Serum ferritin/C-reactive protein ratio is a simple and effective biomarker for diagnosing iron deficiency in the context of systemic inflammation.

BACKGROUND: Diagnosing iron deficiency is challenging in the presence of systemic inflammation. AIM: To investigate the relationship between plasma C-reactive protein (CRP), serum ferritin (SF) and transferrin saturation (TS), with the objective of establishing a straightforward ratio applicable in the presence of inflammatory syndrome. DESIGN: Test prospective cohort and validation retrospective cohort. METHODS: A prospective cohort of inpatients (n = 140) assessed the correlation between CRP and SF/TS levels. The diagnostic performance of a determined ratio was evaluated for identifying iron deficiency (ID) using different definitions and in the presence of inflammation and/or chronic heart and/or kidney failure. A large validation cohort (n = 795) further assessed the predictive power of this ratio. RESULTS: In a training cohort (median age 76 years [57-84]), a linear relation was observed between SF (µg/l) and CRP (mg/l), unlike with TS. The SF/CRP ratio accurately predicted ID, with receiver operating characteristic-area under the curve (ROC-AUC) values ranging from 0.85 to 0.92 for different ID definitions. A threshold of ≤6 demonstrated the highest Youden index (0.61). In the validation cohort (age 72 years [57-84]), the SF/CRP ratio exhibited an ROC-AUC of 0.88 [95% CI: 0.85-0.90], with an odds ratio of 37.9 [95% CI: 20.3-68.9] for the threshold of ≤6. CONCLUSION: In this study, we demonstrated that the SF/CRP ratio, with a threshold of ≤6, is a simple and effective biomarker for ID, even in the presence of systemic inflammation or comorbidities. This ratio could potentially replace the complex set of criteria currently recommended by learned societies.

A rare cause of abdominal pain: Fibromuscular dysplasia.

A 50-year-old man had a sudden severe abdominal pain. An abdominal enhanced computed tomographic scan revealed irregular stenosis of the celiac artery, dissection of the hepatic artery and of the left common iliac artery, and mid stenosis of the right renal artery with extended renal parenchymal hypodensity. After careful evaluation, the patient was diagnosed with fibromuscular dysplasia. Systematic head and neck CT angiography revealed right vertebral arterial stenosis and saccular aneurysm of the left vertebral artery. We have chosen conservative medical treatment for the multifocal fibromuscular dysplasia.

A role for reactive oxygen species in JAK2 V617F myeloproliferative neoplasm progression.

Although other mutations may predate the acquisition of the JAK2(V617F) mutation, the latter is sufficient to drive the disease phenotype observed in BCR-ABL-negative myeloproliferative neoplasms (MPNs). One of the consequences of JAK2(V617F) is genetic instability that could explain JAK2(V617F)-mediated MPN progression and heterogeneity. Here, we show that JAK2(V617F) induces the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment of a knock-in (KI) mouse model and in patients with JAK2(V617F) MPNs. JAK2(V617F)-dependent ROS elevation was partly mediated by an AKT-induced decrease in catalase expression and was accompanied by an increased number of 8-oxo-guanines and DNA double-strand breaks (DSBs). Moreover, there was evidence for a mitotic recombination event in mice resulting in loss of heterozygosity of Jak2(V617F). Mice engrafted with 30% of Jak2(V617F) KI bone marrow (BM) cells developed a polycythemia vera-like disorder. Treatment with the anti-oxidant N-acetylcysteine (NAC) substantially restored blood parameters and reduced damages to DNA. Furthermore, NAC induced a marked decrease in splenomegaly with reduction in the frequency of the Jak2(V617F)-positive hematopoietic progenitors in BM and spleen. Altogether, overproduction of ROS is a mediator of JAK2(V617F)-induced DNA damages that promote disease progression. Targeting ROS accumulation might prevent the development of JAK2(V617F) MPNs.

Zyxin is up-regulated during megakaryocytic differentiation of human UT-7/c-mpl cells.

To characterize genes involved in megakaryocytic commitment, we compared expression profiles of bipotent cells (UT-7/c-mpl) with those of the same cells induced to differentiate towards megakaryopoiesis in the presence of TPO. Using cDNA arrays, we showed that 12 out of 2260 genes changed their expression level after 6h of TPO stimulation. One of these genes encodes for zyxin, a cytoskeleton protein component. Zyxin is up-regulated at the mRNA and protein levels in UT-7/c-mpl cells in response to TPO confirming the reliability of the cDNA array technology. Similarly, when CD34 positive cells were induced to differentiate into megakaryocytes, zyxin mRNA was accumulated. Furthermore, when megakaryocytes were allowed to spread on fibrinogen, formation of stress fibers and lamellipodia was induced and zyxin was localized at the picks of actin stress fibers. These results suggest an important role for zyxin during megakaryocytic differentiation and more precisely in the regulation of the integrin mediated adhesion process in megakaryocytes.

