright © 2021 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

β-thalassemia major (β-TM) is an inherited hemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of hemoglobin, leading to the accumulation of free a-globin chains that aggregate and cause ineffective erythropoiesis. We have previously demonstrated that terminal erythroid maturation requires a transient activation of caspase-3 and that the chaperone Heat Shock Protein 70 (HSP70) accumulates in the nucleus to protect GATA-1 transcription factor from caspase-3 cleavage. This nuclear accumulation of HSP70 is inhibited in human β-TM erythroblasts due to HSP70 sequestration in the cytoplasm by free a-globin chains, resulting in maturation arrest and apoptosis. Likewise, terminal maturation can be restored by transduction of a nuclear-targeted HSP70 mutant. Here we demonstrate that in normal erythroid progenitors, HSP70 localization is regulated by the exportin-1 (XPO1), and that treatment of β-thalassemic erythroblasts with an XPO1 inhibitor increased the amount of nuclear HSP70, rescued GATA-1 expression and improved terminal differentiation, thus representing a new therapeutic option to ameliorate ineffective erythropoiesis of β-TM.

The most common treatment for patients with sickle cell disease (SCD) is the chemotherapeutic hydroxyurea, a therapy with pleiotropic effects, including increasing fetal hemoglobin (HbF) in red blood cells and reducing adhesion of white blood cells to the vascular endothelium. Hydroxyurea has been proposed to mediate these effects through a mechanism of increasing cellular cGMP levels. An alternative path to increasing cGMP levels in these cells is through the use of phosphodiesterase-9 inhibitors that selectively inhibit cGMP hydrolysis and increase cellular cGMP levels. We have developed a novel, potent and selective phosphodiesterase-9 inhibitor (IMR-687) specifically for the treatment of SCD. IMR-687 increased cGMP and HbF in erythroid K562 and UT-7 cells and increased the percentage of HbF positive erythroid cells generated in vitro using a two-phase liquid culture of CD34 + progenitors from sickle cell blood or bone marrow. Oral daily dosing of IMR-687 in the Townes transgenic mouse SCD model, increased HbF and reduced red blood cell sickling, immune cell activation and microvascular stasis. The IMR-687 reduction in red blood cell sickling and immune cell activation was greater than that seen with physiological doses of hydroxyurea. In contrast to other described phosphodiesterase-9 inhibitors, IMR-687 did not accumulate in the central nervous system, where it would inhibit phosphodiesterase-9 in neurons, or alter rodent behavior. IMR-687 was not genotoxic or myelotoxic and did not impact fertility or fetal development in rodents. These data suggest that IMR-687 may offer a safe and effective oral alternative for hydroxyurea in the treatment of SCD., (Copyright© 2020 Ferrata Storti Foundation.)

Sickle cell disease (SCD), considered the most common monogenic disease worldwide, is a severe hemoglobin disorder. Although the genetic and molecular bases have long been characterized, the pathophysiology remains incompletely elucidated and therapeutic options are limited. It has been increasingly suggested that innate immune cells, including monocytes, neutrophils, invariant natural killer T cells, platelets and mast cells, have a role in promoting inflammation, adhesion and pain in SCD. Here we provide a thorough review of the involvement of these novel, major protagonists in SCD pathophysiology, highlighting recent evidence for innovative therapeutic perspectives., (Copyright© 2020 Ferrata Storti Foundation.)

Vitamin D (VD) is a known differentiating agent, but the role of VD receptor (VDR) is still incompletely described in acute myeloid leukemia (AML), whose treatment is based mostly on antimitotic chemotherapy. Here, we present an unexpected role of VDR in normal hematopoiesis and in leukemogenesis. Limited VDR expression is associated with impaired myeloid progenitor differentiation and is a new prognostic factor in AML. In mice, the lack of Vdr results in increased numbers of hematopoietic and leukemia stem cells and quiescent hematopoietic stem cells. In addition, malignant transformation of Vdr -/- cells results in myeloid differentiation block and increases self-renewal. Vdr promoter is methylated in AML as in CD34 + cells, and demethylating agents induce VDR expression. Association of VDR agonists with hypomethylating agents promotes leukemia stem cell exhaustion and decreases tumor burden in AML mouse models. Thus, Vdr functions as a regulator of stem cell homeostasis and leukemic propagation., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019. Published by Elsevier Inc.)

