Anna Rita Migliaccio

Abstract There is evidence that the total cellular content of placental cord blood (PCB) grafts is related to the speed of engraftment, though the total nucleated cell (TNC) dose is not a precise predictor of the time of neutrophil or platelet engraftment. It is important to understand the reasons for the quantitative association and to improve the criteria for selecting PCB grafts by using indices more precisely predictive of engraftment. The posttransplant course of 204 patients who received grafts evaluated for hematopoietic colony-forming cell (CFC) content among 562 patients reported previously were analyzed using univariate and multivariate life-table techniques to determine whether CFC doses predicted hematopoietic engraftment speed and risk for transplant-related events more accurately than the TNC dose. Actuarial times to neutrophil and platelet engraftment were shown to correlate with the cell dose, whether estimated as TNC or CFC per kilogram of recipient's weight. CFC association with the day of recovery of 500 neutrophils/μL, measured as the coefficient of correlation, was stronger than that of the TNC (R = −0.46 and −0.413, respectively). In multivariate tests of speed of platelet and neutrophil engraftment and of probability of posttransplantation events, the inclusion of CFC in the model displaced the significance of the high relative risks associated with TNC. The CFC content of PCB units is associated more rigorously with the major covariates of posttransplantation survival than is the TNC and is, therefore, a better index of the hematopoietic content of PCB grafts.

Recombinant rat stem cell factor (SCF) was studied for its ability to stimulate the growth of murine hematopoietic progenitor cells and to generate colony-forming cells (CFC) from highly enriched populations of hematopoietic cells. In serum-deprived cultures, SCF alone stimulated few colonies but interacted with a number of other hematopoietic growth factors, particularly interleukin 3, to promote colony formation. The most marked effect was on the generation of mixed-cell colonies. Hematopoietic cells were sorted into wheat-germ agglutinin-negative, monocyte-depleted, rhodamine 123 (Rh123)-bright or Rh123-dull cells. Historically, Rh123-bright cells are capable of short-term (less than 1 mo) marrow engraftment, whereas among Rh123-dull cells are cells capable of long-term marrow engraftment. Enriched cells (2.5 x 10(3) were placed into serum-deprived liquid cultures with various hematopoietic growth factors. Initially, the Rh123-bright and Rh123-dull cells had few CFC but, in the presence of interleukin 3 and SCF, Rh123-bright cells gave rise to greater than 15,000 granulocyte/macrophage CFC, greater than 1500 erythroid burst-forming cells, and greater than 700 mixed-cell CFC by day 5. In contrast, Rh123-dull cells proliferated only in the presence of interleukin 3 and SCF, but total cell numbers rose to a peak of 18,000 by day 21, and one-third of the cells were CFC. Thus, SCF, in combination with other growth factors, can generate large numbers of CFC from pre-CFC and appears to act earlier than hematopoietic growth factors described to date.

Lysyl oxidase (LOX), a matrix cross-linking protein, is known to be selectively expressed and to enhance a fibrotic phenotype. A recent study of ours showed that LOX oxidizes the PDGF receptor-β (PDGFR-β), leading to amplified downstream signaling. Here, we examined the expression and functions of LOX in megakaryocytes (MKs), the platelet precursors. Cells committed to the MK lineage undergo mitotic proliferation to yield diploid cells, followed by endomitosis and acquisition of polyploidy. Intriguingly, LOX expression is detected in diploid-tetraploid MKs, but scarce in polyploid MKs. PDGFR-BB is an inducer of mitotic proliferation in MKs. LOX inhibition with β-aminopropionitrile reduces PDGFR-BB binding to cells and downstream signaling, as well as its proliferative effect on the MK lineage. Inhibition of LOX activity has no influence on MK polyploidy. We next rationalized that, in a system with an abundance of low ploidy MKs, LOX could be highly expressed and with functional significance. Thus, we resorted to GATA-1(low) mice, where there is an increase in low ploidy MKs, augmented levels of PDGF-BB, and an extensive matrix of fibers. MKs from these mice display high expression of LOX, compared with control mice. Importantly, treatment of GATA-1(low) mice with β-aminopropionitrile significantly improves the bone marrow fibrotic phenotype, and MK number in the spleen. Thus, our in vitro and in vivo data support a novel role for LOX in regulating MK expansion by PDGF-BB and suggest LOX as a new potential therapeutic target for myelofibrosis.

Recent advances have increased excitement about the potential for therapeutic production of red blood cells (RBCs) in vitro. However, generation of RBCs in the large numbers required for transfusion remains a significant challenge. In this article, we summarize recent progress in producing RBCs from various cell sources, and discuss the hurdles that remain for translation into the clinical arena.

Proinflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution are yet to be fully elucidated. Previously, we identified a critical role for combined constitutive JAK/STAT and aberrant NF-κB proinflammatory signaling in MF development. Using single-cell transcriptional and cytokine-secretion studies of primary cells from patients with MF and the human MPLW515L (hMPLW515L) murine model of MF, we extend our previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. Hematopoietic stem/progenitor cells from patients with MF are enriched for a CXCL8/CXCR2 gene signature and display enhanced proliferation and fitness in response to an exogenous CXCL8 ligand in vitro. Genetic deletion of Cxcr2 in the hMPLW515L-adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway among patients with MF.

Erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) dependent cell lines have been derived from the murine hematopoietic cell line 32D with a selection strategy involving the culture of the cells in FBS-deprived medium supplemented only with pure recombinant Epo, GM-CSF, or G-CSF. The cells retain the diploid karyotype of the original 32D clone, do not grow in the absence of exogenous growth factor, and do not induce tumors when injected into syngeneic recipients. The morphology of the Epo-dependent cell lines (32D Epo1, -2, and -3) was heterogeneous and evolved with passage. The percent of differentiated cells also was a function of the cell line investigated. Benzidine-positive cells ranged from 1-2% (32D Epo3) to 50-60% (32D Epo1). These erythroid cells expressed carbonic anhydrase I and/or globin mRNA but not carbonic anhydrase II. The GM-CSF- and G-CSF-dependent cell lines had predominantly the morphology of undifferentiated myeloblasts or metamyelocytes, respectively. The GM-CSF-dependent cell lines were sensitive to either GM-CSF or interleukin-3 (IL-3) but did not respond to G-CSF. The G-CSF-dependent cell lines grew to a limited extent in IL-3 but did not respond to GM-CSF. These results indicate that the cell line 32D, originally described as predominantly a basophil/mast cell line, has retained the capacity to give rise to cells which proliferate and differentiate in response to Epo, GM-CSF, and/or G-CSF. These cells represent the first nontransformed cell lines which can be maintained in growth factors other than IL-3 and which differentiate in the presence of physiologic signals. As such, they may represent a model to study the molecular mechanisms underlying the process of hematopoietic differentiation, as well as sensitive targets for bioassays of specific growth factors.

Ex vivo-generated erythroblasts represent alternative transfusion products. However, inclusion of bovine components in media used for their growth precludes clinical use, highlighting the importance of developing culture media based on pharmaceutical grade reagents. In addition, because adult blood generates ex vivo lower numbers of erythroblasts than cord blood, cord blood has been proposed as the source of choice for ex vivo erythroblast production. To clarify the potential of adult blood to generate erythroblasts ex vivo, experiments were designed to identify growth factors [stem cell factor (SCF), interleukin-3 (IL-3), erythropoietin (EPO), and/or thrombopoietin (TPO)] and the optimal concentration and addition schedule of hormones (dexamethasone and estradiol) sustaining maximal erythroid amplification from adult blood mononuclear cells (MNC) using media with serum previously defined as human erythroid massive amplification culture (HEMA ser ). Adult MNC stimulated with SCF and IL-3 in combination with EPO generated a 6–12-fold increase in erythroid cells while TPO was ineffective. Dexamethasone and estradiol (both at 10 −6 M) exerted partially overlapping but nonredundant functions. Dexamethasone was indispensable in the first 10 days of culture while estradiol was required from day 10 on. The growth factor and hormone combinations identified in HEMA ser were then used to formulate a media composed of dialyzed pharmaceutical grade human albumin, human albumin-lipid liposomes, and iron-saturated recombinant human tranferrin (HEMA def ). HEMA def sustained erythroid amplification as efficiently as HEMA ser for cord blood MNC and 10-fold higher than HEMA ser for adult blood MNC. In fact, the numbers of erythroblasts generated in HEMA def by adult MNC were similar to those generated by cord blood MNC. In conclusion, this study identifies growth factors, hormone combinations, and human protein-based media that allow similar levels of ex vivo erythroid expansion from adult and cord blood MNC, paving the way to evaluate adult blood as a source of ex vivo-expanded erythroblasts for transfusion.

Abstract We have studied the effect of recombinant human Stem Cell Factor (SCF) on the growth of human peripheral blood, bone marrow, and cord blood progenitor cells in semisolid medium. While SCF alone had little colony‐stimulating activity under fetal bovine serum (FBS)‐deprived culture conditions, SCF synergized with erythropoietin (Epo), granulocyte/macrophage colony‐stimulating factor (GM‐CSF), and interleukin 3 (IL‐3) to stimulate colony growth. Colony morphology was determined by the late‐acting growth factor added along with SCF. Of all the combinations of growth factors, SCF plus IL‐3 and Epo resulted in the largest number of mixed‐cell colonies‐ a larger number than observed with IL‐3 and Epo alone even in FBS‐supplemented cultures. These results suggest that SCF is a growth factor that more specifically targets early progenitor cells (mixed‐cell colony‐forming cells) and has the capacity to synergize with a wide variety of other hematopoietic growth factors to cause the proliferation and differentiation of committed progenitor cells. Our studies indicate that SCF may be the earliest acting growth factor described to date.

In order to investigate differences in control mechanisms between embryonic and adult hemopoiesis, we have studied the sensitivity of human embryonic progenitors (5–8 weeks postconception) to either positive (erythropoietin (Ep), granulocyte-macrophage colony-stimulating factor (GM-CSF) and insulin-like growth factor 1 (IGF-1)) or negative (tumor necrosis factor (TNF) and interferon-γ (IFN-γ)) in vitro regulators of adult hemopoietic differentiation. Growth stimulators were analyzed under serum-deprived conditions whereas growth inhibitors were investigated in serum-supplemented culture. Formation of granulocyte-macrophage colonies from embryonic progenitors was induced by GM-CSF but inhibited by TNF and IFN-γ. Early erythroid progenitors resemble adult erythroid burst-forming cells (BFU-E) in their sensitivity to Ep and TNF but differ in their lack of response to GM-CSF or other adult sources of burst-promoting activity, and absence of inhibition by IFN-γ. IGF-1 promoted erythroid burst formation in the absence of insulin, but did not have Ep-like activity. These data indicate that embryonic and adult erythroid progenitors differ at least in terms of in vitro sensitivity to GM-CSF and IFN-γ and suggest that different cellular response to control signals may underlie the differences observed in vivo between embryonic and adult hemopoiesis.

Blood transfusion is indispensable for modern medicine. In developed countries, the blood supply is adequate and safe but blood for alloimmunized patients is often unavailable. Concerns are increasing that donations may become inadequate in the future as the population ages prompting a search for alternative transfusion products. Improvements in culture conditions and proof-of-principle studies in animal models have suggested that ex-vivo expanded red cells may represent such a product. Compared to other cell therapies transfusion poses the unique challenge of requiring great cell doses (2.5×10(12) cells vs 10(7) cells). Although production of such cell numbers is theoretically possible, current technologies generate red cells in numbers sufficient only for safety studies. It is conceived that by the time these studies will be completed, technical barriers to mass cell production will have been eliminated making transfusion with ex-vivo generated red cells a reality.

The definition "porcine respiratory disease complex" (PRDC) is used to indicate the current approach for presenting respiratory pathology in modern pig farming. PRDC includes pneumonias with variable pictures, mixed with both aerogenous and hematogenous forms with variable etiology, often multimicrobial, and influenced by environmental and management factors. The notion that many etiological agents of swine respiratory pathology are ubiquitous in the airways is commonly understood; however, their isolation or identification is not always associable with the current pathology. In this complex context, lung lesions registered at slaughterhouse or during necropsy, and supplemented by histological investigations, must be considered as powerful tools for assigning a prominent role to etiologic agents. In recent years, the goal of colocalizing causative agents with the lesions they produce has been frequently applied, and valid examples in routine diagnostics are those that indicate pulmonary involvement during porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) infections.

After the approval of suberoylanilide hydroxamic acid (SAHA, vorinostat, Zolinza) for the treatment of cutaneous T cell lymphoma (CTCL), a number of HDAC inhibitors (HDACi) are currently in Phase II or III clinical trials (alone or in combination) for the treatment of a great number of tumors. In addition to these cancer uses, HDACi can be successfully used in non-cancer diseases. In this review we focused on the uses of HDACi in some infectious diseases and beta-hemoglobinopaties. In C. albicans cultures, HDACi increased the frequency of cell switching (a relevant virulence trait) in the white-to-opaque transition, reduced the azole trailing effect through reduction in azole-dependent upregulation of CDR and ERG genes, and inhibited the fluconazole-dependent resistance induction. Moreover, they inhibited germination in several strains, and caused 90% reduction in the adherence of C. albicans to human cultured pneumocytes. In HIV-1-infected cells, the treatment with HDACi reactivates the HIV-1 expression in latent cellular reservoirs. Thus, the use of HDACi as adjuvant to highly active antiretroviral therapy (HAART) can represent a new potential therapeutic strategy to eradicate the viral infection. A number of HDACi have been reported as active against P. falciparum infection. Two recent papers show some 2-aminosuberic acid-based compounds as well as a series of phenylthiazolyl suberoyl hydroxamates as very potent and selective antimalarial agents. Among the many agents capable to perform post-natal reactivation of fetal hemoglobin production, HDACi for their capacity to de-repress gamma-globin gene expression in adult red cell, are presently considered promising molecules for personalized therapy of beta-hemoglobinopathies.

Glucocorticoid receptor (GR) agonists increase erythropoiesis in vivo and in vitro. To clarify the effect of the dominant negative GRβ isoform (unable to bind STAT-5) on erythropoiesis, erythroblast (EB) expansion cultures of mononuclear cells from 18 healthy (nondiseased) donors (NDs) and 16 patients with polycythemia vera (PV) were studied. GRβ was expressed in all PV EBs but only in EBs from 1 ND. The A3669G polymorphism, which stabilizes GRβ mRNA, had greater frequency in PV (55%; n = 22; P = .0028) and myelofibrosis (35%; n = 20) patients than in NDs (9%; n = 22) or patients with essential thrombocythemia (6%; n = 15). Dexamethasone stimulation of ND cultures increased the number of immature EBs characterized by low GATA1 and β-globin expression, but PV cultures generated great numbers of immature EBs with low levels of GATA1 and β-globin irrespective of dexamethasone stimulation. In ND EBs, STAT-5 was not phosphorylated after dexamethasone and erythropoietin treatment and did not form transcriptionally active complexes with GRα, whereas in PV EBs, STAT-5 was constitutively phosphorylated, but the formation of GR/STAT-5 complexes was prevented by expression of GRβ. These data indicate that GRβ expression and the presence of A3669G likely contribute to development of erythrocytosis in PV and provide a potential target for identification of novel therapeutic agents.

