Decreasing TfR1 expression reverses anemia and hepcidin suppression in ß-thalassemic mice.

Iron availability for erythropoiesis and its dysregulation in ß-thalassemia are incompletely understood. We previously demonstrated that exogenous apotransferrin leads to more effective erythropoiesis, decreasing erythroferrone (ERFE) and derepressing hepcidin in ß-thalassemic mice. Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for cellular iron delivery during erythropoiesis. We hypothesize that apotransferrin's effect is mediated via decreased TfR1 expression and evaluate TfR1 expression in ß-thalassemic mice in vivo and in vitro with and without added apotransferrin. Our findings demonstrate that ß-thalassemic erythroid precursors overexpress TfR1, an effect that can be reversed by the administration of exogenous apotransferrin. In vitro experiments demonstrate that apotransferrin inhibits TfR1 expression independent of erythropoietin- and iron-related signaling, decreases TfR1 partitioning to reticulocytes during enucleation, and enhances enucleation of defective ß-thalassemic erythroid precursors. These findings strongly suggest that overexpressed TfR1 may play a regulatory role contributing to iron overload and anemia in ß-thalassemic mice. To evaluate further, we crossed TfR1+/- mice, themselves exhibiting iron-restricted erythropoiesis with increased hepcidin, with ß-thalassemic mice. Resultant double-heterozygote mice demonstrate long-term improvement in ineffective erythropoiesis, hepcidin derepression, and increased erythroid enucleation in relation to ß-thalassemic mice. Our data demonstrate for the first time that TfR1+/- haploinsufficiency reverses iron overload specifically in ß-thalassemic erythroid precursors. Taken together, decreasing TfR1 expression during ß-thalassemic erythropoiesis, either directly via induced haploinsufficiency or via exogenous apotransferrin, decreases ineffective erythropoiesis and provides an endogenous mechanism to upregulate hepcidin, leading to sustained iron-restricted erythropoiesis and preventing systemic iron overload in ß-thalassemic mice.

A novel flow cytometric application discriminates among the effects of chemical inhibitors on various phases of Babesia divergens intraerythrocytic cycle.

Human babesiosis is a global emerging infectious disease caused by intraerythrocytic parasites of the genus Babesia. Its biology has remained largely unexplored due to a lack of critical tools and techniques required to define the various stages and phases of the parasite's cycle in its host RBC and the interplay between host and parasite. This article presents a powerful set of tools combining stage synchronization of the parasite with a platform that encompasses both a flow cytometric evaluation of the subpopulation structure of the parasite population together with a morphological assessment of the population parasites using light microscopy of conventional Giemsa stained smears. Together, these yield specific information on the effect of any drug/condition of interest and its targeted biological process, allowing the characterization of the adaptive response of parasites to a particular stressor agent. Three inhibitors were used in this study, each targeting a specific phase of the parasite's lifecycle, neuraminidase for host cell invasion, N-acetyl-L-leucyl-L-leucyl-L-norleucinal for parasite development and EGTA for parasite egress from the host cell. Results presented prove the power of this combination platform in discriminating the specific targets among the life-cycle processes of the parasite-invasion, development/proliferation and egress. This will expand the range of queries that can now be successfully addressed in this parasite, opening avenues for the development of new methods to control babesiosis, either by chemicals (screening for new chemotherapy drugs or defining levels of parasite resistance) or physical methods (light irradiation or heat shock used in pathogen reduction/elimination methods). © 2017 International Society for Advancement of Cytometry.

Increased hepcidin in transferrin-treated thalassemic mice correlates with increased liver BMP2 expression and decreased hepatocyte ERK activation.

Iron overload results in significant morbidity and mortality in ß-thalassemic patients. Insufficient hepcidin is implicated in parenchymal iron overload in ß-thalassemia and approaches to increase hepcidin have therapeutic potential. We have previously shown that exogenous apo-transferrin markedly ameliorates ineffective erythropoiesis and increases hepcidin expression in Hbb(th1/th1) (thalassemic) mice. We utilize in vivo and in vitro systems to investigate effects of exogenous apo-transferrin on Smad and ERK1/2 signaling, pathways that participate in hepcidin regulation. Our results demonstrate that apo-transferrin increases hepcidin expression in vivo despite decreased circulating and parenchymal iron concentrations and unchanged liver Bmp6 mRNA expression in thalassemic mice. Hepatocytes from apo-transferrin-treated mice demonstrate decreased ERK1/2 pathway and increased serum BMP2 concentration and hepatocyte BMP2 expression. Furthermore, hepatocyte ERK1/2 phosphorylation is enhanced by neutralizing anti-BMP2/4 antibodies and suppressed in vitro in a dose-dependent manner by BMP2, resulting in converse effects on hepcidin expression, and hepatocytes treated with MEK/ERK1/2 inhibitor U0126 in combination with BMP2 exhibit an additive increase in hepcidin expression. Lastly, bone marrow erythroferrone expression is normalized in apo-transferrin treated thalassemic mice but increased in apo-transferrin injected wild-type mice. These findings suggest that increased hepcidin expression after exogenous apo-transferrin is in part independent of erythroferrone and support a model in which apo-transferrin treatment in thalassemic mice increases BMP2 expression in the liver and other organs, decreases hepatocellular ERK1/2 activation, and increases nuclear Smad to increase hepcidin expression in hepatocytes.