In environments undergoing constant transformation due to human action, such as deforestation and urbanization, the emergence of pests has become a challenge for agriculture and human welfare. In Brazil, over a thousand tonnes of pesticides are used annually, causing serious environmental damage such as the decline of insect populations. It is necessary to search for control alternatives in order to reduce the environmental impact caused by insecticides. This review aims to describe the use of social wasps as agents of biological control, focusing on the perspectives of their use in small farms and urban gardens, and to discuss the benefits of using this method. Studies have shown that 90-95% of the prey captured by wasps in small crops is made of leaf-eating caterpillars. In urban gardens, wasps diversify their prey, among which potential disease vectors, such as dipterans, stand out. We outline techniques for managing social wasp colonies in small farm and urban garden settings, including the use of artificial shelters. Among the advantages of using wasps as control agents, we highlight the practicality of the method, the low operational cost, the absence of prey resistance and the decrease of the use of insecticides., Competing Interests: The author declares no conflict of interest.

Background: The implication of lymphocytes in sickle cell disease pathogenesis is supported by a number of recent reports. These studies provided evidence for the activation of invariant natural killer T (iNKT) cells in adult patients, but did not investigate the involvement of other innate-like T cell subsets so far., Methods: Here we present a monocentric prospective observational study evaluating the number and functional properties of both circulating conventional and innate-like T cells, namely iNKT, Mucosal-Associated Invariant T (MAIT) and gammadelta (γδ) T cells in a cohort of 39 children with sickle cell disease., Results: Relative to age-matched healthy controls, we found that patients had a higher frequency of IL-13- and IL-17-producing CD4+ T cells, as well as higher MAIT cell counts with an increased frequency of IL-17-producing MAIT cells. Patients also presented increased Vδ2 γδ T cell counts, especially during vaso-occlusive crisis, and a lower frequency of IFNγ-producing Vδ2 γδ T cells, except during crisis. iNKT cell counts and the frequency of IFNγ-producing iNKT cells were unchanged compared to controls. Our study revealed positive correlations between 1) the frequency of IFNγ-producing CD4+, CD8+ and Vδ2 γδ T cells and the number of hospitalizations for vaso-occlusive crisis in the previous year; 2) the frequency of IFNγ-producing iNKT cells and patients' age and 3) the frequency of IL-17-producing Vδ2 γδ T cells and hemoglobin S level., Conclusion: These results strongly suggest a role of innate-like T cells in sickle cell disease pathophysiology, especially that of IL-17-producing MAIT and γδ T cells., Competing Interests: The authors have declared that no competing interests exist.

Cyclosporin-A has been known and used for a long time, since its "fast track" approval in the early 80's. This molecule has rapidly demonstrated unexpected immunosuppressive properties, transforming the history of organ transplantation. Cyclosporin's key effect relies on modulation on T-lymphocyte activity, which explains its role in the prevention of graft rejection. However, whether cyclosporin-A exerts other effects on immune system remains to be determined. Until recently, cyclosporin-A was mainly used at a high-dose, but given the drug toxicity and despite the fear of losing its immunosuppressive effects, there is nowadays a tendency to decrease its dose. The literature has been reporting data revealing a paradoxical effect of low dosage of cyclosporin-A. These low-doses appear to have immunomodulatory properties, with different effects from high-doses on CD8+ T lymphocyte activation, auto-immune diseases, graft-vs.-host disease and cancer. The aim of this review is to discuss the role of cyclosporin-A, not only as a consecrated immunosuppressive agent, but also as an immunomodulatory drug when administrated at low-dose. The use of low-dose cyclosporin-A may become a new therapeutic strategy, particularly to treat cancer.

Renal transplants remain a medical challenge, because the parameters governing allograft outcome are incompletely identified. Here, we investigated the role of serum iron in the sterile inflammation that follows kidney ischemia-reperfusion injury. In a retrospective cohort study of renal allograft recipients ( n =169), increased baseline levels of serum ferritin reliably predicted a positive outcome for allografts, particularly in elderly patients. In mice, systemic iron overload protected against renal ischemia-reperfusion injury-associated sterile inflammation. Furthermore, chronic iron injection in mice prevented macrophage recruitment after inflammatory stimuli. Macrophages cultured in high-iron conditions had reduced responses to Toll-like receptor-2, -3, and -4 agonists, which associated with decreased reactive oxygen species production, increased nuclear localization of the NRF2 transcription factor, increased expression of the NRF2-related antioxidant response genes, and limited NF- κ B and proinflammatory signaling. In macrophage-depleted animals, the infusion of macrophages cultured in high-iron conditions did not reconstitute AKI after ischemia-reperfusion, whereas macrophages cultured in physiologic iron conditions did. These findings identify serum iron as a critical protective factor in renal allograft outcome. Increasing serum iron levels in patients may thus improve prognosis of renal transplants., (Copyright © 2017 by the American Society of Nephrology.)