The granulocyte colony-stimulating factor receptor supports erythroid differentiation in the absence of the erythropoietin receptor or Stat5.

To evaluate the functional conservation of signal transduction mechanisms between haematopoietic receptors and to characterize the molecules activated in this phenomenon, we introduced granulocyte colony-stimulating factor receptor (G-CSFR) cDNA into mouse fetal liver cells using a retroviral vector. In semi-solid medium assays, G-CSFR-infected cells gave rise to all types of colonies [granulocyte-macrophage (GM), megakaryocyte (MK) and mixed lineage (GEMM) colony-forming units (CFU) and erythroid burst-forming units (BFU-E)] in the presence of G-CSF alone. The direct effect of G-CSF on erythroid differentiation of G-CSFR-transduced erythroid progenitors was demonstrated by the development of erythroid colonies using G-CSFR-expressing Lin- cells cloned at one cell per well in liquid culture in the presence of G-CSF. Interestingly, while Stat5, but not Stat3, was activated in erythroid cells in response to erythropoietin (EPO), both were activated in erythroid and granulocytic cells stimulated by G-CSF. Furthermore, G-CSF induced the growth of erythroid colonies from G-CSFR-expressing fetal liver cells from EPO receptor-/- (EPO-R-/-) or Stat5a-/- Stat5b-/- mice, demonstrating that erythroid differentiation can occur in the absence of EPO-R or Stat5. These data show that forced expression of G-CSFR allows G-CSF-dependent multilineage proliferation and differentiation of haematopoietic progenitors and rescues EPO-R-/- erythroid cells. While G-CSF induces Stat5 activation in G-CSFR-expressing erythroid cells, this activation is not necessary for the terminal erythroid differentiation induced by G-CSF.

Inhibition of erythroid differentiation and induction of megakaryocytic differentiation by thrombopoietin are regulated by two different mechanisms in TPO-dependent UT-7/c-mpl and TF-1/c-mpl cell lines.

Thrombopoietin (TPO) regulates megakaryocytic (MK) maturation and platelet production. Molecular and cellular mechanisms of the TPO-induced MK differentiation are not totally understood. In order to develop cellular models to study these mechanisms, we introduced c-mpl into UT-7 and TF-1 cells by means of a retroviral vector and compared the effects of TPO on these two cell lines. UT-7 and TF-1 cell lines are two factor-dependent leukemic cell lines with an erythroid and MK phenotype. They proliferate in response to IL-3, GM-CSF and EPO, but not to TPO. The erythroid differentiation of both cell lines can be markedly increased by EPO. Several UT-7/c-mpl and TF-1/c-mpl cell clones which express different levels of the c-mpl protein (Mpl) were obtained and all became TPO-dependent for their proliferation. The UT-7/c-mpl clones, but not the TF-1/c-mpl clones, were capable of undergoing MK differentiation in response to TPO. This was demonstrated by the increase in MK markers (GPIIb, GPIIIa, GPIb alpha, GPIX and vWF), the appearance of cytoplasmic alpha-granules, intracellular membranes resembling demarcation membranes which were immunologically labeled with an GPIIb/IIIa anti-antibody, and a small percentage of polyploid cells (8N and 16N). In contrast, TPO inhibited the erythroid program of differentiation (glycophorin A, beta-globin and EPO receptor) as well as the differentiative activity of EPO in both UT-7/c-mpl and TF-1/c-mpl clones. It is noteworthy that the differentiative effect of EPO in TF-1/c-mpl cells was associated with an increase in GATA-1 transcripts which was totally suppressed by TPO. Overall the effects of TPO are the same as those of phorbol myristate acetate (PMA) which also induces MK differentiation and inhibits erythroid differentiation. These results suggest that: (1) Mpl expression is necessary but not sufficient for induction of MK differentiation; and (2) induction of Mk differentiation and inhibition of erythroid differentiation by TPO involve different signaling pathways; the pathway involved in the inhibition of erythroid differentiation might be related to a downregulation of GATA-1 expression in TF-1 cells.