Abstract The frequency of A3669G single nucleotide polymorphism (SNP) of human glucocorticoid receptor has been reported increased in polycythemia vera. We investigated the frequency of A3669G SNP and its impact on disease phenotype and progression in 499 patients with primary myelofibrosis (PMF). The distribution of the A3669G allele differed between PMF patients and 2 healthy control populations (odds ratio, 1.6 and 1.8). The variant allele at the homozygous state (G/G) was associated with higher white blood cell count, larger spleen index, and higher frequency of circulating CD34+ cells at diagnosis. The latter association remained significant after correction for the JAK2V617F genotype. In patients JAK2V617F mutated, the G/G genotype was associated with shorter overall survival (77.6 months vs 298 months, P = .049) and blast transformation (BT)–free survival (76.7 months vs 261 months; P = .018). The latter association remained significant after correction for the known BT risk factors, such as age, sex, white blood cell count, percentage of blasts, IPSS prognostic score, and homozygosity for JAK2V617F (hazard ratio = 3.3; P = .006). In conclusion, the glucocorticoid receptor A3669G is a susceptibility allele for PMF: it contributes to confer the phenotype of excess myeloproliferation, and it cooperates with the JAK2V617F mutation in determining BT.

Placental/umbilical cord blood (PCB) is a source of hematopoietic stem cells for bone marrow reconstitution. Engraftment speed and survival are related to the total nucleated cell (TNC) dose of the graft. This study explored the possible influence on engraftment of nucleated red blood cells (NRBCs) in the graft. Automated hematology analyzers were used to enumerate TNCs. NRBCs were counted by visual examination or by using an automated analyzer. Hematopoietic progenitor cells were enumerated as either colony-forming cells or CD34+ cells. Transplant centers reported on transplant outcome in 1112 patients given PCB grafts through September 2001. NRBCs correlated with progenitor cell numbers. Both white blood cell and NRBC dose were independently predictive of myeloid engraftment speed. Because NRBC dose predicted engraftment speed, inclusion of NRBCs in the TNC count does not reduce the effectiveness of the prefreezing TNC count as an index of the quality of a PCB unit as a graft. The correlation between the number of NRBCs and the number of hematopoietic progenitor cells probably reflects the involvement of early stem cells in erythroid responses.

Calreticulin is a Ca2+-binding chaperone protein, which resides mainly in the endoplasmic reticulum but also found in other cellular compartments including the plasma membrane. In addition to Ca2+, calreticulin binds and regulates almost all proteins and most of the mRNAs deciding their intracellular fate. The potential functions of calreticulin are so numerous that identification of all of them is becoming a nightmare. Still the recent discovery that patients affected by the Philadelphia-negative myeloproliferative disorders essential thrombocytemia or primary myelofibrosis not harboring JAK2 mutations carry instead calreticulin mutations disrupting its C-terminal domain has highlighted the clinical need to gain a deeper understanding of the biological activity of this protein. However, by contrast with other proteins, such as enzymes or transcription factors, the biological functions of which are strictly defined by a stable spatial structure imprinted by their amino acid sequence, calreticulin contains intrinsically disordered regions, the structure of which represents a highly dynamic conformational ensemble characterized by constant changes between several metastable conformations in response to a variety of environmental cues. This article will illustrate the Theory of calreticulin as an intrinsically disordered protein and discuss the Hypothesis that the dynamic conformational changes to which calreticulin may be subjected by environmental cues, by promoting or restricting the exposure of its active sites, may affect its function under normal and pathological conditions.

Myelofibrosis (MF) is characterized by hyperactivation of thrombopoietin (TPO) signaling, which induces a RPS14 deficiency that de-regulates GATA1 in megakaryocytes by hampering its mRNA translation. As mice carrying the hypomorphic Gata1low mutation, which reduces the levels of Gata1 mRNA in megakaryocytes, develop MF, we investigated whether the TPO axis is hyperactive in this model. Gata1low mice contained two times more Tpo mRNA in liver and TPO in plasma than wild-type littermates. Furthermore, Gata1low LSKs expressed levels of Mpl mRNA (five times greater than normal) and protein (two times lower than normal) similar to those expressed by LSKs from TPO-treated wild-type mice. Gata1low marrow and spleen contained more JAK2/STAT5 than wild-type tissues, an indication that these organs were reach of TPO-responsive cells. Moreover, treatment of Gata1low mice with the JAK inhibitor ruxolitinib reduced their splenomegaly. Also in Gata1low mice activation of the TPO/MPL axis was associated with a RSP14 deficiency and a discordant microarray ribosome signature (reduced RPS24, RPS26 and SBDS expression). Finally, electron microscopy revealed that Gata1low megakaryocytes contained poorly developed endoplasmic reticulum with rare polysomes. In summary, Gata1low mice are a bona fide model of MF, which recapitulates the hyperactivation of the TPO/MPL/JAK2 axis observed in megakaryocytes from myelofibrotic patients.