Alterations in metabolic activities are cancer hallmarks that offer a wide range of new therapeutic opportunities. Here we decipher the interplay between mTORC1 activity and glucose metabolism in acute myeloid leukemia (AML). We show that mTORC1 signaling that is constantly overactivated in AML cells promotes glycolysis and leads to glucose addiction. The level of mTORC1 activity determines the sensitivity of AML cells to glycolysis inhibition as switch-off mTORC1 activity leads to glucose-independent cell survival that is sustained by an increase in mitochondrial oxidative phosphorylation. Metabolic analysis identified the pentose phosphate pathway (PPP) as an important pro-survival pathway for glucose metabolism in AML cells with high mTORC1 activity and provided a clear rational for targeting glucose-6-phosphate dehydrogenase (G6PD) in AML. Indeed, our analysis of the cancer genome atlas AML database pinpointed G6PD as a new biomarker in AML, as its overexpression correlated with an adverse prognosis in this cohort. Targeting the PPP using the G6PD inhibitor 6-aminonicotinamide induces in vitro and in vivo cytotoxicity against AML cells and synergistically sensitizes leukemic cells to chemotherapy. Our results demonstrate that high mTORC1 activity creates a specific vulnerability to G6PD inhibition that may work as a new AML therapy.

Background: In the SOD1(G93A) mutant rat model of amyotrophic lateral sclerosis (ALS), neuronal death and rapid paralysis progression are associated with the emergence of activated aberrant glial cells that proliferate in the degenerating spinal cord. Whether pharmacological downregulation of such aberrant glial cells will decrease motor neuron death and prolong survival is unknown. We hypothesized that proliferation of aberrant glial cells is dependent on kinase receptor activation, and therefore, the tyrosine kinase inhibitor masitinib (AB1010) could potentially control neuroinflammation in the rat model of ALS., Methods: The cellular effects of pharmacological inhibition of tyrosine kinases with masitinib were analyzed in cell cultures of microglia isolated from aged symptomatic SOD1(G93A) rats. To determine whether masitinib prevented the appearance of aberrant glial cells or modified post-paralysis survival, the drug was orally administered at 30 mg/kg/day starting after paralysis onset., Results: We found that masitinib selectively inhibited the tyrosine kinase receptor colony-stimulating factor 1R (CSF-1R) at nanomolar concentrations. In microglia cultures from symptomatic SOD1(G93A) spinal cords, masitinib prevented CSF-induced proliferation, cell migration, and the expression of inflammatory mediators. Oral administration of masitinib to SOD1(G93A) rats starting after paralysis onset decreased the number of aberrant glial cells, microgliosis, and motor neuron pathology in the degenerating spinal cord, relative to vehicle-treated rats. Masitinib treatment initiated 7 days after paralysis onset prolonged post-paralysis survival by 40 %., Conclusions: These data show that masitinib is capable of controlling microgliosis and the emergence/expansion of aberrant glial cells, thus providing a strong biological rationale for its use to control neuroinflammation in ALS. Remarkably, masitinib significantly prolonged survival when delivered after paralysis onset, an unprecedented effect in preclinical models of ALS, and therefore appears well-suited for treating ALS.

Fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is frequently detected in acute myeloid leukemia (AML) patients and is associated with a dismal long-term prognosis. FLT3 tyrosine kinase inhibitors provide short-term disease control, but relapse invariably occurs within months. Pim protein kinases are oncogenic FLT3-ITD targets expressed in AML cells. We show that increased Pim kinase expression is found in relapse samples from AML patients treated with FLT3 inhibitors. Ectopic Pim-2 expression induces resistance to FLT3 inhibition in both FLT3-ITD-induced myeloproliferative neoplasm and AML models in mice. Strikingly, we found that Pim kinases govern FLT3-ITD signaling and that their pharmacological or genetic inhibition restores cell sensitivity to FLT3 inhibitors. Finally, dual inhibition of FLT3 and Pim kinases eradicates FLT3-ITD(+) cells including primary AML cells. Concomitant Pim and FLT3 inhibition represents a promising new avenue for AML therapy.

