Polycations and polyanions in SARS-CoV-2 infection.

We hypothesize that polycations, such as nuclear histones, released by neutrophils COVID-19 aggravate COVID-19 by multiple mechanisms: (A) Neutralization of the electrostatic repulsion between the virus particles and the cell membrane, thereby enhancing receptor-mediated entry. (B) Binding to the virus particles, thereby inducing opsonin-mediated endocytosis. (C) Adding to the cytotoxicity, in conjunction with oxidants, cytokines and other pro-inflammatory substances secreted by cells of the innate immunity system. These effects may be alleviated by the administration of negatively charged polyanions such as heparins and heparinoids.

The Antioxidant Effect of Fermented Papaya Preparation in the Oral Cavity.

Oxidative stress has been recognized to play important roles in various diseases, including of the oral cavity. However, nutritional supplementation of antioxidants to ameliorate the consequences of oxidative stress is debatable. One caveat is that oxidative status is often measured under non-physiological conditions. Here, we investigated the antioxidant potential of fermented papaya preparation (FPP), a product of yeast fermentation of Carica papaya Linn, under conditions that prevail in the oral cavity. Employing highly sensitive luminol-dependent chemiluminescence assays, we show that its antioxidant capacity was augmented by saliva (up to 20-fold, p < 0.0001, at 10 mg) and its components (mucin, albumin) as well as by red blood cells (RBC) and microorganisms present in the normal and pathological environment of the oral cavity. Polyphenols are major plant antioxidants. Using the Folin-Ciocalteu's assay, a very low amount of phenols was measured in FPP suspended in a salt solution. However, its suspension in saliva, albumin, mucin or RBC produced up to sixfold increase, p < 0.001, compared with the sum of polyphenols assayed separately. The results suggested that these enhancing effects were due to the solubilization of antioxidant polyphenols in FPP by saliva proteins and the binding to RBC and microorganisms, thus increasing their availability and activity. Copyright © 2015 John Wiley & Sons, Ltd.

The antioxidant effect of fermented papaya preparation involves iron chelation.

Iron-overload is a major clinical problem in various diseases. Under this condition, serum iron which surpasses the binding capacity of transferrin is present as non-transferrin bound iron and cellular unbound Labile Iron Pool (LIP) is increased. LIP participates in the generation of free radicals, including reactive oxygen species (ROS). Increased ROS, with concomitant decrease in anti-oxidants, results in oxidative stress and toxicity to the liver, heart and other tissues, causing serious morbidity and eventually mortality. Therapeutic iron chelation reduces the LIP and thereby ameliorates oxidative stress-mediated toxicity. Many food-derived antioxidants have the capacities to scavenge ROS and chelate iron. We have reported that fermented papaya preparation (FPP) has ROS scavenging effect on blood cells in vitro or in vivo (in thalassemic patients and experimental animals). We now investigated FPP's iron chelating effect - its ability to prevent (and revert) LIP accumulation. Liver- and heart-derived cells, and RBCs were exposed to non-transferrin bound iron in the form of ferrous ammonium sulfate and the effect of FPP on their LIP content and ROS generation was measured by flow-cytometry. The results indicate that FPP reduces LIP and ROS, and suggests that its antioxidant mechanism is related, at least in part, to iron chelation.

The antiepileptic and anticancer agent, valproic acid, induces P-glycoprotein in human tumour cell lines and in rat liver.