•Treatment with RB40.34 targeting murine P-selectin and ruxolitinib, in combination, is more effective than either drug alone in rescuing myelofibrosis in Gata1low mice.•This drug combination reduced fibrosis, bone deposition and neoangiogenesis while restoring hematopoiesis in bone marrow and reducing hematopoiesis in spleen.•Mechanistically, the drug combination may have exerted their effects by reducing the TGF-β and CXCL1 content of the bone marrow.•These data provide pre-clinical evidence that P-selectin antibodies and Ruxolitinib in combination may be more effective than Ruxulotinib alone to treat myelofibrosis. The bone marrow (BM) and spleen from patients with myelofibrosis (MF), as well as those from the Gata1low mouse model of the disease contain increased number of abnormal megakaryocytes. These cells express high levels of the adhesion receptor P-selectin on their surface, which triggers a pathologic neutrophil emperipolesis, leading to increased bioavailability of transforming growth factor-β (TGF-β) in the microenvironment and disease progression. With age, Gata1low mice develop a phenotype similar to that of patients with MF, which is the most severe of the Philadelphia-negative myeloproliferative neoplasms. We previously demonstrated that Gata1low mice lacking the P-selectin gene do not develop MF. In the current study, we tested the hypothesis that pharmacologic inhibition of P-selectin may normalize the phenotype of Gata1low mice that have already developed MF. To test this hypothesis, we have investigated the phenotype expressed by aged Gata1low mice treated with the antimouse monoclonal antibody RB40.34, alone and also in combination with ruxolitinib. The results indicated that RB40.34 in combination with ruxolitinib normalizes the phenotype of Gata1low mice with limited toxicity by reducing fibrosis and the content of TGF-β and CXCL1 (two drivers of fibrosis in this model) in the BM and spleen and by restoring hematopoiesis in the BM and the architecture of the spleen. In conclusion, we provide preclinical evidence that treatment with an antibody against P-selectin in combination with ruxolitinib may be more effective than ruxolitinib alone to treat MF in patients. The bone marrow (BM) and spleen from patients with myelofibrosis (MF), as well as those from the Gata1low mouse model of the disease contain increased number of abnormal megakaryocytes. These cells express high levels of the adhesion receptor P-selectin on their surface, which triggers a pathologic neutrophil emperipolesis, leading to increased bioavailability of transforming growth factor-β (TGF-β) in the microenvironment and disease progression. With age, Gata1low mice develop a phenotype similar to that of patients with MF, which is the most severe of the Philadelphia-negative myeloproliferative neoplasms. We previously demonstrated that Gata1low mice lacking the P-selectin gene do not develop MF. In the current study, we tested the hypothesis that pharmacologic inhibition of P-selectin may normalize the phenotype of Gata1low mice that have already developed MF. To test this hypothesis, we have investigated the phenotype expressed by aged Gata1low mice treated with the antimouse monoclonal antibody RB40.34, alone and also in combination with ruxolitinib. The results indicated that RB40.34 in combination with ruxolitinib normalizes the phenotype of Gata1low mice with limited toxicity by reducing fibrosis and the content of TGF-β and CXCL1 (two drivers of fibrosis in this model) in the BM and spleen and by restoring hematopoiesis in the BM and the architecture of the spleen. In conclusion, we provide preclinical evidence that treatment with an antibody against P-selectin in combination with ruxolitinib may be more effective than ruxolitinib alone to treat MF in patients. Myelofibrosis (MF) is the most severe of Philadelphia chromosome–negative myeloproliferative neoplasms (MPN). The complex phenotype of patients with MF includes fibrosis and hematopoietic failure in bone marrow (BM), stem/progenitor cell mobilization, and development of extramedullary hematopoiesis with splenomegaly, and their clinical course is associated with an increased risk of thrombosis, bleeding, and evolution to acute leukemia [1Zahr AA Salama ME Carreau N et al.Bone marrow fibrosis in myelofibrosis: pathogenesis, prognosis and targeted strategies.Haematologica. 2016; 101: 660-671Crossref PubMed Scopus (94) Google Scholar, 2Barbui T Tefferi A Vannucchi AM et al.Philadelphia chromosome-negative classical myeloproliferative neoplasms: revised management recommendations from European LeukemiaNet.Leukemia. 2018; 32: 1057-1069Crossref PubMed Scopus (336) Google Scholar, 3Marcellino BK Verstovsek S Mascarenhas J. The myelodepletive phenotype in myelofibrosis: clinical relevance and therapeutic implication.Clin Lymphoma Myeloma Leuk. 2020; 20: 415-421Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 4Dunbar AJ Rampal RK Levine R. Leukemia secondary to myeloproliferative neoplasms.Blood. 2020; 136: 61-70Crossref PubMed Scopus (65) Google Scholar]. MF may be driven by gain of function mutations in several genes of the thrombopoietin axes, such as MPL, the thrombopoietin receptor; JAK2, the first element of the MPL signaling; and calreticulin, a chaperon protein, which when mutated binds MPL on the cell surface, inducing conformational changes, leading to ligand-independent constitutive activation of the receptor [5Vainchenker W Constantinescu SN. JAK/STAT signaling in hematological malignancies.Oncogene. 2013; 32: 2601-2613Crossref PubMed Scopus (391) Google Scholar]. Regardless of the driver mutation, it has been recognized that MF has a distinctive cellular signature. In fact, both the BM and spleen of such patients contain numerous clusters of morphologically immature megakaryocytes (MKs) [6Malara A Abbonante V Zingariello M Migliaccio A Balduini A. Megakaryocyte contribution to bone marrow fibrosis: many arrows in the quiver.Mediterr J Hematol Infect Dis. 2018; 10e2018068Crossref PubMed Google Scholar] endowed with great proliferation potential [7Schmitt A Jouault H Guichard J Wendling F Drouin A Cramer EM. Pathologic interaction between megakaryocytes and polymorphonuclear leukocytes in myelofibrosis.Blood. 2000; 96: 1342-1347Crossref PubMed Google Scholar,8Centurione L Di Baldassarre A Zingariello M et al.Increased and pathologic emperipolesis of neutrophils within megakaryocytes associated with marrow fibrosis in GATA-1low mice.Blood. 2004; 104: 3573-3580Crossref PubMed Scopus (105) Google Scholar]. In MF, MKs are retained immature by a mutation-driven RSP14 ribosomopathy that impairs the translation of the mRNA for GATA1 [9Vannucchi AM Pancrazzi A Guglielmelli P et al.Abnormalities of GATA-1 in megakaryocytes from patients with idiopathic myelofibrosis.Am J Pathol. 2005; 167: 849-858Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar,10Gilles L Arslan AD Marinaccio C et al.Downregulation of GATA1 drives impaired hematopoiesis in primary myelofibrosis.J Clin Invest. 2017; 127: 1316-1320Crossref PubMed Scopus (54) Google Scholar], the transcription factor that plays a pivotal role in the progression of MK maturation [11Crispino JD Weiss MJ. Erythro-megakaryocytic transcription factors associated with hereditary anemia.Blood. 2014; 123: 3080-3088Crossref PubMed Scopus (46) Google Scholar]. The causative role of the resulting abnormal MKs in the pathogenesis of this disease is strongly supported by experiments in mice, indicating that those carrying a hypomorphic mutation, which selectively reduces GATA1 in MKs (Gata1low mice), develop MF with age [12Vannucchi AM Bianchi L Cellai C et al.Development of myelofibrosis in mice genetically impaired for GATA-1 expression (GATA-1(low) mice).Blood. 2002; 100: 1123-1132Crossref PubMed Scopus (198) Google Scholar], whereas transgenic mice expressing JAK2V617F, one of the driver mutations of the disease [1Zahr AA Salama ME Carreau N et al.Bone marrow fibrosis in myelofibrosis: pathogenesis, prognosis and targeted strategies.Haematologica. 2016; 101: 660-671Crossref PubMed Scopus (94) Google Scholar, 2Barbui T Tefferi A Vannucchi AM et al.Philadelphia chromosome-negative classical myeloproliferative neoplasms: revised management recommendations from European LeukemiaNet.Leukemia. 2018; 32: 1057-1069Crossref PubMed Scopus (336) Google Scholar, 3Marcellino BK Verstovsek S Mascarenhas J. The myelodepletive phenotype in myelofibrosis: clinical relevance and therapeutic implication.Clin Lymphoma Myeloma Leuk. 2020; 20: 415-421Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 4Dunbar AJ Rampal RK Levine R. Leukemia secondary to myeloproliferative neoplasms.Blood. 2020; 136: 61-70Crossref PubMed Scopus (65) Google Scholar], only in MKs develop MF even if their hematopoietic stem cells (HSCs) are normal [13Woods B Chen W Chiu S et al.Activation of JAK/STAT signaling in megakaryocytes sustains myeloproliferation in vivo.Clin Cancer Res. 2019; 25: 5901-5912Crossref PubMed Scopus (37) Google Scholar,14Zhang Y Lin CHS Kaushansky K Zhan H. JAK2V617F megakaryocytes promote hematopoietic stem/progenitor cell expansion in mice through thrombopoietin/MPL signaling.Stem Cells. 2018; 36: 1676-1684Crossref PubMed Scopus (22) Google Scholar]. As first hypothesized by Schmitt et al. [7Schmitt A Jouault H Guichard J Wendling F Drouin A Cramer EM. Pathologic interaction between megakaryocytes and polymorphonuclear leukocytes in myelofibrosis.Blood. 2000; 96: 1342-1347Crossref PubMed Google Scholar], mechanistically, malignant MKs are thought to drive MF by engaging in a pathologic process of emperipolesis with the neutrophils, increasing the bioavailability of transforming growth factor-β (TGF-β) and possibly other proinflammatory cytokines in the BM of patients with MF and animal models [8Centurione L Di Baldassarre A Zingariello M et al.Increased and pathologic emperipolesis of neutrophils within megakaryocytes associated with marrow fibrosis in GATA-1low mice.Blood. 2004; 104: 3573-3580Crossref PubMed Scopus (105) Google Scholar,15Ciurea SO Merchant D Mahmud N et al.Pivotal contributions of megakaryocytes to the biology of idiopathic myelofibrosis.Blood. 2007; 110: 986-993Crossref PubMed Scopus (163) Google Scholar, 16Campanelli R Rosti V Villani L et al.Evaluation of the bioactive and total transforming growth factor β1 levels in primary myelofibrosis.Cytokine. 2011; 53: 100-106Crossref PubMed Scopus (24) Google Scholar, 17Chagraoui H Komura E Tulliez M Giraudier S Vainchenker W Wendling F. Prominent role of TGF-beta 1 in thrombopoietin-induced myelofibrosis in mice.Blood. 2002; 100: 3495-3503Crossref PubMed Scopus (202) Google Scholar]. The pathobiological role of TGF-β in the development of MF has been further confirmed by the observation that in animal models, the development of MF is prevented by genetic ablation of the TGF-β gene [17Chagraoui H Komura E Tulliez M Giraudier S Vainchenker W Wendling F. Prominent role of TGF-beta 1 in thrombopoietin-induced myelofibrosis in mice.Blood. 2002; 100: 3495-3503Crossref PubMed Scopus (202) Google Scholar,18Gastinne T Vigant F Lavenu-Bombled C et al.Adenoviral-mediated TGF-beta1 inhibition in a mouse model of myelofibrosis inhibit bone marrow fibrosis development.Exp Hematol. 2007; 35: 64-74Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar] and reverted by treating MF mice with small TGF-β receptor-1 kinase inhibitors [19Zingariello M Martelli F Ciaffoni F et al.Characterization of the TGF-beta1 signaling abnormalities in the Gata1low mouse model of myelofibrosis.Blood. 2013; 121: 3345-3363Crossref PubMed Scopus (76) Google Scholar] or with the TGF-β trap AVID200 [20Varricchio L Iancu-Rubin C Upadhyaya B et al.TGF-β1 protein trap AVID200 beneficially affects hematopoiesis and bone marrow fibrosis in myelofibrosis.JCI Insight. 2021; 6e145651Crossref PubMed Scopus (26) Google Scholar]. The TGF-β trap AVID200 is currently in phase I/II clinical trials for patients with MF who are resistant to therapy with the JAK1/2 inhibitor ruxolitinib (Rux) [21Gerds AT Vannucchi AM Passamonti F et al.Duration of response to luspatercept in patients (Pts) requiring red blood cell (RBC) transfusions with myelofibrosis (MF) – updated data from the Phase 2 ACE-536-MF-001 Study.Blood. 2020; 136: 47-48Crossref Google Scholar]. Later studies have indicated that the adhesion receptor P-selectin (P-SEL) may represent an element upstream to TGF-β in the pathobiological pathway leading to MF. In fact, the MK abnormalities observed in this disease include abnormal cytoplasmic trafficking of P-SEL, which was displayed on the cell surface instead of being partitioned into granules [22Zetterberg E Verrucci M Martelli F et al.Abnormal P-selectin localization during megakaryocyte development determines thrombosis in the gata1low model of myelofibrosis.Platelets. 2014; 25: 539-547Crossref PubMed Scopus (14) Google Scholar]. It has been hypothesized that P-SEL, which is found in high levels on the cell surface, interacting with its ligand P-selectin glycoprotein ligand-1 (PSGL-1) expressed by the neutrophils [23Moore KL Stults NL Diaz S et al.Identification of a specific glycoprotein ligand for P-selectin (CD62) on myeloid cells.J Cell Biol. 1992; 118: 445-456Crossref PubMed Scopus (424) Google Scholar,24Evangelista V Manarini S Sideri R et al.Platelet/polymorphonuclear leukocyte interaction: P-selectin triggers protein-tyrosine phosphorylation-dependent CD11b/CD18 adhesion: role of PSGL-1 as a signaling molecule.Blood. 1999; 93: 876-885Crossref PubMed Google Scholar], drives a process of pathologic emperipolesis between the neutrophils and the MKs, which leads to the death of the MKs by para-apoptosis and release of TGF-β in the microenvironment [25Thiele J Lorenzen J Manich B Kvasnicka HM Zirbes TK Fischer R. Apoptosis (programmed cell death) in idiopathic (primary) osteo-/myelofibrosis: naked nuclei in megakaryopoiesis reveal features of para-apoptosis.Acta Haematol. 1997; 97: 137-143Crossref PubMed Scopus (22) Google Scholar,26Zingariello M Ruggeri A Martelli F et al.A novel interaction between megakaryocytes and activated fibrocytes increases TGF-β bioavailability in the Gata1low mouse model of myelofibrosis.Am J Blood Res. 2015; 5: 34-61PubMed Google Scholar].This hypothesis has been mechanistically demonstrated by the observation that the TGF-β bioavailability in the BM of Gata1low mice lacking the P-sel gene is normal and that these mice do not develop MF with age and live on average 2 months longer than their Gata1low littermates [27Spangrude GJ Lewandowski D Martelli F et al.P-Selectin sustains extramedullary hematopoiesis in the Gata1low model of myelofibrosis.Stem Cells. 2016; 34: 67-82Crossref PubMed Scopus (25) Google Scholar]. These findings support the hypothesis that in MF, the disease is established, and it progresses because of a pathologic P-SEL/TGF-β circuit established by the malignant MKs [28Ceglia I Dueck AC Masiello F et al.Preclinical rationale for TGF-β inhibition as a therapeutic target for the treatment of myelofibrosis.Exp Hematol. 2016; 44: 1138-1155Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar]. Whether inhibition of P-SEL would also be effective in reverting to normal MF once the disease is established has not been demonstrated as yet. Recently, the P-SEL inhibitor crizanlizumab (SEG101) has been demonstrated to reduce the frequency of vaso-occlusive crises in patients with sickle cell disease with limited toxicity [29Ataga KI Kutlar A Kanter J et al.Crizanlizumab for the prevention of pain crises in sickle cell disease.N Engl J Med. 2017; 376: 429-439Crossref PubMed Scopus (430) Google Scholar]. Based on these observations, in November 2019, the Federal Drug Administration approved the use of crizanlizumab for the treatment of pain crises in sickle cell disease. The rationale for the clinical study with crizanlizumab had been provided by a preclinical study that evaluated the effects of the commercially available monoclonal antibody RB40.34 targeting the murine P-SEL as an antithrombotic agent in a mouse model of sickle cell disease [30Embury SH Matsui NM Ramanujam S et al.The contribution of endothelial cell P-selectin to the microvascular flow of mouse sickle erythrocytes in vivo.Blood. 2004; 104: 3378-3385Crossref PubMed Scopus (112) Google Scholar]. Because crizanlizumab had already been approved for clinical use and conditions for effective treatment of mice with RB40.34 had already been described, in this study, we tested the hypothesis that pharmacologic inhibition of P-SEL with RB40.34, alone and in combination with Rux, is effective in reverting the myelofibrotic phenotype expressed by Gata1low mice. Gata1low mice were bred in the animal facility of Istituto Superiore di Sanità as previously described [31Martelli F Ghinassi B Panetta B et al.Variegation of the phenotype induced by the Gata1low mutation in mice of different genetic backgrounds.Blood. 2005; 106: 4102-4113Crossref PubMed Scopus (30) Google Scholar]. Littermates were genotyped at birth by polymerase chain reaction, and those who were found not to carry the mutation were used as wild type (WT) controls. All the experiments, including the size of the experimental groups, were performed according to the protocols (D9997.121) approved by the Italian Ministry of Health on September 2, 2021, and according to the European Directive 86/609/EEC. A total of 47 Gata1low mice were implanted with 14-mm microchips (one chip per mouse) (AVID) and divided into two separate experimental groups (Supplementary Figure E1). In the first experimental group, 24 11-month-old mice were randomly divided into four groups (three males and three females each), which were treated as follows: Group 1: vehicle (2% v/v Dimethyl sulfoxide by gavage, negative control for groups 3 and 4); Group 2: biotin-conjugated rat anti-mouse CD62P (RB40.34, catalog number 553743, BD Pharmigen; 30 μg/mouse/day × 3 days/week by IV, as described in [30Embury SH Matsui NM Ramanujam S et al.The contribution of endothelial cell P-selectin to the microvascular flow of mouse sickle erythrocytes in vivo.Blood. 2004; 104: 3378-3385Crossref PubMed Scopus (112) Google Scholar], until day 45, and then IP); Group 3: Rux (Novartis Pharma AG; 45 mg/kg twice/day × 5 days a week by gavage as described in [32Zingariello M Sancillo L Martelli F et al.The thrombopoietin/MPL axis is activated in the Gata1low mouse model of myelofibrosis and is associated with a defective RPS14 signature.Blood Cancer J. 2017; 7: 1-11Crossref Scopus (22) Google Scholar]); and Group 4: biotin-labeled RB40.34 and Rux in combination. On Day 5, all the mice were weighed and bled for blood cell count determination and detection of RB34.40 on platelets. The mice were sacrificed on day 5 (males) and day 12 (females), and BM and spleen were collected for cell signaling and histopathologic determinations. In the second experimental group, 23 8-month-old Gata1low mice were divided into the same groups described above and treated for 7 weeks. In this experimental group, we used the purified RB40.34 (catalog number 553742, BD Pharmigen). The treatment was interrupted for 2 weeks during the holiday break (from day 24 to day 43). On day 54, all the mice were weighed, bled for blood count determination, and sacrificed for histopathology observations of their BM and spleen. Mice were topically anesthetized with Novesina (catalog number s01ha02, Novartis; one drop/eye), and blood was collected from the retro-orbital plexus into heparinized microcapillary tubes. Blood counts were evaluated on deidentified samples by an accredited commercial laboratory, which provides diagnostic services for laboratory animals (Plaisant Laboratory). Platelet-enriched plasma was prepared by centrifugation of 200 µL heparinized blood (10,000 rpm for 20 min with the Eppendorf Centrifuge 5425/5425 R, Eppendorf), and the binding of biotinylated RB40.34 to platelets was measured by flow cytometry following incubation with PE–Cy7–streptavidin (catalog number 557598, BD Pharmingen). Platelets were identified based on size (forward scatter) and internal cell complexity (side scatter), as described previously [22Zetterberg E Verrucci M Martelli F et al.Abnormal P-selectin localization during megakaryocyte development determines thrombosis in the gata1low model of myelofibrosis.Platelets. 2014; 25: 539-547Crossref PubMed Scopus (14) Google Scholar]. BM and spleen cells were resuspended in Ca++ Mg++-free PBS containing 0.5% (v/v) fetal bovine serum (catalog number F7524, Sigma-Aldrich) and incubated with PE–CD41, FITC–CD61, and PE–Cy7–streptavidin. Cells were then divided by flow cytometry into four populations, corresponding to non-MK (CD41neg/CD61neg), immature MK (CD41neg/CD61high), mature MK (CD41high/CD61high), and very mature MK (CD41high/CD61neg), as described previously [19Zingariello M Martelli F Ciaffoni F et al.Characterization of the TGF-beta1 signaling abnormalities in the Gata1low mouse model of myelofibrosis.Blood. 2013; 121: 3345-3363Crossref PubMed Scopus (76) Google Scholar]. The levels of PE-Cy7–streptavidin staining were assessed as a measure of biotinylated RB40.34 binding to the MK. Mononuclear BM and spleen suspensions were incubated with a cocktail of antibodies, including APC–CD117, APC–Cy7–Sca1, PE–Cy7–CD150, biotin-labeled anti-mouse CD48, and biotin-labeled anti-lineage antibodies. After 30 min of incubation on ice, cells were washed and incubated with streptavidin–PE–Cy5 (all from BD Pharmingen). Hematopoietic progenitor cells were defined as lineage-negative cells (Lin−). HSCs were defined as LSK (Lin−/CD48neg/c-Kitpos/Sca-1pos), whereas long-term repopulating HSCs were defined as SLAM (Lin−/CD48neg/c-Kitpos/Sca-1pos/CD150pos) as described previously [27Spangrude GJ Lewandowski D Martelli F et al.P-Selectin sustains extramedullary hematopoiesis in the Gata1low model of myelofibrosis.Stem Cells. 2016; 34: 67-82Crossref PubMed Scopus (25) Google Scholar,33Oguro H Ding L Morrison SJ. SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors.Cell Stem Cell. 2013; 13: 102-116Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar]. Nonspecific signals and dead cells were excluded, respectively, by appropriate fluorochrome-conjugated isotype and propidium iodide staining. All the flow cytometry analyses were performed using the Gallios analyzer (Beckman Coulter), and the results were analyzed using the Kaluza analysis program, version 2.1 (Beckman Coulter). BM and spleen of Gata1low mice treated for 5 days were dissolved in lysis buffer containing protease and phosphatase inhibitors and stored at −80°C. Protein extracts were separated by electrophoresis under denaturing conditions using 7.5%–10% mini-Protean TGX precasted gels (Bio-Rad) and transferred to nitrocellulose filters with the Transblot-Turbo system (Bio-Rad). Filters were probed with antibodies against proteins of the canonical (SMAD2/3, catalog number 8685, Cell Signaling), p-SMAD2/3 (catalog number 8828, Cell Signaling), TGF-βRII (catalog number ab186838, Abcam), and noncanonical (p38, catalog number 9212; p-p38, catalog number 4511; ERK1/2, catalog number 9102; and p-ERK1/2, catalog number 9101; all from Cell Signaling) TGF-β signaling and JAK2 (catalog number 3230, Cell Signaling), STAT5 (catalog number sc-74442), pJAK2 (Phospho-Tyr1007/1008 JAK2, catalog number 3771, Cell Signaling), and p-STAT5 (catalog number 9351, Cell Signaling) of JAK/STAT signaling. GAPDH (catalog number G9545, Sigma–Aldrich) was used as a loading control. The bands were quantified using ImageJ 1.52q software (National Institutes of Health) and normalized against GAPDH. Stoichiometry determinations of phosphoprotein levels were obtained by normalizing the content of the phosphoprotein with that of the corresponding total protein. Femurs were fixed in formaldehyde (10% v/v with neutral buffer), treated for 1 hour with a decalcifying kit (Osteodec; Bio-Optica), and embedded in paraffin. Spleens were fixed in formaldehyde and then embedded in paraffin [19Zingariello M Martelli F Ciaffoni F et al.Characterization of the TGF-beta1 signaling abnormalities in the Gata1low mouse model of myelofibrosis.Blood. 2013; 121: 3345-3363Crossref PubMed Scopus (76) Google Scholar]. Paraffin-embedded tissues were cut into consecutive 3-μm sections and stained either with hematoxylin–eosin (H&E; catalog number 01HEMH2500 and 01EOY101000, respectively; Histo-Line Laboratories), Gomori silver, or reticulin staining and Mallory trichrome staining (catalog numbers 04-040801, 04-040802, and 04-020802, respectively; Bio-Optica). For immunofluorescence microscopy, BM sections were incubated with anti-CXCL1 (catalog number ab86436, Abcam) and anti-TGF-β1 (catalog number sc-130348, Santa Cruz Biotechnology) antibodies, and reactions were detected by avidin–biotin immune-peroxidase staining and 3,3′-diaminobenzidine (0.05% w/v) (Vectastain Elite ABC Kit, Vector Laboratories). Slides were counterstained with Papanicolaou's hematoxylin (Histo-Line Laboratories). Images were acquired with the optical microscope Eclipse E600 (Nikon) equipped with the Imaging Source “33” Series USB 3.0 Camera (catalog number DFK 33UX264), and the signal was quantified acquiring at least five different areas/femur/mouse from at least four mice per group using ImageJ program (version 1.52t) (National Institutes of Health), as described previously [34Schneider CA Rasband WS Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis.Nat Methods. 2012; 9: 671-675Crossref PubMed Scopus (37614) Google Scholar,35Zingariello M Verachi P Gobbo F et al.Resident self-tissue of proinflammatory cytokines rather than their systemic levels correlates with development of myelofibrosis in Gata1low mice.Biomolecules. 2022; 12: 234-260Crossref PubMed Scopus (5) Google Scholar]. For immunofluorescence microscopy determinations, 3-µm-thick BM sections were dewaxed in xylene, and antigens were retrieved by treatment with EDTA buffer (pH = 8) for 20 min in a pressure cooker (110°C–120°C, high pressure) and incubated with antibodies against CD42b (catalog number ab183345, Abcam), GATA1 (catalog number sc-265, Santa Cruz), CD3 (catalog number ab16669, Abcam), and CD45R/B220 (catalog number 553085, BD-Pharmingen) overnight at 4°C. Primary antibodies were visualized with the secondary antibody goat anti-rat Alexa Fluor 488 (catalog number ab150165, Abcam) or goat anti-rabbit Alexa Fluor 555 (catalog number ab150078, Abcam). All sections were counterstained with 4',6-Diamidino-2-phenylindole (catalog number D9542-5MG, Sigma–Aldrich), mounted with Fluor-shield histology mounting medium (catalog number F6182-10MG, Sigma–Aldrich), and examined using a Nikon Eclipse Ni microscope equipped with filters appropriate for the fluorochrome to be analyzed. Images were recorded with a Nikon DS-Qi1Nc digital camera and NIS 190 Elements software BR 4.20.01 and quantified with ImageJ program by counting the number of cells that exceeded the intensity set as the threshold, as described previously [35Zingariello M Verachi P Gobbo F et al.Resident self-tissue of proinflammatory cytokines rather than their systemic levels correlates with development of myelofibrosis in Gata1low mice.Biomolecules. 2022; 12: 234-260Crossref PubMed Scopus (5) Google Scholar]. Reconstruction of the image of all the femurs was obtained by combining the entire set of stack images (15 images at 20 × or 34 images at 63 ×) with the Zen Blue software (Zeiss). Microvessel density was determined by incubating BM and spleen sections with anti-CD34 (catalog number MAB7100, AbNova, primary), Alexa Fluor 568-conjugated donkey anti-rat (Invitrogen, secondary), and Hoechst 33342 (ThermoFisher Scientific). Data were analyzed and plotted using GraphPad Prism 8.0.2 software (GraphPad Software) and presented as mean (±SD) or as box charts, as more appropriate. All the data had normal distribution, as assessed using the Shapiro–Wilk t test. Values between two groups were compared using t test, whereas those among multiple groups were compared using Tukey's multiple comparisons test or analysis of variance, as indicated. Kaplan–Meier survival curves were compared by log-rank (Mantel–Cox) test. Differences were considered statistically significant at p < 0.05. Because the underlying fibrosis in the BM of Gata1low mice may restrain the RB40.34 antibody to reach the BM, we conducted a feasibility study to determine whether the biotinylated-RB40.34 was detectable in BM sections of mice treated for 5 days. In addition, because we and others have demonstrated that platelets of Gata1low mice express greater levels of P-SEL on their surface [22Zetterberg E Verrucci M Martelli F et al.Abnormal P-selectin localization during megakaryocyte development determines thrombosis in the gata1low model of myelofibrosis.Platelets. 2014; 25: 539-547Crossref PubMed Scopus (14) Google Scholar,36Vyas P Ault K Jackson CW Orkin SH Shivdasani RA. Consequences of GATA-1 deficiency in megakaryocytes and platelets.Blood. 1999; 93: 2867-2875Crossref PubMed Google Scholar], we determined whether biotinylated-RB40.34 was detectable on platelets present in the blood 5 hours after its administration as control of the persistence of the antibody in the circulation after its injection (Figure 1). Biotin is produced in the liver and is present, albeit at low levels, in several cell types [37Fletcher K Myant NB. Biotin in the synthesis of fatty acid and cholesterol by mammalian liver.Nature. 1960; 188: 585Crossref PubMed Scopus (6) Google Scholar]. Therefore, it is not surprising that the APC–Cy7–streptavidin signal was also detected on platelets and BM sections of mice in the vehicle and Rux groups, which did not re