Cancer cells require glutamine to adapt to increased biosynthetic activity. The limiting step in intracellular glutamine catabolism involves its conversion to glutamate by glutaminase (GA). Different GA isoforms are encoded by the genes GLS1 and GLS2 in humans. Herein, we show that glutamine levels control mitochondrial oxidative phosphorylation (OXPHOS) in acute myeloid leukemia (AML) cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacologic GLS1 inhibition by the drug CB-839 can reduce OXPHOS, leading to leukemic cell proliferation arrest and apoptosis without causing cytotoxic activity against normal human CD34(+) progenitors. Strikingly, GLS1 knockdown dramatically inhibited AML development in NSG mice. The antileukemic activity of CB-839 was abrogated by both the expression of a hyperactive GAC(K320A) allele and the addition of the tricarboxyclic acid cycle product α-ketoglutarate, indicating the critical function of GLS1 in AML cell survival. Finally, glutaminolysis inhibition activated mitochondrial apoptosis and synergistically sensitized leukemic cells to priming with the BCL-2 inhibitor ABT-199. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy for AML., (© 2015 by The American Society of Hematology.)

Mast cells are immune cells that accumulate in the tumors and their microenvironment during disease progression. Mast cells are armed with a wide array of receptors that sense environment modifications and, upon stimulation, they are able to secrete several biologically active factors involved in the modulation of tumor growth. For example, mast cells are able to secrete pro-angiogenic and growth factors but also pro- and anti-inflammatory mediators. Recent studies have allowed substantial progress in understanding the role of mast cells in tumorigenesis/disease progression but further studies are necessary to completely elucidate their impact in the pathophysiology of cancer. Here we review observations suggesting that mast cells could modulate tumor growth in humans. We also discuss the drawbacks related to observations from mast cell-deficient mouse models, which could have consequences in the determination of a potential causative relationship between mast cells and cancer. We believe that the understanding of the precise role of mast cells in tumor development and progression will be of critical importance for the development of new targeted therapies in human cancers.

β-Thalassaemia major (β-TM) is an inherited haemoglobinopathy caused by a quantitative defect in the synthesis of β-globin chains of haemoglobin, leading to the accumulation of free α-globin chains that form toxic aggregates. Despite extensive knowledge of the molecular defects causing β-TM, little is known of the mechanisms responsible for the ineffective erythropoiesis observed in the condition, which is characterized by accelerated erythroid differentiation, maturation arrest and apoptosis at the polychromatophilic stage. We have previously demonstrated that normal human erythroid maturation requires a transient activation of caspase-3 at the later stages of maturation. Although erythroid transcription factor GATA-1, the master transcriptional factor of erythropoiesis, is a caspase-3 target, it is not cleaved during erythroid differentiation. We have shown that, in human erythroblasts, the chaperone heat shock protein70 (HSP70) is constitutively expressed and, at later stages of maturation, translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. The primary role of this ubiquitous chaperone is to participate in the refolding of proteins denatured by cytoplasmic stress, thus preventing their aggregation. Here we show in vitro that during the maturation of human β-TM erythroblasts, HSP70 interacts directly with free α-globin chains. As a consequence, HSP70 is sequestrated in the cytoplasm and GATA-1 is no longer protected, resulting in end-stage maturation arrest and apoptosis. Transduction of a nuclear-targeted HSP70 mutant or a caspase-3-uncleavable GATA-1 mutant restores terminal maturation of β-TM erythroblasts, which may provide a rationale for new targeted therapies of β-TM.