Babesia divergens builds a complex population structure composed of specific ratios of infected cells to ensure a prompt response to changing environmental conditions.

Babesia parasites cause a malaria-like febrile illness by infection of red blood cells (RBCs). Despite the growing importance of this tick-borne infection, its basic biology has been neglected. Using novel synchronization tools, the sequence of intra-erythrocytic events was followed from invasion through development and differentiation to egress. The dynamics of the parasite population were studied in culture, revealing for the first time, the complete array of morphological forms in a precursor-product relationship. Important chronological constants including Babesia's highly unusual variable intra-erythrocytic life cycle, the life span of each population of infected cells and the time required for the genesis of the different parasite stages were elucidated. Importantly, the maintenance of specific ratios of the infected RBC populations was shown to be responsible for the parasites' choice of developmental pathways, enabling swift responses to changing environmental conditions like availability of RBCs and nutrition. These results could impact the control of parasite proliferation and therefore disease.

TIGIT-positive circulating follicular helper T cells display robust B-cell help functions: potential role in sickle cell alloimmunization.

T follicular helper cells are the main CD4(+) T cells specialized in supporting B-cell responses, but their role in driving transfusion-associated alloimmunization is not fully characterized. Reports of T follicular helper subsets displaying various markers and functional activities underscore the need for better characterization/identification of markers with defined functions. Here we show that a previously unidentified subset of human circulating T follicular helper cells expressing TIGIT, the T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory domains, exhibit strong B-cell help functions. Compared to the subset lacking the receptor, T follicular helper cells expressing this receptor up-regulated co-stimulatory molecules and produced higher levels of interleukins (IL-21 and IL-4) critical for promoting B-cell activation/differentiation. Furthermore, this subset was more efficient at inducing the differentiation of B cells into plasmablasts and promoting immunoglobulin G production. Blocking antibodies abrogated the B-cell help properties of receptor-expressing T follicular helper cells, consistent with the key role of this molecule in T follicular helper-associated responses. Importantly, in chronically transfused patients with sickle cell anemia, we identified functional differences of this subset between alloimmunized and non-alloimmunized patients. Altogether, these studies suggest that expression of the T-cell immunoreceptor with Ig and immunoreceptor tyro-sine-based inhibitory domains not only represents a novel circulating T follicular helper biomarker, but is also functional and promotes strong B-cell help and ensuing immunoglobulin G production. These findings open the way to defining new diagnostic and therapeutic strategies in modulating humoral responses in alloimmunization, and possibly vaccination, autoimmunity and immune deficiencies.

Nelfinavir, an HIV protease inhibitor, induces apoptosis and cell cycle arrest in human cervical cancer cells via the ROS-dependent mitochondrial pathway.

HIV protease inhibitors (HIV-PIs) are a class of antiretroviral drugs designed to target the viral protease. Strikingly, these drugs have also been reported to possess antitumor effect. In this study, we evaluated the activity of one HIV-PI, Nelfinavir, against human cervical cancer cells. We found that Nelfinavir inhibited the growth of cervical cancer cell lines at the lowest micromolar concentrations clinically attainable. Nelfinavir promoted apoptosis and arrested the cell cycle at G1 phase. Apoptosis is attributed to the promotion of mitochondrial reactive oxygen species (ROS) production, which results in the translocation of mitochondrial apoptosis inducing factor (AIF) to the nucleus. We further showed that Nelfinavir increased mitochondrial ROS production by decreasing manganese superoxide dismutase (MnSOD) protein levels. Taken together, our results suggest that Nelfinavir can be repositioned as a cervical cancer therapeutic.

Mechanisms underlying reduced apoptosis in neonatal neutrophils.

Apoptosis, which leads to phagocytosis by mononuclear cells, represents the primary mechanism for removing neutrophils from inflamed tissues and minimizing injury. The present studies show that membrane phosphatidylserine turnover and permeability, as well as DNA fragmentation, were reduced in neutrophils from neonates when compared with adults. The activity of caspase 3 and expression of the proapoptotic proteins Bax, Bad, and Bak were also decreased in neonatal relative to adult neutrophils. These findings are consistent with impaired apoptosis in neonatal cells, which may contribute to prolonged inflammation in infants after oxidative stress or infection. Neutrophil apoptosis is induced by endogenous ligands such as Fas (FasL), which engage death receptors of the tumor necrosis factor/nerve growth factor superfamily, including Fas receptor (FasR). We found that expression of FasR was decreased in neonatal when compared with adult cells. Moreover, neonatal neutrophils did not undergo apoptosis in response to anti-FasR antibody and exhibited impaired chemotaxis to soluble FasL. However, in both adult and neonatal cells, p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase inhibitors blocked Fas-induced activity. These data suggest that prolonged survival of neonatal neutrophils at injured sites is due, in part, to reduced responsiveness to FasL. This may be related to decreased expression of both FasR and Bcl-2-family proteins that mediate neutrophil apoptosis.