In gene transfer experiments including gene therapy studies, expression of the integrated transgenes in host cells often declines with time. The molecular basis of this phenomenon is not clearly understood. We have used the Green Fluorescent Protein (GFP) gene as both a selectable marker and a reporter to study long-term transgene integration and expression in K562 cells. Cells transfected with plasmids containing the GFP gene coupled to the HS2 or HS3 enhancer of the human beta-globin Locus Control Region (LCR) or the cytomegalovirus (CMV) enhancer were sorted by either fluorescence-activated-cell-sorting (FACS) alone or FACS combined with drug selection based on a co-integrated drug resistance gene. The two groups of selected cells were subsequently cultured for long periods up to 250 cell generations. Comparison of long-term GFP transgene integration and expression in these two groups of cells revealed that the K562 genome contains two types of transgene integration sites: i) abundant unstable sites that permit transcription but not long-term integration of the transgenes and thus eliminate the transgenes in 60-250 cell generations and ii) rare stable sites that permit both efficient transcription and long-term stable integration of the transgenes for at least 200 cell generations. Our results indicate that extinction of GFP expression with time is due at least in part to elimination of the gene from the host genome and not entirely to transcriptional silencing of the gene. However, long-term, stable expression of the transgene can be achieved in cells containing the transgene integrated into the rare, stable host sites.

Abstract Human cord blood was used as a source of progenitor and stem cells to evaluate the effect of recombinant human stem-cell factor (SCF) on colony formation and the generation of colony-forming cells (CFC) under highly defined, serum-deprived conditions. SCF interacted with a number of hematopoietic growth factors to stimulate colony growth and was particularly effective in stimulating the formation of mixed-cell colonies from CD34+ soybean agglutinin negative (SBA-) cells. In suspension culture of CD34+, SBA- cells, SCF alone was unable to maintain cell numbers or CFC but, in combination with interleukin-3 (IL- 3), increased input numbers of cells by 10-fold and increased CFC of all kinds by nearly 20-fold. This included erythroid burst-forming cells (BFU-E), granulocyte/macrophage (GM) CFC, and mixed-cell CFC. In contrast, CD34- SBA- cells neither gave rise to CFC nor were maintained by combinations of growth factors including SCF. SCF interacted with erythropoietin (Epo) and granulocyte colony-stimulating factor (G-CSF) to maintain large numbers of cells as well as to generate a twofold to threefold increase in CFC in the case of Epo, and a 10-fold increase in CFC in the case of G-CSF. With Epo, the predominant CFC generated were BFU-E and erythroid CFC and many of the cells in suspension were erythroblasts. In contrast, SCF plus G-CSF resulted in large numbers of granulocytes at various stages of maturation and the CFC generated were almost exclusively granulocytic-CFC. IL-1 and IL-6, alone or in combination with SCF, showed little or no ability to increase cell numbers or generate CFC. In summary, SCF interacts with a variety of hematopoietic growth factors to promote colony formation, particularly mixed-cell colony formation, and also, in suspension culture, SCF interacts with IL-3, G-CSF, and Epo to generate large numbers of differentiated cells as well as a variety of CFC for up to 1 month.

The aim of this study was to identify whether the rapid membrane-associated pathway of the glucocorticoid receptor (GR) is active in erythroid cells and plays any role in determining the reversible inhibition on erythroid maturation exerted by GR.First we determined the biological effects (inhibition of apoptosis and induction of beta-globin expression) induced in primary erythroblasts by erythropoietin (EPO) and the GR agonist dexamethasone (DXM), alone and in combination. Next, by biochemical analysis, we determined the association between GR and EPO receptor in proerythroblasts generated in vitro from 10 normal adult donors. These studies also analyzed the levels of signal transducers and activators of transcription-5 (STAT-5) phosphorylation induced when the cells were stimulated with DXM alone or in combination with EPO.DXM antagonized the beta-globin messenger RNA increases, but not the inhibition of apoptosis induced by EPO in primary cells. DXM also antagonized the ability of EPO to induce STAT-5 phosphorylation in these cells. In fact, EPO and DXM alone, but not in combination, induced phosphorylation and nuclear translocation of STAT-5. The inhibition likely occurred through an interaction between the two receptors because GR became associated with the EPO receptor and STAT-5 in cells stimulated with EPO and DXM.These data suggest that glucocorticoids inhibit erythroid maturation not only through a transcriptional mechanism, but also through a rapid membrane-associated pathway that interferes with EPO receptor signaling.

To clarify the role of HDACs in erythropoiesis, expression, activity and function of class I (HDAC1, HDAC2, HDAC3) and class IIa (HDAC4, HDAC5) HDACs during in vitro maturation of human erythroblasts were compared. During erythroid maturation, expression of HDAC1, HDAC2 and HDAC3 remained constant and activity and GATA1 association (its partner of the NuRD complex), of HDAC1 increased. By contrast, HDAC4 content drastically decreased and HDAC5 remained constant in content but decreased in activity. In erythroid cells, pull down experiments identified the presence of a novel complex formed by HDAC5, GATA1, EKLF and pERK which was instead undetectable in cells of the megakaryocytic lineage. With erythroid maturation, association among HDAC5, GATA1 and EKLF persisted but levels of pERK sharply decreased. Treatment of erythroleukemic cells with inhibitors of ERK phosphorylation reduced by >90% the total and nuclear content of HDAC5, GATA1 and EKLF, suggesting that ERK phosphorylation is required for the formation of this complex. Based on the function of class IIa HDACs as chaperones of other proteins to the nucleus and the erythroid-specificity of HDAC5 localization, this novel HDAC complex was named nuclear remodeling shuttle erythroid (NuRSERY). Exposure of erythroid cells to the class II-selective HDAC inhibitor (HDACi) APHA9 increased γ/(γ+β) globin expression ratios (Mai et al., 2007), suggesting that NuRSERY may regulate globin gene expression. In agreement with this hypothesis, exposure of erythroid cells to APHA9 greatly reduced the association among HDAC5, GATA1 and EKLF. Since exposure to APHA9 did not affect survival rates or p21 activation, NuRSERY may represent a novel, possibly less toxic, target for epigenetic therapies of hemoglobinopaties and other disorders.

A major role for human (h)CXCL8 (interleukin-8) in the pathobiology of myelofibrosis (MF) has been suggested by observations indicating that MF megakaryocytes express increased levels of hCXCL8 and that plasma levels of this cytokine in MF patients are predictive of poor patient outcomes. Here, we demonstrate that, in addition to high levels of TGF-β, the megakaryocytes from the bone marrow of the Gata1low mouse model of myelofibrosis express high levels of murine (m)CXCL1, the murine equivalent of hCXCL8, and its receptors CXCR1 and CXCR2. Treatment with the CXCR1/R2 inhibitor, Reparixin in aged-matched Gata1low mice demonstrated reductions in bone marrow and splenic fibrosis. Of note, the levels of fibrosis detected using two independent methods (Gomori and reticulin staining) were inversely correlated with plasma levels of Reparixin. Immunostaining of marrow sections indicated that the bone marrow from the Reparixin-treated group expressed lower levels of TGF-β1 than those expressed by the bone marrow from vehicle-treated mice while the levels of mCXCL1, and expression of CXCR1 and CXCR2, were similar to that of vehicle-treated mice. Moreover, immunofluorescence analyses performed on bone marrow sections from Gata1low mice indicated that treatment with Reparixin induced expression of GATA1 while reducing expression of collagen III in megakaryocytes. These data suggest that in Gata1low mice, Reparixin reduces fibrosis by reducing TGF-β1 and collagen III expression while increasing GATA1 in megakaryocytes. Our results provide a preclinical rationale for further evaluation of this drug alone and in combination with current JAK inhibitor therapy for the treatment of patients with myelofibrosis.

Assuming a threshold of 2 × 107 nucleated cells (NC)/kg body weight required for transplantation and 10 ± 5 × 108 NC per cord blood (CB) unit (n = 1828, July 1997), 100%, 65% and 25% of the CB units stored in the CB Bank Düsseldorf contain sufficient NC to engraft patients of 10 kg, 35 kg and 50–70 kg, respectively. Thus, there is a potential limitation for the use of CB in adults which, however, may be overcome by ex vivo expansion of cells important in the different phases of engraftment. Therefore, four combinations of SCF, Flt3-L, IL-3, erythropoietin and GM-CSF as well as three media were evaluated for their capacity to amplify hematopoietic progenitors. A prerequisite for expansion was the significantly higher recovery of CD34+ cells, colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC) by thawing cryopreserved CB units with an isotonic albumin/dextran solution. When CD34+ CB cells were cultured with the four cytokine combinations in H5100 medium, all combinations promoted an expansion of total cells (43 to 356-fold) and CFC (49 to 462-fold) within 7 days, however, early progenitors as defined by mixed-colony formation (CFU-GEMM) were substantially amplified only with SCF, Flt3-L plus IL-3 (94.3 ± 62.4-fold). H5100 medium or a serum-free medium supplemented with SCF, Flt3-L plus IL-3 were superior to 20% FCS/RPMI-1640 medium in the expansion of all progenitor cell types and were similarly effective in supporting the amplification of total cells, CFC, CFU-GM, BFU-E/CFU-E and LTC-IC (maximum at day 7: 6.7 ± 3.4-fold and 5.5 ± 0.5-fold, respectively). However, the serum-free medium promoted a significantly higher expansion of CFU-GEMM (176.9 ± 81.7-fold) than H5100 medium (83.5 ± 26.2-fold) at day 7 and only under serum-free conditions, CFU-GEMM were maintained over 14 days in tissue culture. These results demonstrate that committed progenitors as well as the more immature CFU-GEMM and LTC-IC can be substantially amplified at the same time without exhausting the proliferative potential.

We have identified two new histone deacetylase (HDAC) inhibitors (9 and 24) capable of inducing the expression of gamma-globin and/or beta-globin promoter-driven reporter genes in a synthetic model of Hb switch. Both compounds also increased, with different mechanisms, the gamma/(gamma+beta) ratio expressed in vitro by normal human erythroblasts. Compound 9 increased the levels of gamma-globin mRNA and the gamma/(gamma+beta) ratio (both by 2-fold). Compound 24 increased by 3-fold the level of gamma-globin and decreased by 2-fold that of beta-globin mRNA, increasing the gamma/(gamma+beta) ratio by 6-fold, and raising (by 50%) the cell HbF content. Both compounds raised the acetylation state of histone H4 in primary cells, an indication that their activity was mediated through HDAC inhibition. Compounds 9 and 24 were also tested as gamma/(gamma+beta) mRNA inducers in erythroblasts obtained from patients with beta(0) thalassemia. Progenitor cells from patients with beta(0) thalassemia generated in vitro morphologically normal proerythroblasts that, unlike normal cells, failed to mature in the presence of EPO and expressed low beta-globin levels but 10 times higher-than-normal levels of the alpha hemoglobin-stabilizing protein (AHSP) mRNA. Both compounds ameliorated the impaired in vitro maturation in beta(0) thalassemic erythroblasts, decreasing AHSP expression to normal levels. In the case of two patients (of five analyzed), the improved erythroblast maturation was associated with detectable increases in the gamma/(gamma+beta) mRNA ratio. The low toxicity exerted by compounds 9 and 24 in all of the assays investigated suggests that these new HDAC inhibitors should be considered for personalized therapy of selected patients with beta(0) thalassemia.

Abstract Histology refers to the study of the morphology of cells within their natural tissue environment. As a bio‐medical discipline, it dates back to the development of first microscopes which allowed to override the physical visual limitation of the human eye. Since the first observations, it was understood that cell shape predicts function and, therefore, shape alterations can identify and explain dysfunction and diseases. The advancements in morphological investigation techniques have allowed to extend our understanding of the shape–function relationships close to the molecular level of organization of tissues, as well as to derive reliable data not only from fixed, and hence static, biological samples but also living cells and tissues and even for extended time periods. These modern approaches, which encompass quantitative microscopy, precision microscopy, and dynamic microscopy, represent the new frontier of morphology. This article summarizes how the microscopy techniques have evolved to properly face the challenges of biomedical sciences, thus transforming histology from a merely qualitative discipline, which played an ancillary role to traditional “major” sciences such as anatomy, to a modern experimental science capable of driving knowledge progress in biology and medicine.