The pathophysiology of ineffective erythropoiesis in β-thalassemia is poorly understood. We report that RAP-011, an activin receptor IIA (ActRIIA) ligand trap, improved ineffective erythropoiesis, corrected anemia and limited iron overload in a mouse model of β-thalassemia intermedia. Expression of growth differentiation factor 11 (GDF11), an ActRIIA ligand, was increased in splenic erythroblasts from thalassemic mice and in erythroblasts and sera from subjects with β-thalassemia. Inactivation of GDF11 decreased oxidative stress and the amount of α-globin membrane precipitates, resulting in increased terminal erythroid differentiation. Abnormal GDF11 expression was dependent on reactive oxygen species, suggesting the existence of an autocrine amplification loop in β-thalassemia. GDF11 inactivation also corrected the abnormal ratio of immature/mature erythroblasts by inducing apoptosis of immature erythroblasts through the Fas-Fas ligand pathway. Taken together, these observations suggest that ActRIIA ligand traps may have therapeutic relevance in β-thalassemia by suppressing the deleterious effects of GDF11, a cytokine which blocks terminal erythroid maturation through an autocrine amplification loop involving oxidative stress and α-globin precipitation.

Cancer cells require nutrients and energy to adapt to increased biosynthetic activity, and protein synthesis inhibition downstream of mammalian target of rapamycin complex 1 (mTORC1) has shown promise as a possible therapy for acute myeloid leukemia (AML). Glutamine contributes to leucine import into cells, which controls the amino acid/Rag/mTORC1 signaling pathway. We show in our current study that glutamine removal inhibits mTORC1 and induces apoptosis in AML cells. The knockdown of the SLC1A5 high-affinity transporter for glutamine induces apoptosis and inhibits tumor formation in a mouse AML xenotransplantation model. l-asparaginase (l-ase) is an anticancer agent also harboring glutaminase activity. We show that l-ases from both Escherichia coli and Erwinia chrysanthemi profoundly inhibit mTORC1 and protein synthesis and that this inhibition correlates with their glutaminase activity levels and produces a strong apoptotic response in primary AML cells. We further show that l-ases upregulate glutamine synthase (GS) expression in leukemic cells and that a GS knockdown enhances l-ase-induced apoptosis in some AML cells. Finally, we observe a strong autophagic process upon l-ase treatment. These results suggest that l-ase anticancer activity and glutamine uptake inhibition are promising new therapeutic strategies for AML.

The serine/threonine kinase mammalian target of rapamycin (mTOR) is crucial for cell growth and proliferation, and is constitutively activated in primary acute myeloid leukemia (AML) cells, therefore representing a major target for drug development in this disease. We show here that the specific mTOR kinase inhibitor AZD8055 blocked mTORC1 and mTORC2 signaling in AML. Particularly, AZD8055 fully inhibited multisite eIF4E-binding protein 1 phosphorylation, subsequently blocking protein translation, which was in contrast to the effects of rapamycin. In addition, the mTORC1-dependent PI3K/Akt feedback activation was fully abrogated in AZD8055-treated AML cells. Significantly, AZD8055 decreased AML blast cell proliferation and cell cycle progression, reduced the clonogenic growth of leukemic progenitors and induced caspase-dependent apoptosis in leukemic cells but not in normal immature CD34+ cells. Interestingly, AZD8055 strongly induced autophagy, which may be either protective or cell death inducing, depending on concentration. Finally, AZD8055 markedly increased the survival of AML transplanted mice through a significant reduction of tumor growth, without apparent toxicity. Our current results strongly suggest that AZD8055 should be tested in AML patients in clinical trials.

IgA nephropathy (IgAN) is a common cause of renal failure worldwide. Treatment is limited because of a complex pathogenesis, including unknown factors favoring IgA1 deposition in the glomerular mesangium. IgA receptor abnormalities are implicated, including circulating IgA-soluble CD89 (sCD89) complexes and overexpression of the mesangial IgA1 receptor, TfR1 (transferrin receptor 1). Herein, we show that although mice expressing both human IgA1 and CD89 displayed circulating and mesangial deposits of IgA1-sCD89 complexes resulting in kidney inflammation, hematuria, and proteinuria, mice expressing IgA1 only displayed endocapillary IgA1 deposition but neither mesangial injury nor kidney dysfunction. sCD89 injection into IgA1-expressing mouse recipients induced mesangial IgA1 deposits. sCD89 was also detected in patient and mouse mesangium. IgA1 deposition involved a direct binding of sCD89 to mesangial TfR1 resulting in TfR1 up-regulation. sCD89-TfR1 interaction induced mesangial surface expression of TGase2 (transglutaminase 2), which in turn up-regulated TfR1 expression. In the absence of TGase2, IgA1-sCD89 deposits were dramatically impaired. These data reveal a cooperation between IgA1, sCD89, TfR1, and TGase2 on mesangial cells needed for disease development. They demonstrate that TGase2 is responsible for a pathogenic amplification loop facilitating IgA1-sCD89 deposition and mesangial cell activation, thus identifying TGase2 as a target for therapeutic intervention in this disease.