Major effects of TPO delivered by a single injection of a recombinant adenovirus on prevention of septicemia and anemia associated with myelosuppression in mice: risk of sustained expression inducing myelofibrosis due to immunosuppression.

Adenoviral vectors may be useful tools to deliver a cytokine in vivo. A single intravenous injection of an adenovirus vector containing the human thrombopoietin (TPO) cDNA (AdRSVhuTPO) was able to induce a thrombocytosis for more than 6 weeks in SCID mice, associated with a megakaryocyte (MK) hyperplasia in different organs. A marrow and spleen fibrosis was observed at 6 weeks. In immunocompetent mice, a single AdRSVhuTPO injection led to a moderate and transient thrombocytosis without myelofibrosis. To evaluate the usefulness of TPO for the prevention of secondary side-effects during an aplastic period, mice were subjected to a myeloablative regimen 7 days after the intravenous AdRSVhuTPO injection. In this setting, TPO prevented mortality by accelerating hematological recovery. Survival was essentially related to an improvement in the leukopenia since all control mice died from septicemia. However, the effects of TPO may be potentiated by the release of inflammatory cytokines following the adenovirus infection; AdRSV beta galactosidase injected-mice had higher numbers of BFU-E and CFU-GM in the marrow than PBS-injected mice. Myelosuppression induced transient immunosuppression responsible for a sustained expression and elevation of platelet numbers for at least 5 months. These results further suggest that TPO may be an effective therapy in diminishing hematological complications related to myeloablative regimens, but emphasize that immunosuppression secondary to myelosuppression may lead to sustained expression associated with a risk of thrombosis and myelofibrosis when delivered by adenovirus vectors.

Thrombopoietin (TPO) knockout phenotype induced by cross-reactive antibodies against TPO following injection of mice with recombinant adenovirus encoding human TPO.

Adenovirus vectors have emerged as potent agents for gene transfer. Immune response against the vector and the encoded protein is one of the major factors in the transient expression following in vivo gene transfer. A single injection of an adenovirus encoding human thrombopoietin (TPO) into mice induced transient thrombocytosis, followed by a chronic immune thrombocytopenia. Thrombocytopenic mice had anti-human TPO Abs of the IgG2a and IgG1 isotypes. Thrombocytopenic mice sera neutralized more efficiently human than murine TPO, and exhibited no detectable anti-murine TPO Abs. Despite their low affinity for murine TPO, anti-TPO Abs induced a TPO knockout-like phenotype, i.e., low number of marrow megakaryocytes and of all kinds of hemopoietic progenitors. Hybridomas derived from a thrombocytopenic mouse revealed cross-reactivity of all of the secreted anti-TPO Ab isotypes. Mice subjected to myelosuppression after virus injection showed that anti-human TPO of IgG1 and IgG2a isotypes disappeared. Thus, sustained human TPO production was responsible for platelet elevation for at least 5 mo. Compelling results showed that elevated IgG2a/IgG2b ratios are always associated with thrombocytopenia, whereas low ratios are associated with tolerance or normal platelet counts. Finally, we hypothesize that in humans some chronic thrombocytopenia associated with a low TPO plasma level are due to anti-TPO Abs.

Thrombopoietin does not induce lineage-restricted commitment of Mpl-R expressing pluripotent progenitors but permits their complete erythroid and megakaryocytic differentiation.