Abstract TGFβ plays a pivotal role in the pathobiology of myelofibrosis (MF) by not only promoting bone marrow fibrosis (BMF) but also by enhancing the dormancy of normal but not MF hematopoietic stem cells (HSCs). TGFβ has also previously been reported to inhibit normal megakaryocyte (MK) production (Bruno et al Blood 1998). TGFβ1 promotes the synthesis of collagen by normal human mesenchymal stromal cells (MSCs). Treatment of MSCs with AVID200, a potent TGFβ1/3 protein trap, significantly decreased MSC proliferation, phosphorylation of SMAD2, and collagen expression. Robust expression of pSMAD2 was observed in the absence of exogenous TGFβ in normal donor or MF-MKs, Addition of AVID200 to MKs decreased pSMAD2 without affecting total SMAD2/3 and led to increased numbers of MKs. Treatment of MF MNCs with AVID200 also led to increased numbers of progenitor cells with wild type JAK2 and a reduction of mutated colonies. A phase 1b trial of AVID200 (NCT03895112) was performed and completed in INT-2/high risk MF patients resistant/intolerant to ruxolitinib (rux); baseline platelet count of ≥ 25 x 10 9/L, and grade 2/3 BMF. Subjects received AVID200 intravenously on Day 1 of a 21 day cycle. Response was assessed by IWG/ELN criteria after 6 cycles of AVID200. Subjects attaining at least a CI or SD with a decrease in BMF by ≥1 grade, continued AVID200. We previously presented the results of the dose escalation study (Mascarenhas ASH 2020) demonstrating that AVID200 was well tolerated without dose limiting toxicities at 3 tested dose levels (Lots A and B) in dose cohorts of 180 mg (A), 550 mg (A)/70 mg (B), and 180 mg (B). Here we report updated safety and efficacy results of the phase 1b dose expansion stage at the two highest doses tested (70 mg (B) and 180 mg (B). Twenty-two subjects were enrolled (1 withdrew before receiving treatment) and 9 were treated with AVID200 in the dose escalation phase and 12 in the dose expansion phase [Table1]. Median time after rux discontinuation was 7.4 months (0.5-59.9). The most common mutations observed at baseline in this cohort included JAK2V617F (71%), TET2 (29%) ASXL1 (24%) and CALR (19%). (Fig 1) No DLTs were observed and Grade 3/4 AEs were observed in 16 (76.2%) subjects. Grade 3/4 non-hematologic AEs were observed in 8 (38.1%) subjects and included one subject in each case (epistaxis, mucositis, extraocular muscle paresis, fatigue, rash, duodenal hemorrhage, gastric hemorrhage, urinary tract infection, and syncope). Grade 3/4 hematologic AEs were anemia (6; 28.3%) and thrombocytopenia (2; 14.3%) [Table 2]. No fatal events were observed. The median number of cycles received was 5 (range 2 - 13) and 7 (33%) patients received more than 6 cycles. For dose levels 2-3 at cycle 7, a CI was attained in one subject at dose level 2 [anemia, spleen and TSS], 5 subjects had SD, 3 subjects had PD and two subjects with 10% and 15% blasts at screening developed MPN-BP while on study based on central review. Reasons for discontinuation by local PI included PD (n=8), lack of response (n=5), study completed (n=2), other (n=2), patient decision (n=1). Median % change in palpable spleen length was +10% (range -70% to +150%) and TSS change was -50% (-100% to +185.7%) The median platelet count at baseline was 114 x 10 9/L (range: 28-695) and 215 x 10 9/L (range: 66-263) after cycle 6 in 7 evaluable subjects (Fig 2A). Notably, 17 subjects had an increase in platelets from baseline during treatment and two subjects normalized their platelet counts. Maximum changes in platelets from baseline across all cycles was +63.8% [range -15.7%, +505.5%] (Fig 2B). Paired bone marrow biopsy pathology samples for 12 subjects were available for central review and showed no significant changes in BMF score or MK histo topography at end of treatment compared to baseline. All patients had elevated plasma levels of TGF β1, but not TGFβ2/β3 levels as detected by ELISA, which were dramatically reduced 21 days after the last dose of AVID200. AVID200 a TGFβ1/3 protein trap is well tolerated and clinical responses at cycle 7 of therapy in this advanced MF patient population were limited as judged by IWG/MRT response criteria. However, AVID200 therapy resulted in significant reduction in serum TGFβ levels and improvements in platelet counts indicating that TGF β1 plays a pivotal role in MF leading to thrombocytopenia which can be reversed with AVID200 therapy. We conclude that AVID200 may best be employed in combination therapy approaches in thrombocytopenic MF patients. Figure 1 Figure 1. Disclosures Mascarenhas: Constellation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Promedior: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Geron: Consultancy, Research Funding; Forbius: Research Funding; Genentech/Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene/BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Galecto: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Prelude: Consultancy; Kartos: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CTI Biopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Geron: Consultancy; Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merus: Research Funding. Palmer: PharmaEssentia: Research Funding; Sierra Oncology: Consultancy, Research Funding; Incyte: Research Funding; CTI BioPharma: Consultancy, Research Funding; Protagonist: Consultancy, Research Funding. Kuykendall: Celgene/BMS: Honoraria; Pharmaessentia: Honoraria; Novartis: Honoraria, Speakers Bureau; Protagonist: Consultancy, Research Funding; Incyte: Consultancy; Abbvie: Honoraria; Blueprint: Honoraria. Mesa: Genentech: Research Funding; Promedior: Research Funding; Samus: Research Funding; Gilead: Research Funding; CTI: Research Funding; Abbvie: Research Funding; Sierra Oncology: Consultancy, Research Funding; Celgene: Research Funding; Novartis: Consultancy; Pharma: Consultancy; CTI: Research Funding; Constellation Pharmaceuticals: Consultancy, Research Funding; AOP: Consultancy; La Jolla Pharma: Consultancy; Incyte Corporation: Consultancy, Research Funding. Rampal: Stemline: Consultancy, Research Funding; Memorial Sloan Kettering: Current Employment; BMS/Celgene: Consultancy; Abbvie: Consultancy; CTI: Consultancy; Novartis: Consultancy; Disc Medicine: Consultancy; Blueprint: Consultancy; Pharmaessentia: Consultancy; Incyte: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; Constellation: Research Funding; Kartos: Consultancy; Sierra Oncology: Consultancy. Gerds: PharmaEssentia Corporation: Consultancy; Sierra Oncology: Consultancy; CTI BioPharma: Research Funding; Constellation: Consultancy; Celgene/Bristol Myers Squibb: Consultancy; AbbVie: Consultancy; Novartis: Consultancy. Yacoub: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CTI Biopharma: Membership on an entity's Board of Directors or advisory committees; ACCELERON PHARMA: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Dynavex: Current equity holder in publicly-traded company; Cara: Current equity holder in publicly-traded company; Ardelyx: Current equity holder in publicly-traded company; Seattle Genetics: Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria, Speakers Bureau; Hylapharm: Current equity holder in publicly-traded company. Talpaz: Imago: Consultancy; Constellation: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Other: Grant/research support ; Celgene: Consultancy. Komrokji: Acceleron: Consultancy; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Geron: Consultancy; Jazz: Consultancy, Speakers Bureau. Kremyanskaya: Astellas: Research Funding; Astex: Research Funding; Chimerix: Research Funding; Bristol Myers Squibb: Research Funding; Constellation: Research Funding; Protagonist Therapeutics: Consultancy, Research Funding; Incyte: Research Funding. Salama: Mayo Clinic: Current Employment, Other: Mayo Clinic had the contractual work for the central pathology review for this study and I was one of the reviewing pathologists; Constellation Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Hoffman: Kartos Therapeutics, Inc.: Research Funding; Protagonist Therapeutics, Inc.: Consultancy; Novartis: Other: Data Safety Monitoring Board, Research Funding; AbbVie Inc.: Other: Data Safety Monitoring Board, Research Funding. OffLabel Disclosure: AVID200 is a TGFb trap and is in clinical testing for fibrotic diseases. It does not have an approved indication at this time.

Real-time in vivo imaging provides an essential window into the spatiotemporal cellular events contributing to tissue development and pathology. By coupling longitudinal intravital imaging with genetic lineage tracing, here we capture the earliest cellular events arising in response to active Wnt/β-catenin signaling and the ensuing impact on the organization and differentiation of the mammary epithelium. This enables us to interrogate how Wnt/β-catenin regulates the dynamics of distinct subpopulations of mammary epithelial cells in vivo and in real time. We show that β-catenin stabilization, when targeted to either the mammary luminal or basal epithelial lineage, leads to cellular rearrangements that precipitate the formation of hyperplastic lesions that undergo squamous transdifferentiation. These results enhance our understanding of the earliest stages of hyperplastic lesion formation in vivo and reveal that, in mammary neoplastic development, β-catenin activation dictates a hair follicle/epidermal differentiation program independently of the targeted cell of origin.

It is commonly accepted that some form of skin barrier dysfunction is present in canine atopic dermatitis (AD), one of the most common cutaneous pruritic inflammatory diseases of dogs. The impaired skin barrier function facilitates the penetration of allergens and subsequently stronger sensitization responses. The role of the endocannabinoid system (ECS) in the physiology and pathology of the skin is becoming increasingly established. It has been demonstrated that cannabinoid receptors are expressed in healthy and diseased skin and, based on current knowledge, it could be stated that cannabinoids are important mediators in the skin. The present study has been designed to immunohistochemically investigate the expression of the cannabinoid receptors type 1 (CB1R) and 2 (CB2R) and the cannabinoid-related receptors G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), peroxisome proliferator-activated receptors alpha (PPARα), and serotoninergic receptor 1a (5-HT1aR) in keratinocytes of healthy dogs and of dogs with AD. Samples of skin tissues were collected from 7 healthy controls (CTRL-dogs) and from 8 dogs with AD (AD-dogs). The tissue samples were processed using an immunofluorescence assay with commercially available antibodies, and the immunolabelling of the receptors studied was quantitatively evaluated. The keratinocytes of the CTRL- and the AD-dogs showed immunoreactivity for all the receptors investigated with a significant upregulation of CB2R, TRPA1, and 5-HT1aR in the epidermis of the AD-dogs. The presence of cannabinoid and cannabinoid-related receptors in healthy keratinocytes suggested the possible role of the ECS in canine epidermal homeostasis while their overexpression in the inflamed tissues of the AD-dogs suggested the involvement of the ECS in the pathogenesis of this disease, having a possible role in the related skin inflammation and itching. Based on the present findings, the ECS could be considered a potential therapeutic target for dogs with AD.

Rubinstein, Pablo; Carrier, Carmelita; Scaradavou, Andromachi; Kurtzberg, Joanne; Adamson, John; Migliaccio, Anna Rita; Berkowitz, Richard L.; Cabbad, Michael; Dobrila, N. Ludy; Taylor, Patricia E.; Rosenfield, Richard E.; Stevens, Cladd E. Author Information

Serum levels of inflammatory cytokines are currently investigated as prognosis markers in myelofibrosis, the most severe Philadelphia-negative myeloproliferative neoplasm. We tested this hypothesis in the Gata1low model of myelofibrosis. Gata1low mice, and age-matched wild-type littermates, were analyzed before and after disease onset. We assessed cytokine serum levels by Luminex-bead-assay and ELISA, frequency and cytokine content of stromal cells by flow cytometry, and immunohistochemistry and bone marrow (BM) localization of GFP-tagged hematopoietic stem cells (HSC) by confocal microscopy. Differences in serum levels of 32 inflammatory-cytokines between prefibrotic and fibrotic Gata1low mice and their wild-type littermates were modest. However, BM from fibrotic Gata1low mice contained higher levels of lipocalin-2, CXCL1, and TGF-β1 than wild-type BM. Although frequencies of endothelial cells, mesenchymal cells, osteoblasts, and megakaryocytes were higher than normal in Gata1low BM, the cells which expressed these cytokines the most were malignant megakaryocytes. This increased bioavailability of proinflammatory cytokines was associated with altered HSC localization: Gata1low HSC were localized in the femur diaphysis in areas surrounded by microvessels, neo-bones, and megakaryocytes, while wild-type HSC were localized in the femur epiphysis around adipocytes. In conclusion, bioavailability of inflammatory cytokines in BM, rather than blood levels, possibly by reshaping the HSC niche, correlates with myelofibrosis in Gata1low mice.

We have measured the number of progenitor cells circulating in fetal (17-32 weeks of gestation), perinatal (36 weeks of gestation), and adult (30-50 years old) blood. The progenitor cells at each ontogenetic stage were also characterized in terms both of the minimal combinations of growth factors they required to form maximal numbers of colonies in vitro and of their self-replication potential, as measured by the number of secondary and tertiary progenitor cells each could generate. The number of progenitor cells circulating in fetal and perinatal blood can be measured by directly plating the unfractionated blood. In this assay, fetal blood contains half the number of progenitor cells detected in perinatal blood (18.0 +/- 16.4 versus 40.88 +/- 0.63, p < 0.01), and the number of progenitor cells in adult blood is below the level of detection of the assay (< 1/8 microliter of blood). To compare the number of progenitor cells in all three stages of human development, progenitor cell counts were performed on blood mononuclear cells enriched by density separation. In this case, the light density cell fractions from fetal and neonatal blood contained the same number of progenitor cells (300/10(5) cells), numbers that were 10-fold higher than those observed with adult blood (30/10(5) cells). Circulating fetal-neonatal erythroid and multipotential progenitor cells were found to differ from their adult counterparts in terms of their response to growth factors and their self-renewal ability. In fact, the number of cytokines required to observe maximal colony formation increased with the ontogenetic stage of the cells. No differences were found in the frequency of primary colonies containing progenitor cells or in the mean number of secondary progenitor cells per primary colony in cultures of fetal, neonatal, or adult blood. Differences between the three ontogenetic stages, however, were found with respect to the number of sequential replatings that were possible. In fact, although both secondary granulocyte-macrophage (GM) and mixed-cell colonies derived from fetal cells gave rise to tertiary colonies, only perinatal secondary mixed-cell colonies grew in tertiary cultures, and no growth was observed in tertiary cultures of adult cells. These results suggest that the greater amplification of progenitor cells observed in liquid culture of fetal/neonatal versus adult blood is due both to a higher proliferative capacity of neonatal progenitor cells (up to two replatings versus one) and to a higher frequency in these samples of mixed-cell colony-forming cells (CFC) (37.7 +/- 7.3 versus 2.0 +/- 0.7/10(5) light density cells, respectively). Because of the high numbers of progenitor cells circulating in the fetus, as well as their high proliferative capacity, it is predicted that if blood could be harvested directly in utero, fetal blood would be as good a source of stem cells for transplantation as perinatal placental/cord blood. Circulating fetal stem cells would, therefore, represent an ideal target for gene therapy and in utero autologous transplantation.