Anemia because of insufficient production of and/or response to erythropoietin (Epo) is a major complication of chronic kidney disease and cancer. The mechanisms modulating the sensitivity of erythroblasts to Epo remain poorly understood. We show that, when cultured with Epo at suboptimal concentrations, the growth and clonogenic potential of erythroblasts was rescued by transferrin receptor 1 (TfR1)-bound polymeric IgA1 (pIgA1). Under homeostatic conditions, erythroblast numbers were increased in mice expressing human IgA1 compared to control mice. Hypoxic stress of these mice led to increased amounts of pIgA1 and erythroblast expansion. Expression of human IgA1 or treatment of wild-type mice with the TfR1 ligands pIgA1 or iron-loaded transferrin (Fe-Tf) accelerated recovery from acute anemia. TfR1 engagement by either pIgA1 or Fe-Tf increased cell sensitivity to Epo by inducing activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. These cellular responses were mediated through the TfR1-internalization motif, YXXΦ. Our results show that pIgA1 and TfR1 are positive regulators of erythropoiesis in both physiological and pathological situations. Targeting this pathway may provide alternate approaches to the treatment of ineffective erythropoiesis and anemia.

Vascular calcifications can occur in the elderly and in patients suffering from various diseases. Interestingly, depending on the pathology, different regions of the arterial system can be affected. Embryonic observations have clearly indicated that vascular smooth muscle cell (VSMC) origin is notably heterogeneous. For instance, in the aorta, VSMCs colonizing the aortic arch region derive from cardiac neural crest cells, whereas those populating the descending aorta derive from the mesoderm. We examined here whether the embryonic origin of aortic VSMCs would correlate with their ability to mineralize. Under hyperphosphatemic conditions that induce vascular calcifications, we performed ex vivo aortic explant cultures as well as in vitro VSMC cultures from wild-type mice. Our data showed that VSMC embryonic origin affects their ability to mineralize. Indeed, the aortic arch media made up of VSMCs of neural crest origin calcifies significantly earlier than the descending aorta composed of VSMCs, which are mesoderm-derived. Similar results were obtained with cultured VSMCs harvested from both aortic regions. We also demonstrated that in a mouse model deficient in matrix Gla protein, a potent calcification inhibitor, developing extensive and spontaneous medial calcifications of the aorta, lesions initiate in the aortic arch. Subsequently, calcifications progress outside the aortic arch region and ultimately spread all over the entire arterial tree, including the descending aorta. Altogether, our results support an unsuspected correlation between VSMCs of embryonic origin and the timing of appearance of calcifications., (Copyright © 2011 American Society for Bone and Mineral Research.)

Finding an effective treatment for acute myeloid leukemia (AML) remains a challenge, and all cellular processes that are deregulated in AML cells should be considered in the design of targeted therapies. We show in our current study that the LKB1/AMPK/TSC tumor suppressor axis is functional in AML and can be activated by the biguanide molecule metformin, resulting in a specific inhibition of mammalian target of rapamycin (mTOR) catalytic activity. This induces a multisite dephosphorylation of the key translation regulator, 4E-BP1, which markedly inhibits the initiation step of mRNA translation. Consequently, metformin reduces the recruitment of mRNA molecules encoding oncogenic proteins to the polysomes, resulting in a strong antileukemic activity against primary AML cells while sparing normal hematopoiesis ex vivo and significantly reducing the growth of AML cells in nude mice. The induction of the LKB1/AMPK tumor-suppressor pathway thus represents a promising new strategy for AML therapy.