BACKGROUND AND PURPOSE: The antiepileptic drug valproic acid, a histone deacetylase (HDAC) inhibitor, is currently being tested as an anticancer agent. However, HDAC inhibitors may interact with anticancer drugs through induction of P-glycoprotein (P-gp, MDR1) expression. In this study we assessed whether valproic acid induces P-gp function in tumour cells. We also investigated effects of valproic acid on the mRNA for P-gp and the cytochrome P450, CYP3A, in rat livers. EXPERIMENTAL APPROACH: Effects of valproic acid on P-gp were assessed in three tumour cell lines, SW620, KG1a and H4IIE. Accumulation of acetylated histone H3 in rats' livers treated for two or seven days with valproic acid was evaluated using a specific antibody. Hepatic expression of the P-gp genes, mdr1a, mdr1b and mdr2, was determined by real-time polymerase chain reaction. The effects of valproic acid on CYP3A were assessed by Northern blot analysis and CYP3A activity assays. KEY RESULTS: Valproic acid (0.5-2.0 mM) induced P-gp expression and function up to 4-fold in vitro. The effect of a series of valproic acid derivatives on P-gp expression in SW620 and KG1a cells correlated with their HDAC inhibition potencies. Treatment of rats with 1 mmol kg(-1) valproic acid for two and seven days increased hepatic histone acetylation (1.3- and 3.5-fold, respectively) and the expression of mdr1a and mdr2 (2.2-4.1-fold). Valpromide (0.5-2.0 mM) did not increase histone acetylation or P-gp expression in rat livers, but induced CYP3A expression. CONCLUSIONS: Valproic acid increased P-gp expression and function in human tumour cell lines and in rat liver. The clinical significance of this increase merits further investigation.

Macrophages promote development of human erythroid precursors in transferrin-free culture medium.

Iron is mandatory for cell growth and development. Erythroid precursors need iron to a greater degree for hemoglobinization. Culturing erythroid precursors under serum and transferrin-free conditions resulted in their death, whereas under the same conditions, but in the presence of macrophages erythroid cell growth and development was evident as measured by hemoglobin (Hb)-specific cytochemical staining, flow cytometric immuno-staining of glycophorin A and Hb quantitation by a spectrophotometric method as well as by high performance liquid chromatography. Thus, macrophages support erythroid cell growth in the absence of transferrin, most likely by providing iron.

Differentiation of human erythroid cells in culture.

A culture procedure for growing erythroid progenitors in liquid medium is described. The procedure is divided into two phases. The first is an erythropoietin (EPO)--independent phase in which peripheral blood mononuclear cells are isolated and cultured in the presence of a combination of growth factors, but in the absence of EPO. During this phase, early erythroid-committed progenitors proliferate and differentiate into more mature progenitors. In the second phase, the latter cells, cultured in an EPO-supplemented medium, continue to proliferate and mature into hemoglobin-containing nucleated erythroid cells. The culture procedure yields populations that are large, relatively pure, and synchronized (in terms of differentiation), and which recapitulate in vivo erythropoiesis. Since the cells are grown in suspension, samples of cells can be withdrawn at any time, without disturbing the cultures, and assayed for various parameters, e.g., morphology, size, number, viability, apoptosis, cell cycle, surface antigens, or gene expression. Several procedures for analyzing the cultured cells with respect to their hemoglobin content during their differentiation are included.

Flow cytometric analysis of fetal hemoglobin in erythroid precursors of beta-thalassemia.

Fetal hemoglobin (HbF), the major hemoglobin species in fetal life, drops to <1% in normal adults, where it is restricted to a few 'F-cells', which may increase in various acquired and genetic conditions, including thalassemia. Using flow cytometry, we studied the percentage of HbF-containing cells and their HbF content in RBC, reticulocytes (retics) and normoblasts (NRBC) present in the peripheral blood of patients with beta-thalassemia. Thiazol orange, a nucleic acid-specific dye, and anti-CD45 antibodies identified the various blood cells and antihuman HbF antibodies quantitated HbF. The results indicated that F-RBC were more numerous in beta-thalassemic (both transfused and nontransfused) patients than in normal donors, but, in most cases, their HbF content was comparable, suggesting that increased HbF in thalassemia is mainly due to higher %F-cells rather than an increased HbF per cell. Among the retics, the %F-cells and their HbF content were highest in immature retics and decreased with maturation to levels of RBC. This may reflect preferential maturation of F-retics into RBC in the circulation. The NRBC population contained the lowest %F-cells. This could be due to preferential maturation of F-NRBC, having more normal phenotype than non-F-NRBC, in the bone marrow into F-retics, while non-F-NRBC enter the circulation.