Two murine bipotent erythroid/megakaryocytic cells, the progenitor (MEP) and precursor (PEM) cells, recently have been identified on the basis of the phenotypes of linnegc-kitposSca-1neg CD16/CD32lowCD34low and TER119pos4A5pos or 2D5pos, respectively. However, the functional relationship between these two subpopulations and their placement in the hemopoietic hierarchy is incompletely understood. We compared the biological properties of these subpopulations in marrow and spleen of mice with and without acute or chronic erythroid stress. MEP cells, but not PEM cells, express c-kit, respond to stem cell factor in vitro, and form spleen colonies in vivo. PEM cells comprise up to 50%-70% of the cells in BFU-E-derived colonies but are not present among the progeny of purified MEP cells cultured under erythroid and megakaryocytic permissive conditions. PEM cells increase 10- to 20-fold under acute and chronic stress, whereas MEP cell increases (21%-84%) are observed only in acutely stressed animals. These data suggest that MEP and PEM cells represent distinct cell populations that may exist in an upstream-downstream differentiation relationship under conditions of stress. Whereas the dynamics of both populations are altered by stress induction, the differential response to acute and chronic stress suggests different regulatory mechanisms. A model describing the relationship between MEP, PEM, and common myeloid progenitor cells is presented.

In Human Erythroid Massive Amplification (HEMA) cultures, AB mononuclear cells (MNC) generate 1-log more erythroid cells (EBs) than the corresponding <svg style="vertical-align:-0.17555pt;width:53.275002px;" id="M1" height="11.8375" version="1.1" viewBox="0 0 53.275002 11.8375" width="53.275002" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,11.8375)"> <g transform="translate(72,-62.53)"> <text transform="matrix(1,0,0,-1,-71.95,62.75)"> <tspan style="font-size: 12.50px; " x="0" y="0">C</tspan> <tspan style="font-size: 12.50px; " x="8.3395014" y="0">D</tspan> <tspan style="font-size: 12.50px; " x="17.366667" y="0">3</tspan> <tspan style="font-size: 12.50px; " x="23.618168" y="0">4</tspan> </text> <text transform="matrix(1,0,0,-1,-42.08,67.92)"> <tspan style="font-size: 8.75px; " x="0" y="0">p</tspan> <tspan style="font-size: 8.75px; " x="4.3759999" y="0">o</tspan> <tspan style="font-size: 8.75px; " x="8.7519999" y="0">s</tspan> </text> </g> </g> </svg> cells, suggesting that MNC may also contain <svg style="vertical-align:-0.17555pt;width:53.887501px;" id="M2" height="11.8375" version="1.1" viewBox="0 0 53.887501 11.8375" width="53.887501" xmlns="http://www.w3.org/2000/svg"> <g transform="matrix(1.25,0,0,-1.25,0,11.8375)"> <g transform="translate(72,-62.53)"> <text transform="matrix(1,0,0,-1,-71.95,62.75)"> <tspan style="font-size: 12.50px; " x="0" y="0">C</tspan> <tspan style="font-size: 12.50px; " x="8.3395014" y="0">D</tspan> <tspan style="font-size: 12.50px; " x="17.366667" y="0">3</tspan> <tspan style="font-size: 12.50px; " x="23.618168" y="0">4</tspan> </text> <text transform="matrix(1,0,0,-1,-42.08,67.92)"> <tspan style="font-size: 8.75px; " x="0" y="0">n</tspan> <tspan style="font-size: 8.75px; " x="4.3759999" y="0">e</tspan> <tspan style="font-size: 8.75px; " x="8.2618876" y="0">g</tspan> </text> </g> </g> </svg> HPC. To clarify the phenotype of AB HPC which generate EBs in these cultures, flow cytometric profiling for CD34/CD36 expression, followed by isolation and functional characterization (colony-forming-ability in semisolid-media and fold-increase in HEMA) were performed. 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To the editor: The human glucocorticoid receptor (GR) is encoded by GR/NR3C1 located in the 5q31-32 cytoband of chromosome 5, which is deleted in patients with 5q− myelodysplastic syndrome.[1][1] The gene is highly polymorphic, containing single nucleotide polymorphisms (SNPs) both in the coding

Although human cell cultures stimulated with dexamethasone suggest that the glucocorticoid receptor (GR) activates stress erythropoiesis, the effects of GR activation on erythropoiesis in vivo remain poorly understood. We characterized the phenotype of a large cohort of patients with Cushing disease, a rare condition associated with elevated cortisol levels. Results from hypercortisolemic patients with active Cushing disease were compared with those obtained from eucortisolemic patients after remission and from volunteers without the disease. Patients with active Cushing disease exhibited erythrocytosis associated with normal hemoglobin F levels. In addition, their blood contained elevated numbers of GR-induced CD163+ monocytes and a unique class of CD34+ cells expressing CD110, CD36, CD133 and the GR-target gene CXCR4. When cultured, these CD34+ cells generated similarly large numbers of immature erythroid cells in the presence and absence of dexamethasone, with raised expression of the GR-target gene GILZ. Of interest, blood from patients with Cushing disease in remission maintained high numbers of CD163+ monocytes and, although their CD34+ cells had a normal phenotype, these cells were unresponsive to added dexamethasone. Collectively, these results indicate that chronic exposure to excess glucocorticoids in vivo leads to erythrocytosis by generating erythroid progenitor cells with a constitutively active GR. Although remission rescues the erythrocytosis and the phenotype of the circulating CD34+ cells, a memory of other prior changes is maintained in remission.

Atopic dermatitis (AD) is one of the most common cutaneous inflammatory and pruritic diseases in dogs. Considering its multifactorial nature, AD can be a challenging disease to manage, and the therapeutic strategy must often be multimodal. In recent years, research has been moving toward the use of natural products which have beneficial effects on inflammation and itching, and no side effects. Cannabinoid receptors have been demonstrated to be expressed in healthy and diseased skin; therefore, one of the potential alternative therapeutic targets for investigating AD is the endocannabinoid system (ECS).To immunohistochemically investigate the expression of the cannabinoid receptor type 2 (CB2R), and the cannabinoid-related receptors G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) in mast cells (MCs), macrophages, dendritic cells (DCs), T cells, and neutrophils of the skin of dogs with AD.Samples of skin tissues were collected from eight dogs with AD (AD-dogs).The immunofluorescent stained cryosections of the skins of 8 dogs with AD having antibodies against CB2R, GPR55, TRPV1, TRPA1 were semiquantitatively evaluated. The inflammatory cells were identified using antibodies against tryptase (mast cells), ionized calcium binding adaptor molecule 1 (IBA1) (macrophages/DCs), CD3 (T cells), and calprotectin (neutrophils). The proportions of MCs, macrophages/DCs, T cells, and neutrophils expressing CB2R, GPR55, TRPV1 and TRPA1 were evaluated.The cells of the inflammatory infiltrate showed immunoreactivity (IR) for all or for some of the cannabinoid and cannabinoid-related receptors studied. In particular, MCs and macrophages/DCs showed CB2R-, GPR55-, TRPA1-, and TRPV1-IR; T cells showed CB2R-, GPR55- and TRPA1-IR, and neutrophils expressed GPR55-IR. Co-localization studies indicated that CB2R-IR was co-expressed with TRPV1-, TRPA1-, and GPR55-IR in different cellular elements of the dermis of the AD-dogs.Cannabinoid receptor 2, and cannabinoid-related receptors GPR55, TRPV1 and TRPA1 were widely expressed in the inflammatory infiltrate of the AD-dogs. Based on the present findings, the ECS could be considered to be a potential therapeutic target for dogs with AD, and may mitigate itch and inflammation.

Background Cultured red blood cells ( cRBC s) from cord blood ( CB ) have been proposed as transfusion products. Whether buffy coats discarded from blood donations (adult blood [ AB ]) may be used to generate cRBC s for transfusion has not been investigated. Study Design and Methods Erythroid progenitor cell content and numbers and blood group antigen profiles of erythroblasts ( ERYs ) and cRBC s generated in human erythroid massive amplification ( HEMA ) culture by CB (n = 7) and AB (n = 33, three females, three males, one AB with rare blood antigens cryopreserved using CB protocols) were compared. Results Variability was observed both in progenitor cell content (twofold) and number of ERYs generated (1 log) by CB and AB in HEMA . The average progenitor cell contents of the subset of AB and CB analyzed were similar. AB generated numbers of ERY s three times lower (p &lt; 0.01) than CB in HEMA containing fetal bovine serum but similar to CB in HEMA containing human proteins. Female AB contained two times fewer (p &lt; 0.05) erythroid progenitor cells but generated numbers of ERY s similar to those generated by male AB . Cryopreserved AB with a rare blood group phenotype and shipped to another laboratory generated great numbers of ERY s, 90% of which matured into cRBC s. Blood group antigen expression was consistent with the donor genotype for ERY s generated both by CB and AB but concordant with that of native RBC s only for cells derived from AB . Conclusion Buffy coats from regular donors, including a donor with rare phenotypes stored under conditions established for CB , are not inferior to CB for the generation of cRBC s.

Equine Asthma Syndrome (EAS) is a common respiratory problem that affects horses of any age. In the severe EAS form (historically referred to as recurrent airway obstruction or RAO) the diagnosis, based mainly on history and clinical signs, it is definitively confirmed by the cytological examination of the bronchoalveolar lavage (BAL), also very useful in monitoring lower airway inflammation in response to environmental management and medication. Cytocentrifugated preparation is usually the staging method for the BAL cytological interpretation. To evaluate whether the BAL cytology in horses with severe EAS under different environmental conditions and before and after treatment can undergo significant changes, and at the same time to investigate whether in the cytological evaluation of the BAL there are interpretative differences between two methods of smear preparation of the collected fluid, a study was carried out on a series of BAL samples (n=48) collected in 8 withdrawals from 6 EAS-affected horses subjected to process exacerbation through environmental stimuli and then to pharmacological treatment. Each BAL fluid collected was equally divided into duplicate portions: one set up by cytocentrifugation and one by sediment from simple centrifugation. The cytological examination revealed any significant difference between the EAS-affected horses in all experimental phases (asymptomatic, early exacerbation phase, late exacerbation phase, and remission phase). Diagnostic interpretive comparison between the two BAL preparations then showed no significant differences in results, suggesting how they can be used indifferently in the evaluation of BAL under conditions of airway inflammation in the horse. Thus, the concentrated smear preparation appears to be an equally diagnostically useful method in conditions where there is no possibility of using an appropriate cytocentrifuge.

Immuno-oncology research has brought to light the paradoxical role of immune cells in the induction and elimination of cancer. Programmed cell death protein 1 (PD1), expressed by tumor-infiltrating lymphocytes, and programmed cell death ligand 1 (PDL1), expressed by tumor cells, are immune checkpoint proteins that regulate the antitumor adaptive immune response. This study aimed to validate commercially available PDL1 antibodies in canine tissue and then, applying standardized methods and scoring systems used in human pathology, evaluate PDL1 immunopositivity in different types of canine tumors. To demonstrate cross-reactivity, a monoclonal antibody (22C3) and polyclonal antibody (cod. A1645) were tested by western blot. Cross-reactivity in canine tissue cell extracts was observed for both antibodies; however, the polyclonal antibody (cod. A1645) demonstrated higher signal specificity. Canine tumor histotypes were selected based on the human counterparts known to express PDL1. Immunohistochemistry was performed on 168 tumors with the polyclonal anti-PDL1 antibody. Only membranous labeling was considered positive. PDL1 labeling was detected both in neoplastic and infiltrating immune cells. The following tumors were immunopositive: melanomas (17 of 17; 100%), renal cell carcinomas (4 of 17; 24%), squamous cell carcinomas (3 of 17; 18%), lymphomas (2 of 14; 14%), urothelial carcinomas (2 of 18; 11%), pulmonary carcinomas (2 of 20; 10%), and mammary carcinomas (1 of 31; 3%). Gastric (0 of 10; 0%) and intestinal carcinomas (0 of 24; 0%) were negative. The findings of this study suggest that PDL1 is expressed in some canine tumors, with high prevalence in melanomas.

BACKGROUND: Ex vivo generated erythroblasts are being evaluated for transfusion. Expression of balanced levels of globin mRNA is essential for normal red blood cell function and survival but it is unknown whether the expression of the globin genes in ex vivo expanded cells is balanced. STUDY DESIGN AND METHODS: Immature erythroblasts (IEs) were expanded in human erythroid massive amplification cultures from blood mononuclear cells of 19 normal donors and four β 0 ‐thalassemia patients (for comparison) and induced to mature for 4 days in the presence of erythropoietin. mRNA was prepared from IEs and mature erythroblasts to evaluate the expression of α‐, β‐, and γ‐globin genes and of adult hemoglobin‐stabilizing protein (AHSP) and BCL11A, two proteins directly controlling globin function and/or production. Results were analyzed using Pearson's correlation coefficient, the Wilcoxon signed rank, and the Mann‐Whitney rank sum tests. RESULTS: The absolute levels of globin, AHSP, and BCL11A mRNA expressed by erythroblasts generated ex vivo from normal donors were distributed along a 2‐log range. With maturation, the levels of γ‐globin and BCL11A mRNA did not decrease while those of α‐globin, γ + β‐globins, and AHSP mRNA greatly increased. In normal cells, the modest imbalance (two‐ to fourfold) observed between α‐ and γ + β‐globin mRNA was fully compensated by AHSP expression. Thus, the levels of α‐globin mRNA were correlated with those of γ + β‐globin (R 2 = 0.93, p &lt; 0.0001) and AHSP (R 2 = 0.86, p &lt; 0.0001). CONCLUSIONS: Ex vivo expanded erythroblasts from normal donors express modestly imbalanced levels of α‐globin and γ + β‐globin fully compensated by AHSP expression, likely ensuring normal function and survival.

To identify the regulatory sequences driving Gata1 expression in conventional dendritic cells (cDC).The number and expression levels of Gata1, Gata1-target genes and hypersensitive site (HS) 2 (the eosinophil-specific enhancer)-driven green fluorescent protein (GFP) reporter of cDCs from mice lacking HS1 (the erythroid/megakaryocytic-specific enhancer, Gata1(low) mutation) and wild-type littermates, as well as the response to lipopolysaccharide of ex vivo-generated wild-type and Gata1(low) DCs were investigated.cDC maturation was associated with bell-shaped changes in Gata1 expression that peaked in cDCs precursors from blood. The Gata1(low) mutation did not affect Gata1 expression in cDC precursors and these cells expressed the HS2-driven reporter, indicating that Gata1 expression is HS2-driven in these cells. By contrast, the Gata1(low) mutation reduced Gata1 expression in mature cDCs and these cells did not express GFP, indicating that mature cDCs express Gata1 driven by HS1. In blood, the number of cDC precursors expressing CD40/CD80 was reduced in Gata1(low) mice, while CD40(pos)/CD80(pos) cDC precursors from wild-type mice expressed the HS2-GFP reporter, suggesting that Gata1 expression in these cells is both HS1- and HS2-driven. In addition, the antigen and accessory molecules presentation process induced by lipopolysaccharide in ex vivo-generated wild-type DC was associated with increased acetylated histone 4 occupancy of HS1, while ex vivo-generated Gata1(low) cDCs failed to respond to lipopolysaccharide, suggesting that HS1 activation is required for cDC maturation.These results identify a dynamic pattern of Gata1 regulation that switches from an HS1 to an HS2-dependent phase during the maturation of cDCs associated with the antigen-presentation process in the blood.