Presently, little is understood about how endometriosis is established or maintained, or how genetic factors can predispose women to the disease. Because of the crucial role that the progesterone receptor polymorphism PROGINS plays in predisposing women to the development of endometriosis, we hypothesized that this variant may influence critical steps during endometrial cell metabolism that are involved in the pathogenesis of endometriosis. Eutopic endometria were collected from three sources: women with endometriosis who had a single PROGINS allele (from the progesterone receptor gene); women with endometriosis who had the wild-type progesterone receptor allele; and women without endometriosis who had the wild-type allele. Cells prepared from the eutopic endometria of these women were stimulated with both estradiol and progesterone, and then examined for cell proliferation, viability, and apoptosis. The cells from women with endometriosis that carried the PROGINS allele demonstrated increased proliferation, greater viability, and decreased apoptosis following progesterone treatment. In general, these parameters were very different as compared with those of women with endometriosis but without the PROGINS allele and women in the control group. This result indicates there is a reduced level of progesterone responsiveness in women who carry the PROGINS polymorphism. Because progesterone responsiveness is known to be an important characteristic of women with endometriosis, these data support the contention that the PROGINS polymorphism enhances the endometriosis phenotype.

Amitriptyline is a pleiotropic tricyclic antidepressant, which has anti-oxidant and anti-inflammatory properties. We tested whether amitriptyline might be useful in the treatment of chronic renal disease using the mouse model of unilateral ureteral obstruction. Amitriptyline caused a significant reduction of interstitial fibrosis, determined by Masson's staining, with minimal myofibroblast formation and macrophage infiltration following ureteral obstruction. Using quantitative PCR we found that this treatment significantly reduced the expression of key molecular markers of progressive tubulointerstitial injury such as osteopontin, MCP-1, ICAM-1, and TGF-beta1 compared to their level in a saline-treated control group. Sublethal X-irradiation or mycophenolate mofetil, treatments that reduce inflammation, were comparable to amitriptyline in the reduction of interstitial fibrosis and macrophage infiltration. These studies in animals suggest that amitriptyline is worth testing as a therapeutic agent that might preserve renal function by blocking inflammation and renal fibrosis.

Background: Extracellular matrix accumulation, epithelial-to-mesenchymal transition, tubular atrophy and loss of peritubular capillary network are hallmarks of tubulointerstitial injury in progressive renal diseases. In this study, we analyzed endostatin expression in kidneys subjected to unilateral ureteral obstruction (UUO)., Methods: Collagen XVIII mRNA expression was evaluated by real-time polymerase chain reaction (PCR). Endostatin and CD31 protein levels were analyzed by Western blot and immunohistochemistry. In vitro quantification of collagen XVIII and fibrosis-related genes in HK2 cells was performed by real-time PCR., Results: UUO significantly increased collagen XVIII mRNA expression and released a 30-kDa endostatin fragment. Immunohistochemistry revealed endostatin expression increased in injured tissue, mainly on tubular cells. Of interest, expression of CD31 was significantly reduced by UUO. Endostatin administration in vitro did not modify the expression of genes related to fibrosis development. However, in vitro TGF-beta1 administration induced expression of collagen XVIII/endostatin mRNA in human tubular cells., Conclusion: Endostatin is expressed during the progression of renal fibrosis in vitro and in vivo, suggesting a role for endostatin in development of tubulointerstitial injury.

Hyperactivation of systemic renin-angiotensin system (RAS) during sepsis is well documented. However, the behavior of intrarenal RAS in the context of endotoxemia is yet to be defined. The present study evaluates the direct effect of Escherichia coli lipopolysaccharide (LPS) on immortalized human mesangial cell (HMC) RAS. Quiescent HMC were incubated with vehicle or LPS (1-100 microg/ml), and levels of angiotensin I and II (Ang I and II) and their metabolites were analyzed by high-performance liquid chromatography. In addition, angiotensin-converting enzyme (ACE) and renin activity were also investigated. Cell lysate and extracellular medium levels of Ang II were rapidly reduced (1 h) in a time- and concentration-dependent manner, reaching a significant -9 fold-change (P<0.001) after 3 h of LPS incubation. Similar results were obtained for Ang I levels (-3 fold-change, P<0.001). We ruled out Ang I and II degradation, as levels of their metabolic fragments were also significantly decreased by LPS. ACE activity was slightly increased following LPS incubation. On the other hand, renin activity was significantly inhibited, as Ang I concentration elevation following exogenous angiotensinogen administration was blunted by LPS (-60% vs vehicle, P<0.001). Renin and angiotensinogen protein levels were not affected by LPS according to Western blot analysis. Taken together, these data demonstrate for the first time that LPS significantly downregulates HMC RAS through inhibition of renin or renin-like activity. These findings are potentially related to the development of and/or recovery from acute renal failure in the context of sepsis.