In this study, we examined the in vitro and in vivo effects of forced expression of Mpl-R (the thrombopoietin receptor) on the progeny of murine hematopoietic stem cells. Bone marrow cells from 5-FU-treated mice were transduced with retroviral vectors containing the human Mpl-R cDNA, or the neomycine gene as a control. After 7 days cocultivation on virus-producer cells, GpE86-Mpl-R or Gp86-Neo, the types of hematopoietic progenitor cells responding to thrombopoietin (TPO) were studied by clonogenic assays. Mpl-R-infected cells gave rise to CFU-GEMM, BFU-E, CFU-MK, but not CFU-GM while Neo-infected cells produced only megakaryocytic colonies. In addition, when nonadherent cells from GpE86-Mpl-R cocultures were grown with TPO as the only stimulus for 7 days, a marked expansion of CFU-GEMM, BFU-E, and CFU-MK was observed, while no change in CFU-GM number was seen. Erythroid and megakaryocytic maturation occurred in the presence of TPO while a block in granulocytic differentiation was observed at the myeloblast stage. The direct effects of TPO on Mpl-R-transduced progenitor cells were demonstrated by single cell cloning experiments. To analyze the effects of the constitutive expression of Mpl-R on the determination of multipotent progenitors (CFU-S) and long-term repopulating stem cells, Mpl-R- or Neo-infected cells were injected into lethally irradiated recipient mice. No difference was seen in (1) the number of committed progenitor cells contained in individual CFU-S12 whether colonies arose from noninfected or Mpl-R-infected CFU-S; (2) the mean numbers of progenitor cells per leg or spleen of mice reconstituted with Mpl-R- or Neo-infected cells, 1 or 7 months after the graft; and (3) the blood parameters of the two groups of animals, with the exception of a 50% reduction in circulating platelet counts after 7 months in mice repopulated with Mpl-R-infected bone marrow cells. These results indicate that retrovirus-mediated expression of Mpl-R in murine stem cells does not modify their ability to reconstitute all myeloid lineages of differentiation and does not result in a preferential commitment toward the megakaryocytic lineage.

Ectopic expression of the erythropoietin receptor in a murine interleukin-6-dependent plasmacytoma cell line (TEPC-2027) confers proliferative responsiveness to erythropoietin.

To compare the signal transduction pathways used by erythropoietin (Epo) and interleukin-6 (IL-6), the cDNA for the murine Epo receptor (Epo-R) was introduced into an IL-6-responsive plasmacytoma cell line (TEPC-2027) by retrovirally mediated gene transfer. G418-resistant clones were amplified in IL-6 and studied for their ability to grow and differentiate in response to Epo. Epo-R synthesized from the viral gene showed the same affinity for Epo as did the receptor on erythroid cells; however, the numbers of Epo receptors expressed on the cell membrane varied among clones. After a delay of 3 to 5 days in the presence of Epo, all the clones studied proliferated as well in response to Epo as in response to IL-6. In response to IL-6, Stat3 was activated and JunB mRNA was accumulated, whereas in response to Epo, Jak2 and Stat5 were activated and JunB mRNA was not accumulated in Epo-R-expressing TEPC (Epo-R/TEPC) cells. These results suggest that Epo and IL-6 transduced their proliferative signals through different pathways. Further studies showed that, in Epo-R/TEPC cells, Epo neither induces the synthesis of erythroid-specific mRNA nor modifies the synthesis of gamma 1 lg heavy chain, suggesting that ectopic expression of the Epo-R in plasmacytoma cells does not modify their differentiative potential. The data show that Epo induces a proliferative response without differentiation providing a new cellular model for evaluating molecular events specific for proliferation.

Stromal cells maintain the radioprotective capacity of CFU-S during retroviral infection.

Retroviral vectors provide an efficient means to introduce genes into hematopoietic stem cells. In order to develop retroviral infection protocols which preserve the radioprotective capacity of CFU-S, we designed a clonal hematopoietic reconstitution assay. In this assay, single CFU-S-derived derived colonies from bone marrow cells of 5-FU-treated mice were tested for their capacity to prevent radiation-induced mortality. Three parameters which may modify stem cell potential were tested in infection protocols using a retroviral vector containing the gene for neomycin resistance: (1) the partition of stem cells between the adherent and nonadherent fraction; (2) the replacement of the packaging cell line by a "competent' stromal cell line; and (3) the effects of G418 selection. All CFU-S having radioprotective capacity were found in the adherent fraction when the packaging cell line or the stromal cell line (MS-5) chosen for its capacity to maintain long-term bone marrow culture were used during the co-culture. The neo resistance gene was transduced into CFU-S with the same efficiency using co-culture with the packaging cell line or co-culture with the MS-5 cell line plus viral supernatant. However, in the presence of MS-5, a much higher proportion of CFU-S (70% versus 30%) had radioprotective properties, suggesting an important role for the stromal cells in the maintenance of hematopoietic reconstituting ability. Finally, G418 selection, even for a limited period (24 h), significantly decreased the radioprotective capacities of CFU-S (56% versus 18%). Subsequently, hematopoietic reconstitution by single CFU-S was quantified in recipient mice. The progeny of CFU-S were found at a significant level in the blood, spleen and bone marrow in 38% and 15% of mice, 1 and 3 months after transplantation, respectively. These results demonstrate that we have substantially improved the infection protocol. Under these conditions of infection, it is possible to conserve CFU-S properties and to transduce a gene into a stem cell with short-term hematopoietic reconstitution potential.