Pre-clinical development of cord blood-derived progenitor cell graft expanded ex vivo with cytokines and the polyamine copper chelator tetraethylenepentamine.

BACKGROUND: We have previously demonstrated that the copper chelator tetraethylenepentamine (TEPA) enables preferential expansion of early hematopoietic progenitor cells (CD34+CD38-, CD34+CD38-Lin-) in human umbilical cord blood (CB)-derived CD34+ cell cultures. This study extends our previous findings that copper chelation can modulate the balance between self-renewal and differentiation of hematopoietic progenitor cells. METHODS: In the present study we established a clinically applicative protocol for large-scale ex vivo expansion of CB-derived progenitors. Briefly, CD133+ cells, purified from CB using Miltenyi Biotec's (Bergisch Gladbach, Germany) CliniMACS separation device and the anti-CD133 reagent, were cultured for 3 weeks in a clinical-grade closed culture bag system, using the chelator-based technology in combination with early-acting cytokines (SCF, thrombopoietin, IL-6 and FLT-3 ligand). This protocol was evaluated using frozen units derived from accredited cord blood banks. RESULTS: Following 3 weeks of expansion under large-scale culture conditions that were suitable for clinical manufacturing, the median output value of CD34+ cells increase by 89-fold, CD34+CD38- increase by 30-fold and CFU cells (CFUc) by 172-fold over the input value. Transplantation into sublethally irradiated non-obese diabetic (NOD/SCID) mice indicated that the engraftment potential of the ex vivo expanded CD133+ cells was significantly superior to that of unexpanded cells: 60+/-5.5% vs. 21+/-3.5% CD45+ cells, P=0.001, and 11+/-1.8% vs. 4+/-0.68% CD45+CD34+ cells, P=0.012, n=32, respectively. DISCUSSION: Based on these large-scale experiments, the chelator-based ex vivo expansion technology is currently being tested in a phase 1 clinical trial in patients undergoing CB transplantation for hematological malignancies.

On the mechanism of post-splenectomy leukocytosis in mice.

BACKGROUND: Increased number of peripheral white blood cells (PWBCs) has been noted after removal of the spleen. DESIGN: To clarify the possible mechanisms by which splenectomy affects the PWBC number, the percentage of apoptotic PWBCs, the number and migration rate of peritoneal cells, as well as the 3H-TdR incorporation into PWBCs, were examined in splenectomized, sham-operated and control mice. In addition, the effect of control plasma injected to splenectomized animals on the number of PWBCs was examined. RESULTS: One and two months after splenectomy the PWBC counts significantly increased, whereas the percentage of apoptotic PWBCs and the number of cells in the peritoneal cavity decreased in comparison with that of the control and sham-operated mice. Seventeen days after injection of carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labelled peritoneal cells into the peritoneal cavity of the animals, their number was significantly higher in the peripheral blood and lower in the peritoneal cavity of the splenectomized animals in comparison with that of the control and sham-operated mice. Injection of control plasma into the splenectomized mice prevented the development of postsplenectomy leukocytosis. Finally, 3H-TdR incorporation into nonstimulated and Con A stimulated PBMCs from the splenectomized mice was higher as compared with cells from the control and sham-operated mice. CONCLUSIONS: The results of the study present several mechanisms that may clarify the cause of postsplenectomy leukocytosis.

Developing human erythroid cells grown in transferrin-free medium utilize iron originating from extracellular ferritin.

In addition to transferrin, ferritin can also function as a source of iron for heme synthesis (Gelvin D, et al. Blood 1996;88:3200-3207; Meyron-Holtz EG, et al. Blood 1999;94:3205-3211). In the present study we investigate the effect of external ferritin on the proliferation and hemoglobinization of human erythroid precursors grown in transferrin-free cultures.

Developmental regulation of DNA replication timing at the human beta globin locus.

The human beta globin locus replicates late in most cell types, but becomes early replicating in erythroid cells. Using FISH to map DNA replication timing around the endogenous beta globin locus and by applying a genetic approach in transgenic mice, we have demonstrated that both the late and early replication states are controlled by regulatory elements within the locus control region. These results also show that the pattern of replication timing is set up by mechanisms that work independently of gene transcription.