Dry eye disease (DED) is a multifactorial disease where ocular surface inflammation and damage play key etiological roles.To compare a combination of 3% trehalose (T) and 0.15% hyaluronic acid (HA) (Thealoz duo®, T/HA) with a tear substitute containing 0.001% hydrocortisone (I) and 0.2% HA (Idroflog®, I/HA), with respect to changes on signs and inflammatory markers in a mouse DED model.Thirty 12-week-old C57BL/6 mice were exposed in a controlled-environment chamber as a desiccating stress model of DED for 35 days. At day 14 (T1), administration of 5 µL T or I in the right eye (RE) or NaCl 0.9% in the left eye (LE) started, twice a day. Animals were sacrificed after 7 (T2), 14 (T3), 21 (T4, endpoint) days from the beginning of treatment. Corneal fluorescein staining ratio (Image J), histological and histochemical assessment of ocular surface tissues (goblet cell GC density and characterization -PAS, Alcian blue pH 2.5, pH 1.0, and MUC4 expression-in the superior and inferior conjunctiva), and levels of inflammatory markers HLA-DR, IL-1β and TNF-α in cornea and conjunctiva were measured.No animal fully recovered from DED signs at the endpoint. Difference between arms was observed at T3 and T4, with T treated eyes showing a higher corneal damage reduction, PAS-positive GC recovery, lower inflammatory marker expression as compared to the I treated ones.Data suggest that 21 days of treatment with T/HA improved signs, GC recovery and inflammatory markers in a DED mouse model, to a greater extent as compared to I/HA. Data suggest that 21 days of treatment with T/HA improved signs, GC recovery and inflammatory markers in a DED mouse model, to a greater extent as compared to I/HA.

Splenectomized mice express progressively increased numbers of platelets in the blood and reduced numbers of megakaryocytes in the marrow with age. The megakaryocytes in the marrow of these animals express reduced levels of Gata1, a transcription factor necessary for their maturation. In addition, the marrow from these animals expresses greater levels of cytokines (TGF‐β, PDGF‐α, and VEGF) known to be produced at high levels by megakaryocytes expressing reduced levels of Gata1. This high level of cytokine expression is in turn associated with active osteoblast proliferation localized to areas of the femur, where megakaryocytes expressing reduced Gata1 levels are also found. These results confirm the role of megakaryocytes as regulator of bone formation in mice and suggest that a cross‐talk between the spleen and marrow may regulate the total numbers of hemopoietic niches present in an animal.

Abstract PKCα was found to be expressed (mRNA and protein) throughout the in vitro maturation of primary human erythroblasts but its activity (phosphorylation levels and nuclear localization) was consistently higher in cells derived from human neonatal rather than adult blood. Since the γ/γ + β globin expression ratio represented the major difference between neonatal and adult erythroblasts (58 ± 12 vs. 7 ± 3, respectively), we tested the hypothesis that PKCα might affect γ‐globin expression by measuring the levels of A γ‐ or β‐promoter‐driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual µLCRβprRluc A γprFluc reporter in the presence of transient expression of either the constitutively active (sPKCα) or catalytically inactive (iPKCα) PKCα. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 µM). In all the cells analyzed, sPKCα significantly increased (by two‐ to sixfold) the levels of luciferase activity driven by the A γ‐promoter and the A γ‐F/( A γ‐F + 2β‐R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the A γ‐driven luciferase activity and the A γ‐F/( A γ‐F + 2β‐R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic‐specific functions in erythropoiesis and that modulation of PKCα might affect the activity of A γ‐promoter‐driven reporters. J. Cell. Biochem. 101: 411–424, 2007. © 2007 Wiley‐Liss, Inc.

Abstract In 2002, we discovered that mice carrying the hypomorphic Gata1 low mutation that reduces expression of the transcription factor GATA1 in megakaryocytes ( Gata1 low mice) develop myelofibrosis, a phenotype that recapitulates the features of primary myelofibrosis (PMF), the most severe of the Philadelphia‐negative myeloproliferative neoplasms (MPNs). At that time, this discovery had a great impact on the field because mutations driving the development of PMF had yet to be discovered. Later studies identified that PMF, as the others MPNs, is associated with mutations activating the thrombopoietin/JAK2 axis raising great hope that JAK inhibitors may be effective to treat the disease. Unfortunately, ruxolitinib, the JAK1/2 inhibitor approved by FDA and EMEA for PMF, ameliorates symptoms but does not improve the natural course of the disease, and the cure of PMF is still an unmet clinical need. Although GATA1 is not mutated in PMF, reduced GATA1 content in megakaryocytes as a consequence of ribosomal deficiency is a hallmark of myelofibrosis (both in humans and mouse models) and, in fact, a driving event in the disease. Conversely, mice carrying the hypomorphic Gata1 low mutation express an activated TPO/JAK2 pathway and partially respond to JAK inhibitors in a fashion similar to PMF patients (reduction of spleen size but limited improvement of the natural history of the disease). These observations cross‐validated Gata1 low mice as a bona fide animal model for PMF and prompted the use of this model to identify abnormalities that might be targeted to cure the disease. We will summarize here data generated in Gata1 low mice indicating that the TGF‐β/P‐selectin axis is abnormal in PMF and represents a novel target for its treatment.

Cultured human erythroid cells derived in vitro may represent alternative transfusion products. It is unknown, however, if these ex vivo expanded erythroid cells remain functional or develop genetic abnormalities after storage.Using mononuclear cells from four adult blood donors, erythroblasts were generated ex vivo in expansion cultures supplemented with stem cell factor, interleukin-3, erythropoietin (EPO), dexamethasone, and estradiol. The viability and in vitro function of freshly expanded or short (1-2 months)- and long (8 years)-term-stored erythroblasts cryopreserved in dimethyl sulfoxide were compared. Erythroblast function was defined as ability to proliferate in expansion media and mature in response to EPO. Cell number was determined manually and expressed as fold increase. Viability was assessed by trypan blue and propidium iodide exclusion. Maturation was evaluated by morphologic analyses and CD36/CD235a expression profiling. Cytogenetic evaluation included karyotype and multicolor fluorescence in situ hybridization analyses.Equivalent numbers (>80%) of erythroblasts were viable after short- and long-term storage. Freshly expanded and short- and long-term-stored erythroblasts equally doubled in number (fold increase, 2.4) retaining an immature phenotype (23% of the cells were CD36(high)CD235a(neg)) when cultured for 4 days under expansion conditions. The numbers of freshly expanded and short-term-stored erythroblasts that matured when exposed for 4 days to EPO were also similar (approx. 22% of the cells became CD36(neg)CD235a(high)). In spite of the massive amplification, ex vivo generated erythroblasts demonstrated a normal (46,XY) karyotype with no obvious genomic rearrangements.Ex vivo expanded erythroblasts remain functional and genetically normal after long-term storage.

P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are major actors in multidrug resistance (MDR) phenomenon in both human and canine mammary carcinomas (CMCs). The aim of this study was to investigate an association between the intrinsic expression of P-gp and BCRP compared to the immunophenotypes and outcome in CMCs. Fifty CMCs were evaluated at immunohistochemistry (IHC) for P-gp, BCRP, Estrogen receptor alpha (ER), Progesterone receptors (PR), Human Epidermal Growth Factor Receptor type 2 (HER2), basal cytokeratins 5/6 (CK5/6), Epidermal Growth Factor Receptor 1 (EGFR), and Ki67 proliferation index. P-gp and BCRP positive cases were, respectively, 52% and 74.5%, with a significantly higher expression of BCRP than P-gp. Five immunophenotypes were defined in 37 out of 50 CMCs: 9 (24.3%) Luminal A, 5 (13.5%) Luminal B, 9 (24.3%) HER2 overexpressing, 9 (24.3%) Triple-negative basal-like, and 5 (13.5%) Triple-negative non-basal-like. In all CMCs at least one marker was expressed. Follow-up data were available for 25 animals. The average cancer-specific survival was 739 ± 444 days. A number of CMCs bear a high expression of P-gp and BCRP but no significant association was found between their expression and the immunophenotypes, Ki67 index, the histological grade, and tumor-related death.

Thrombocytopoiesis is a complex process beginning at the level of hematopoietic stem cells, which ultimately generate megakaryocytes, large marrow cells with a distinctive morphology, and then, through a process of terminal maturation, megakaryocytes shed thousands of platelets into the circulation.This process is controlled by intrinsic and extrinsic factors.Emerging data indicate that an important intrinsic control on the late stages of thrombopoiesis is exerted by integrins, a family of transmembrane receptors composed of one α and one β subunit.One β subunit expressed by megakaryocytes is the β1 integrin, the role of which in the regulation of platelet formation is beginning to be clarified.Here, we review recent data indicating that activation of β1 integrin by outside-in and inside-out signaling regulates the interaction of megakaryocytes with the endosteal niche, which triggers their maturation, while its inactivation by galactosylation determines the migration of these cells to the perivascular niche, where they complete their terminal maturation and release platelets in the bloodstream.Furthermore, β1 integrin mediates the activation of transforming growth factor β (TGF-β), a protein produced by megakaryocytes that may act in an autocrine fashion to halt their maturation and affect the composition of their surrounding extracellular matrix.These findings suggest that β1 integrin could be a therapeutic target for inherited and acquired disorders of platelet production.

This issue of the Stem Cell International journal contains papers from many of the leading scientists in the emerging field: ex vivo expansion of hematopoietic progenitor cells into erythrocytes for transfusion. Blood transfusion, the first form of successful cell therapy and, at least to some, “transplantation”, was inspired by the discovery of the circulation by Richard Harvey in the 1600s [1] and begun in earnest later in that century. The development of this clinical practice into the safe and routine therapy we all know today has been both an exciting scientific adventure and the foundation for a number of other scientific disciplines. More specifically, the immunology of transfusion and transplantation began with the discovery of the heterogeneity of human blood group antigens by Dr. Karl Landsteiner in 1901 (recognized with a Nobel Prize in 1930). The discovery of clinically relevant infectious diseases transmitted by transfusion played an important role in the development and advancement of virology. The inheritance of certain form of anemias was discovered during blood transfusion practice and led to development of the genetics of human red cell disorders. In the 1940–1950s, the establishment of blood banks followed by the development of rigorous donation criteria and standardization of blood manufacturing processes has made transfusion safe and widely available and has provided a paradigm for the development of emerging therapies using ex vivo expansion and differentiation of many cell types. An example of one such therapy is represented by the tumor immunotherapy described by Lapteva and Vera. The blood supply of industrialized countries is adequate overall. Nearly one hundred million donations are made every year worldwide (http://www.who.int/mediacentre/factsheets/fs279/en/index.html). The availability of blood and blood products in these nations has permitted the development and implementation of numerous life-saving surgical procedures (open heart surgery, organ transplantation, damage control resuscitation for trauma, and others) and cancer treatments which were not even imaginable without assurance that blood for transfusion would be readily available and safe. However, blood is not an unlimited resource and its potential need as the world rapidly develops requires a significant increase in blood donation. By some estimates (CDH), given the world's population and given the per capita transfusion of Canada as a utilization benchmark, nearly 250 million whole blood donations would be needed. Furthermore, and despite its high level of safety, human donated, unit-by-unit-derived blood donation/transfusion (i.e., without batched blood manufacturing into an aliquoted and homogenized pharmaceutical product), still leads to morbidity and mortality of its own accord and has significant variation from product to product based on the nature of the collection, manufacturing and storage processes, and the antigenic variation of any given donor, amongst others. Finally, it is not known what effect the aging of the world's population will have both on per capita utilization and on the ability of the smaller, younger populations to donate [2]. These issues, and the nearly 20-year-old search for alternative products to meet the transfusion need are discussed in the paper by Whitsett et al. Scientific research is inspired by the prospect of a clinical goal. In recent years, a revolution in stem cell biology has occurred that has far reaching implications, specifically, the discovery that it is possible to generate a potentially unlimited supply of stem cells by epigenetic/genetic treatments of somatic cells (T cells, fibroblasts, others) from any individual (see Pourcher et al., Hyroyama et al., and Chang et al.). In addition, techniques have been discovered to reprogram any cell into another cell type avoiding the induction of pluripotency. These techniques are fascinating though there are numerous scientific, safety, and scaling-up issues to be resolved before cells which have been genetically “altered” in the laboratory may be considered ready for widespread clinical use. As red blood cells do not have a nucleus, it is possible that they will be accepted as genetically safe. Indeed, it is this notion that supports that red blood cells from Hematopoietic stem/progenitor cell expansion or redifferentiation may represent the first therapeutic product to be generated by genomic reprogramming technology. Reprogramming technology is still under development. Therefore, red blood cells expanded ex vivo from primary stem cell sources currently discarded (buffy coats produced during the blood manufacturing processes and low-volume umbilical cord blood) are being considered for first-in-man studies. Tirelli et al. identify the cell populations present in adult blood which are responsible for massive production of red blood cells ex vivo. The first-in-man proof-of-principle study for the use of in vitro expanded red blood cells for transfusion was reported on September 1st 2011, by Luc Douay and colleagues [3], who have also coauthored Pourcher et al. This paper reported that red blood cells generated in vitro from mobilized CD34pos cells collected by apheresis have normal survival (determined by 51Cr labeling) when transfused into an autologous recipient [3]. This first-in-man autologous transfusion described also what would be the most likely safety data necessary for a larger clinical study with such products [in vitro characterization (blood group antigen expression profiling, deformability, hemoglobin content and O2 dissociation curves) and in vivo functional studies in animal models (survival and morphology); http://www.clinicaltrials.gov/ct2/show/NCT00929266]. In vivo functional studies of human red blood cells in animal models will likely allow more complete characterization in many ways [4]. Ghinassi et al. describe an improved animal model which allows in vivo imaging and cell fate determination of human erythroid cells by labeling the cells before transfusion with a fluorescent reporter gene by retroviral technology. Although red blood cells do not have nuclei, their immediate precursors the erythroblasts do. The terminal maturation of erythroblasts into functional red cells requires a complex remodeling process which ends with extrusion of the nucleus and the formation of an enucleated red blood cell [5]. These late stages of maturation are intrinsically controlled by epigenetic/genetic expression programs of the erythroblast itself. Cell reprogramming methodologies may (and at present appear to) disrupt these programs, leading to inefficient enucleation. Keerthivasan et al. discuss novel insights into the critical mechanisms of terminal maturation of a red blood cell and strategies to improve the efficiency of these processes. As represented by all the information, data, and in fact vision contained in this issue, we are clearly at the beginning of a rapidly expanding field. The papers herein provide a broad and comprehensive overview of the most relevant areas of research which have been pursued and are needed to advance the field. Still, as state of the art as this issue is presently, the field is moving so rapidly that one may predict that new knowledge will rapidly follow. Anna Rita Migliaccio Giuliano Grazzini Christopher D. Hillyer

A cute or chronic blood loss reduces the numbers of red blood cells (RBCs) in the circulation, reducing oxygen delivery and producing tissue hypoxia. One of the first cellular responses to hypoxia is increased production of reactive oxygen species (ROS) by mitochondria. With evolution, organisms

Airway remodeling encompass structural changes that occur as the result of chronic injury and lead to persistently altered airway structure and function. Although this process is known in several human respiratory conditions such as asthma and chronic obstructive pulmonary disease (COPD), airway remodeling is poorly characterized in the feline counterpart. In this study, we describe the spontaneous pulmonary changes in 3 cats paralleling the airway remodeling reported in humans. We observed airway smooth muscle cells (ASMCs) hyperplasia (peribronchial and interstitial), airway subepithelial and interstitial fibrosis, and vascular remodeling by increased number of vessels in the bronchial submucosa. The hyperplastic ASMCs co-expressed α-SMA, vimentin and desmin suggesting that vimentin, which is not normally expressed by ASMCs, may play a role in airway thickening, and remodeling. ASMCs had strong cytoplasmic expression of TGFβ-1, which is known to contribute to tissue remodeling in asthma and in various bronchial and interstitial lung diseases, suggesting its involvement in the pathogenesis of ASMCs hyperplasia. Our findings provide histologic evidence of airway remodeling in cats. Further studies on larger caseloads are needed to support our conclusions on the value of this feline condition as an animal model for nonspecific airway remodeling in humans.