Pluripotent stem cells constitutively expressing a normal erythropoietin receptor give rise to normal hematopoiesis in lethally irradiated recipient mice.

The cellular mechanism by which the stem cell differentiates toward an individual myeloid lineage is unknown. To determine whether lineage-specific cytokines are involved in stem cell determination, murine bone marrow cells were infected with a retroviral vector carrying a murine erythropoietin receptor (EpoR) cDNA. Infected marrow cells were transplanted into lethally irradiated syngeneic recipient mice, and the effect of Epo was studied on EpoR-expressing pluripotent stem cell determination. The graft contained, among myeloid cells, around 100 CFU-S12, half of which were retrovirally infected. One month after grafting, the bone marrow of mice reconstituted with EpoR-infected cells contained 50 times more infected multipotent progenitors than mice reconstituted with control bone marrow cells. However, this number returned to normal 45 days after the graft. No variation was observed in peripheral blood, bone marrow, and spleen cellularities or in committed progenitors in the bone marrow and in the spleen when Neo or EpoR reconstituted mice were assayed. When Epo was delivered into reconstituted mice one month after grafting, Epo had no differential effect in EpoR or Neo reconstituted mice. This study emphasizes the in vivo Epo proliferative response of multipotent progenitors expressing a normal EpoR gene and shows that, in vivo as in vitro, the differentiation of these multipotent progenitors is not preferentially oriented toward erythropoiesis.

Murine pluripotent hematopoietic progenitors constitutively expressing a normal erythropoietin receptor proliferate in response to erythropoietin without preferential erythroid cell differentiation.

Erythropoietin (EPO) is a prime regulator of the growth and differentiation of erythroid blood cells. The EPO receptor (EPO-R) is expressed in late erythroid progenitors (mature BFU-E and CFU-E), and EPO induces proliferation and differentiation of these cells. By introducing, with a retroviral vector, a normal EPO-R cDNA into murine adult bone marrow cells, we showed that EPO is also able to induce proliferation in pluripotent progenitor cells. After 7 days of coculture with virus-producing cells, bone marrow cells were plated in methylcellulose culture in the presence of EPO, interleukin-3, or Steel factor alone or in combination. In the presence of EPO alone, EPO-R virus-infected bone marrow cells gave rise to mixed colonies comprising erythrocytes, granulocytes, macrophages and megakaryocytes. The addition of interleukin-3 or Steel factor to methylcellulose cultures containing EPO did not significantly modify the number of mixed colonies. The cells which generate these mixed colonies have a high proliferative potential as shown by the size and the ability of the mixed colonies to give rise to secondary colonies. Thus, it appears that EPO has the same effect on EPO-R-expressing multipotent cell proliferation as would a combination of several growth factors. Finally, our results demonstrate that inducing pluripotent progenitor cells to proliferate via the EPO signaling pathway has no major influence on their commitment.

Infection with a Kirsten-retrovirus can induce a multiplicity of tumorigenic phenotypes in the interleukin-3-dependent FDC-P1 cells.

The identification of ras oncogenes in both human and animal tumors as well as in preleukemic and precancerous lesions suggests that activated ras genes participate in neoplastic development, yet the precise role of ras oncogenes in leukemogenesis is not clear. To assess the functional role of ras genes in tumorigenesis, we introduced with a retroviral vector either a wild-type (Gly-12) or a mutant (Val-12) Kirsten ras cDNA into the cells of a factor-dependent myeloid cell line, FDC-P1. FDC-P1 cells are nontumorigenic and their proliferation is dependent on either interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). The Ki-Val 12-infected FDC-P1 cell population is still strictly IL-3-dependent but has acquired the ability to survive up to 72 hours in the absence of growth factor and to form tumors in nude mice. These tumors are easily established into cell lines that are clonal and show a multiplicity of phenotypes with respect to their growth factor dependence. These results suggest that, in contrast with the overexpression of a normal Ki-ras, Ki-ras oncogene can efficiently promote the tumorigenic conversion of FDC-P1 cells. However, the clonality of the tumors as well as the distinct phenotypes indicates that other genetic events are required for tumorigenicity. Therefore, in FDC-P1 cells, an activated ras gene acts as a dominant oncogene through the induction of tumor progression. Finally, in this simple experimental system we observed a multiplicity of tumorigenic phenotypes which are reminiscent of those observed in patients with acute myeloid leukemia.