Cell culture and animal models to screen for promising fetal hemoglobin-stimulating compounds.

Sickle cell anemia (SCA) and the thalassemias are globally the most common class of inherited single-gene disorders. Current treatment options are limited, especially in developing countries. More practical and cheaper therapies are urgently needed. Since high fetal hemoglobin (HbF) levels ameliorate the clinical symptoms of these diseases, one current approach is to use pharmacological agents to reactivate the gamma-globin genes and stimulate the production of HbF. Several in vitro and in vivo experimental models developed for this purpose are the subject of this review. The models include in vitro established erythroid-like cell lines and primary cultures (both in semisolid and liquid media) of erythroid progenitor cells obtained from normal donors and patients with SCA and beta-thalassemia, as well as in vivo models in genetically modified (transgenic) and unmodified animals. These experimental systems are useful for large-scale screening of compounds for HbF-stimulating potential, for determining the mechanism of action of potent compounds at the cellular and molecular levels, and for studying the pharmacology, pharmacokinetics, and toxicology of the drugs. These models are essential to find, test, and develop new drugs that will be effective and safe for clinical use in adults and children.

Accumulation of gamma-globin mRNA and induction of erythroid differentiation after treatment of human leukaemic K562 cells with tallimustine.

Human leukaemic K562 cells can be induced in vitro to erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine, cytosine arabinoside, mithramycin and chromomycin, cisplatin and cisplatin analogues. Differentiation of K562 cells is associated with an increase of expression of embryo-fetal globin genes, such as the zeta-, epsilon- and gamma-globin genes. The K562 cell line has been proposed as a very useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation stimulating gamma-globin synthesis could be considered for possible use in the therapy of haematological diseases associated with a failure in the expression of normal beta-globin genes. We have analysed the effects of tallimustine and distamycin on cell growth and differentiation of K562 cells. The results demonstrated that tallimustine is a potent inducer, while distamycin is a weak inducer, of K562 cell erythroid differentiation. Erythroid differentiation was associated with an increase of accumulation of gamma-globin mRNA and of production of both haemoglobin (Hb) Gower 1 and Hb Portland. In addition, tallimustine-mediated erythroid induction occurred in the presence of activation of the apoptotic pathway. The reasons for proposing tallimustine as an inducer of gamma-globin gene expression are strongly sustained by the finding that this compound stimulates fetal haemoglobin production in human erythroid precursor cells from normal subjects.

Prodrugs of butyric acid. Novel derivatives possessing increased aqueous solubility and potential for treating cancer and blood diseases.

The synthesis and biological activities of acidic, basic and neutral types of butyric acid (BA) prodrugs possessing increased aqueous solubility are described. The compounds are butyroyloxyalkyl derivatives of carboxylic acids, which possess functionalities suitable for aqueous solubilization. The anticancer activity of the prodrugs in vitro was evaluated by examining their effect on the growth of human colon, breast and pancreatic carcinoma cell lines, and their solubility in aqueous media was determined. The most promising compounds, with respect to activity and solubility, were found to be the butyroyloxymethyl esters of glutaric 2a and nicotinic acids 4a and phosphoric acid as its diethyl ester 10a, which displayed IC(50) values of 100 microM or lower. These prodrugs are expected to release formaldehyde upon metabolic hydrolysis. The corresponding butyroyloxyethyl esters (2b, 4b and 10b) that release acetaldehyde upon metabolism were significantly less potent. A similar correlation was observed for growth inhibition of the human prostate carcinoma cell lines PC-3 and LnCap and for induction of differentiation and apoptosis in the human myeloid leukemia cell line HL-60. The higher biological activity of the formaldehyde-releasing prodrugs 2a and 10a was further confirmed when induction of hemoglobin (Hb) synthesis in the human erythroleukemic cell line K562 was measured. Moreover, a therapeutic index (IC(50)/ED(50)) of ca. 5 was observed. The acute i.p. toxicity LD(50) in mice for 2a, 2b, 10a and 10b was similar and in the range of 400-600 mg kg(-1). The results obtained support the potential use of the butyric acid prodrugs for the treatment of neoplastic diseases and beta-globin disorders.

Ferritin expression in maturing normal human erythroid precursors.

We studied the expression of H- and L-ferritin subunits at sequential stages of maturation of normal human erythroid precursors. The erythroid cells developed in liquid culture and were purified immunomagnetically before analysis. It was found that the content of both ferritin subunits decreased exponentially with maturation: the decrease was rapid when cellular haemoglobin was low, and it slowed down when the haemoglobin was increased. This mode of decline was especially pronounced for the L-subunits. The H-/L-subunit ratio did not change significantly during the investigated period. The synthesis of both subunits was equal at each given developmental stage, and declined significantly with maturation. However, this decline was just slightly faster than that of total protein synthesis. The data indicated that the degradation of H- and L-ferritin also declined as maturation proceeded. No decrease was observed in mRNA levels of either ferritin subunit. Thus, the ferritin content and turnover were maximal at the beginning of haemoglobin accumulation and diminished later. As the rate of ferritin turnover determines the rate of incorporation and release of its iron, the results presented suggest that ferritin mediates cellular iron transport and donates iron for haem synthesis, mainly at the beginning of haemoglobin accumulation. The synthesis of both ferritin subunits is regulated during erythroid maturation at the post-transcriptional level.

Two-phase liquid culture system models normal human adult erythropoiesis at the molecular level.

We have studied the patterns of expression of various genes during maturation of normal human adult erythroid precursors cultured in a two-phase liquid culture method. In the first phase, peripheral blood mononuclear cells are cultured for one week in the presence of a combination of growth factors, but not erythropoietin (Epo). In Phase II, Epo is included in the medium. Cell samples were taken throughout phase II, and expression of globins, transcription factors, and cytokine receptors was assayed by RT-PCR and quantified by phosphor imaging. We have divided phase II into stages: early (days 0-5), intermediate (days 6-10) and late (days 11-15) and measured maximum expression of each gene. During early phase II, gamma-globin, Spl, and GATA-2 mRNAs were expressed at their highest levels. As the cells matured during the intermediate period, GATA-2 levels remained high, and then declined, while the transcription factors GATA-1, EKLF, NF-E2, and the Epo receptor (EpoR) reached maximum expression. In late phase II, beta-globin increased and reached its maximum level of expression. This erythroid culture system appears to recapitulate normal adult erythropoiesis at the molecular level, and thus may be a suitable model to examine the molecular basis of severe congenital or acquired disorders of erythropoiesis.

Flow cytometric analysis of autonomous growth of erythroid precursors in liquid culture detects occult polycythemia vera in the Budd-Chiari syndrome.

BACKGROUND/AIMS: Hepatic vein thrombosis (Budd-Chiari syndrome) is associated with various hypercoagulable states, such as polycythemia vera (PV), presence of the lupus anticoagulant, paroxysmal nocturnal hemoglobinuria (PNH) and deficiencies of antithrombin III, protein C and protein S. In recent years, it has become evident that patients with the Budd-Chiari syndrome may have more than one risk factor that may cause a state of hypercoagulability. The aim of the current study was to assess the prevalence of occult PV in patients with Budd-Chiari syndrome using a novel method for the detection of spontaneous erythroid growth. METHODS: Twenty-two patients with Budd-Chiari syndrome were evaluated. As controls, we studied normal donors and four patients with liver cirrhosis and five patients with right-side heart failure, two conditions that in part mimic Budd-Chiari syndrome. The presence of PV was determined by flow cytometric analysis of autonomous growth of erythroid precursors. Patients were considered as having occult PV if they had spontaneous erythroid cell growth in the absence of erythropoietin and with no features of overt PV. RESULTS: Cells from ten patients with Budd-Chiari syndrome demonstrated spontaneous erythroid cell growth; eight patients (32%) were found to have occult PV and two patients had overt PV. None of the controls had spontaneous erythroid growth. Of the eight Budd-Chiari patients with occult PV, six had one or more additional recognized hypercoagulable states. Seven patients (32%) had protein C deficiency, six patients (27%) had activated protein C resistance, five (23%) had anti-cardiolipin antibodies, five (23%) had antithrombin III deficiency, and four patients (18%) had protein S deficiency. Three patients (14%) were homozygous to methyltetra hydrofolate reductase and ten (45.5%) were heterozygous. One patient had PNH. Overall, in 12 patients there were two or more combined risk factors. CONCLUSIONS: Using a flow cytometric analysis of autonomous growth of erythroid precursors we found a clear correlation between Budd-Chiari syndrome and occult PV.

Regulation of intracellular iron metabolism in human erythroid precursors by internalized extracellular ferritin.

Human erythroid precursors grown in culture possess membrane receptors that bind and internalize acid isoferritin. These receptors are regulated by the iron status of the cell, implying that ferritin iron uptake may represent a normal physiologic pathway. The present studies describe the fate of internalized ferritin, the mechanisms involved in the release of its iron, and the recognition of this iron by the cell. Normal human erythroid precursors were grown in a 2-phase liquid culture that supports the proliferation, differentiation, and maturation of erythroid precursors. At the stage of polychromatic normoblasts, cells were briefly incubated with (59)Fe- and/or (125)I-labeled acid isoferritin and chased. The (125)I-labeled ferritin protein was rapidly degraded and only 50% of the label remained in intact ferritin protein after 3 to 4 hours. In parallel, (59)Fe decreased in ferritin and increased in hemoglobin. Extracellular holoferritin uptake elevated the cellular labile iron pool (LIP) and reduced iron regulatory protein (IRP) activity; this was inhibited by leupeptin or chloroquine. Extracellular apoferritin taken up by the cell functioned as an iron scavenger: it decreased the level of cellular LIP and increased IRP activity. We suggest that the iron from extracellular is metabolized in a similar fashion by developing erythroid cells as is intracellular ferritin. Following its uptake, extracellular ferritin iron is released by proteolytic degradation of the protein shell in an acid compartment. The released iron induces an increase in the cellular LIP and participates in heme synthesis and in intracellular iron regulatory pathways.

ABL1 methylation is a distinct molecular event associated with clonal evolution of chronic myeloid leukemia.

Methylation of the proximal promoter of the ABL1 oncogene is a common epigenetic alteration associated with clinical progression of chronic myeloid leukemia (CML). In this study we queried whether both the Ph'-associated and normal ABL1 alleles undergo methylation; what may be the proportion of hematopoietic progenitors bearing methylated ABL1 promoters in chronic versus acute phase disease; whether methylation affects the promoter uniformly or in patches with discrete clinical relevance; and, finally, whether methylation of ABL1 reflects a generalized process or is gene-specific. To address these issues, we adapted the techniques of methylation-specific PCR and bisulfite-sequencing to study the regulatory regions of ABL1 and other genes with a role in DNA repair or genotoxic stress response. In cell lines established from CML blast crisis, which only carry a single ABL1 allele nested within the BCR-ABL fusion gene, ABL1 promoters were universally methylated. By contrast, in clinical samples from patients at advanced stages of disease, both methylated and unmethylated promoter alleles were detectable. To distinguish between allele-specific methylation and a mixed cell population pattern, we studied the methylation status of ABL1 in colonies derived from single hematopoietic progenitors. Our results showed that both methylated and unmethylated promoter alleles coexisted in the same colony. Furthermore, ABL1 methylation was noted in the vast majority of colonies from blast crisis, but not chronic-phase CML. Both cell lines and clinical samples from acute-phase CML showed nearly uniform hypermethylation along the promoter region. Finally, we showed that ABL1 methylation does not reflect a generalized process and may be unique among DNA repair/genotoxic stress response genes. Our data suggest that specific methylation of the Ph'-associated ABL1 allele accompanies clonal evolution in CML.