Human epidermal growth factor receptor 2 (HER2) is a tyrosine kinase receptor that promotes tumor cell growth and is implicated in the pathogenesis of human breast cancer. The role of HER2 in canine mammary carcinomas (CMCs) is not clear. Therefore, this study aimed to examine the protein expression and cytogenetic changes of HER2 and their correlation with other clinical-pathological parameters in CMC. We retrospectively selected 112 CMCs. HER2, ER, and Ki67 were assessed by immunohistochemistry. HER2 antibody validation was investigated by immunoblot on mammary tumor cell lines. Fluorescence in situ hybridization (FISH) was performed with probes for HER2 and CRYBA1 (control gene present on CFA9). HER2 protein overexpression was detected in 15 carcinomas (13.5%). A total of 90 carcinomas were considered technically adequate by FISH, and 8 out of 90 CMC (10%) were HER2 amplified, 3 of which showed a cluster-type pattern. HER2 overexpression was correlated with an increased number of HER2 gene copies (p = 0.01; R = 0.24) and overall survival (p = 0.03), but no correlation with ER, Ki67, grade, metastases, and tumor-specific survival was found. Surprisingly, co-amplification or polysomy was identified in three tumors, characterized by an increased copy number of both HER2 and CRYBA1. A morphological translocation-fusion pattern was recognized in 20 carcinomas (22%), with a co-localized signal of HER2 and CRYBA1. HER2 is not associated with clinical-pathological parameters of increased malignancy in canine mammary tumors, but it is suitable for studying different amplification patterns.

Here it is analyzed the expression of a mini locus dual reporter construct composed by a micro-LCR and by the promoters for Aγ- and β-globin gene, each one linked to a different Luciferase, in stably transfected GM979 cells for as long as 1–2 years from transfection. The transfected GM979 cells rapidly (within 1 month) evolved into a stable population which expresses constant levels of reporters for more than a year of continuous bulk culture. No silencing of the inserted construct was observed over time. In contrast, after 1 month, the reporter activity (both from Aγ- and β-promoter) expressed per cell increased over time. The analysis of the Luciferase contained in single cell clones indicated that the higher reporter activity was due to increased gene expression per cell rather than to clonal selection of the most expressing clones. Since the activity driven by the β-promoter increased 10-fold more than that driven by the Aγ one, the ratio between Aγ-driven/(Aγ-driven + β-driven) reporter activity in the cells decreased after 1 month and became similar to the γ/(γ + β) globin mRNA ratio expressed by adult erythroid cells. Moreover, although both cells from early and late bulk culture responded to incubation with butyric acid, a known inducer of fetal globin gene expression, by increasing the reporter activity driven by the Aγ-promoter, only cells from late bulk culture decreased, as normal primary erythroblasts do, the activity of the reporter driven by the β-promoter. These results suggest that the rapid changes in activity driven by the Aγ- and β-globin promoters occurring during the first month after transfection may represent a novel in vitro model to study epigenetic regulation of the Aγ- and β-promoter during the fetal to adult hemoglobin switch and confirm GM979 cells stably transfected with the dual reporter construct as a reliable assay for automated screening of chemical inducers of fetal globin gene activation.

In this issue of Blood , Chen and colleagues [1][1] identify the 11-kDa nonstructural protein of the parvovirus B19 as the main culprit for induction of apoptosis in primary human erythroblasts through activation of caspase 10. Infection with B19 parvovirus, usually spread by the respiratory route

NR3C1, the gene encoding the glucocorticoid receptor, is polymorphic presenting numerous single nucleotide polymorphisms (SNPs) some of which are emerging as leading cause in the variability of manifestation and/or response to glucocorticoids in human diseases. Since 60–80% of patients with Diamond Blackfan anemia (DBA), an inherited pure red cell aplasia induced by mutations in ribosomal protein genes became transfusion independent upon treatment with glucocorticoids, we investigated whether clinically relevant NR3C1 SNPs are associated with disease manifestation in DBA. The eight SNPs rs10482605, rs10482616, rs7701443, rs6189/rs6190, rs860457, rs6198, rs6196, and rs33388/rs33389 were investigated in a cohort of 91 European DBA patients. Results were compared with those observed in healthy volunteers ( n =37) or present in public genome databases of Italian and European populations. Although, cases vs. control analyses suggest that the frequency of some of the minor alleles is significantly altered in DBA patients with respect to healthy controls or to the Italian or other European registries, lack of consistency among the associations across different sets suggests that overall the frequency of these SNPs in DBA is not different from that of the general population. Demographic data (47 females and 31 males) and driver mutations (44 S and 29 L genes and eight no-known mutation) are known for 81 patients while glucocorticoid response is known, respectively, for 81 (36 responsive and 45 non-responsive) and age of disease onsets for 79 (55 before and 24 after 4months of age) patients. Neither gender nor leading mutations were associated with the minor alleles or with disease manifestation. In addition, none of the SNPs met the threshold in the response vs. non-responsive groups. However, two SNPs (rs6196 and rs860457) were enriched in patients manifesting the disease before 4months of age. Although the exact biomechanistical consequences of these SNPs are unknown, the fact that their configuration is consistent with that of regulatory regions suggests that they regulate changes in glucocorticoid response during ontogeny. This hypothesis was supported by phosphoproteomic profiling of erythroid cells expanded ex vivo indicating that glucocorticoids activate a ribosomal signature in cells from cord blood but not in those from adult blood, possibly providing a compensatory mechanism to the driving mutations observed in DBA before birth.

SUMMARY Pro-inflammatory signaling is a hallmark feature of human cancer, including in myeloproliferative neoplasms (MPNs), most notably myelofibrosis (MF). Dysregulated inflammatory signaling contributes to fibrotic progression in MF; however, the individual cytokine mediators elicited by malignant MPN cells to promote collagen-producing fibrosis and disease evolution remain yet to be fully elucidated. Previously we identified a critical role for combined constitutive JAK/STAT and aberrant NF-κB pro-inflammatory signaling in myelofibrosis development. Using single-cell transcriptional and cytokine-secretion studies of primary MF patient cells and two separate murine models of myelofibrosis, we extend this previous work and delineate the role of CXCL8/CXCR2 signaling in MF pathogenesis and bone marrow fibrosis progression. MF patient hematopoietic stem/progenitor cells are enriched in a CXCL8/CXCR2 gene signature and display dose-dependent proliferation and fitness in response to exogenous CXCL8 ligand in vitro . Genetic deletion of Cxcr2 in the hMPL W515L adoptive transfer model abrogates fibrosis and extends overall survival, and pharmacologic inhibition of the CXCR1/2 pathway improves hematologic parameters, attenuates bone marrow fibrosis, and synergizes with JAK inhibitor therapy. Our mechanistic insights provide a rationale for therapeutic targeting of the CXCL8/CXCR2 pathway in MF patients at risk for continued fibrotic progression.

SUMMARY Although mechanisms controlling differentiation of hemopoietic stem and early progenitor cells are still poorly understood, it is generally conceded that a pivotal role is played by hemopoietic growth factors (HGFs). 1,2 However, in‐vitro analysis of their action on early progenitors may be obscured by cell‐cell interaction, as well as by the presence of fetal bovine serutn (FBS). To overcome these limitations, we investigated the action of pure multipotent or lineage‐specific HGFs on purified progenitors grown in FBS‐free cultures. In the murine system, highly purified progenitors were cultured in the presence of multipotent colony‐stimulating factor (multi‐CSF, also termed interleukin‐3), erythropoietin (Ep) and macrophagic‐CSF (M‐CSF). Each HGF was unable by itself to induce significant colony growth. However, combined addition of multi‐CSF and either Ep or M‐CSF gave rise only to pure erythroid or macrophagic colonies, respectively. Partly purified human progenitors were challenged by human granulomonocytic‐CSF (GM‐CSF), pluripotent CSF (PPO, also termed granulocytic‐CSF, G‐CSF) and Ep. Here again, each HGF was unable per se to promote colony growth, but combined addition of GM‐CSF or PPO and Ep gave rise only to pure erythroid colonies. These results support a model of early hemopoietic differentiation according to which multi‐lineage HGFs represent “competence” GFs, the action of which is complemented by lineage‐specific “progression” HGFs.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disorder with limited therapeutic options. Insufficient understanding of driver mutations and poor fidelity of currently available animal models has limited the development of effective therapies. Since GATA1 deficient megakaryocytes sustain myelofibrosis, we hypothesized that they may also induce fibrosis in lungs. We discovered that lungs from IPF patients and Gata1low mice contain numerous GATA1negative immune-poised megakaryocytes that, in mice, have defective RNA-seq profiling and increased TGF-β1, CXCL1 and P-selectin content. With age, Gata1low mice develop fibrosis in lungs. Development of lung fibrosis in this model is prevented by P-selectin deletion and rescued by P-selectin, TGF-β1 or CXCL1 inhibition. Mechanistically, P-selectin inhibition decreases TGF-β1 and CXCL1 content and increases GATA1positive megakaryocytes while TGF-β1 or CXCL1 inhibition decreased CXCL1 only. In conclusion, Gata1low mice are a novel genetic-driven model for IPF and provide a link between abnormal immune-megakaryocytes and lung fibrosis.

Equine asthma is a common respiratory disease that may affect horses of any age. The diagnosis of severe equine asthma (SEA) (historically referred as recurrent airway obstruction or RAO) is based mainly on the history of the animal and clinical signs, which are further supported by the cytological examination of the bronchoalveolar lavage (BAL). This can also be helpful in monitoring the inflammation of the lower airways in response to environmental management and medication. The cytocentrifugated preparation is usually considered the method of choice for BAL cytological interpretation. The aim of this study was to compare the results in terms of differential cell counts (DCC) in BAL cytology performed on sedimented smears and cytocentrifugated preparations. To carry this out, 48 BAL samples were collected from six horses with SEA that were subjected to a process of exacerbation of the disease by environmental stimuli, which was later followed by the appropriate treatment. Each collected BAL fluid was equally divided into duplicate portions: one processed by cytocentrifugation and one by sediment smear from simple centrifugation. Cytologic examination of all BAL by both methods showed poor concordance in DCC, although it was still able to allow diagnostic recognition of severe lung neutrophilic disorders. These results suggest that sediment smear preparation, although remaining a useful method in general equine practice associated with clinical assessments in the diagnosis of SEA under conditions where there is no possibility of using a cytocentrifuge, cannot be considered a comparable alternative.

Cannabinoid receptors are expressed in human and animal trigeminal sensory neurons; however, the expression in the equine trigeminal ganglion is unknown. Ten trigeminal ganglia from five horses were collected post-mortem from an abattoir. The expression of cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and the cannabinoid-related receptors like transient receptor potential vanilloid type 1 (TRPV1), peroxisome proliferator-activated receptor gamma (PPARɣ), and G protein-related receptor 55 (GPR55) in the trigeminal ganglia (TG) of the horse were studied, using immunofluorescence on cryosections and formalin-fixed paraffin-embedded (FFPE) sections. Neurons and glial cells were identified using fluorescent Nissl staining NeuroTrace® and an antibody directed against the glial marker glial fibrillary acidic protein (GFAP), respectively. Macrophages were identified by means of an antibody directed against the macrophages/microglia marker ionized calcium-binding adapter molecule 1 (IBA1). The protein expression of CB1R, CB2R, TRPV1, and PPARɣ was found in the majority of TG neurons in both cryosections and FFPE sections. The expression of GPR55 immunoreactivity was mainly detectable in FFPE sections, with expression in the majority of sensory neurons. Some receptors were also observed in glial cells (CB2R, TRPV1, PPARγ, and GPR55) and inflammatory cells (PPARγ and GPR55). These results support further investigation of such receptors in disorders of equine trigeminal neuronal excitability.

Abstract Background Three-dimensional (3D) cell cultures are the new frontier for reproducing the tumor micro-environment in vitro. The aims of the study were (1) to establish primary 3D cell cultures from canine spontaneous neoplasms and (2) to demonstrate the morphological, phenotypic and genotypic similarities between the primary canine neoplasms and the corresponding 3D cultures, through the expression of tumor differentiation markers. Results Seven primary tumors were collected, including 4 carcinomas and 3 soft tissue sarcomas. 3D cell cultures reproduced the morphological features of the primary tumors and showed an overlapping immunophenotype of the primary epithelial tumors. Immunohistochemistry demonstrated the growth of stromal cells and macrophages admixed with the neoplastic epithelial component, reproducing the tumor microenvironment. Mesenchymal 3D cultures reproduced the immunophenotype of the primary tumor completely in 2 out of 3 examined cases while a discordant expression was documented for a single marker in one case. No single nucleotide variants or small indel were detected in TP53 or MDM2 genes, both in primary tumors and in 3D cell cultures specimens. In one sample, MDM2 amplicons were preferentially increased in number compared to TP53 ones, indicating amplification of MDM2 , detectable both in the primary tumor and in the corresponding cell culture specimen. Conclusion Here we demonstrate a good cell morphology, phenotype and genetic profile overlap between primary tumors and the corresponding 3D cultures grown in a repeatable system.

We have studied the frequency of colony forming cells (CFC) in fetal and neonatal blood in comparison with adult blood and marrow. Fetal/neonatal blood contains at least as many CFC as adult marrow and higher numbers of the more primitive CFC—those CFC giving rise to colonies composed of erythroid and myeloid cells. CD34+ cord blood cells (selected either by sorting, panning or affinity chromatography) proliferate in culture over time and generate more CFC (from pre-CFC) and differentiated cells in response to Steel factor plus different hematopoietic growth factors. Steel factor is unable to stimulate cell growth by itself under serum-deprived conditions and requires the synergistic action of erythropoietin (Epo), granulocyte colony stimulating factor (G-CSF) or interleukin 3 (IL-3). In the presence of Epo or G-CSF, CFC and differentiated cells are generated for 15 days and are mainly erythroid or granulocytic, respectively. In contrast, Steel factor plus IL-3 generates multilineage CFC and differentiated cells for more than one month. When the conditions for these long-term suspension cultures were optimized (37°C, regular refeeding with fresh growth factors and media without changing the flask), CFC and differentiated cells were generated for more than two months. At this time, CFC were no longer detectable and all cells had a mast cell phenotype. These cells have been maintained and propagated for more than eight months in the presence of IL-3 and Steel factor and may represent a useful tool to study human mast cell differentiation. Finally, the addition of oligonucleotides antisense to c-kit, the receptor for Steel factor, selectively suppresses the generation of erythroid cells, indicating that Steel factor/c-kit interaction plays a major role in the process of erythroid commitment.

Immunohistochemistry (IHC) is a widely used technique in diagnostic pathology, but the simultaneous analysis of more than one antibody at a time with different chromogens is rather complex, time-consuming, and quite expensive. In order to facilitate the identification of mast cells (MCs) during immunohistochemical analysis of membrane and/or nuclear markers, we propose a new staining method that includes the association of IHC and toluidine blue as a counterstain. To achieve this goal, we tested c-kit, Ki67, and cannabinoid receptor 2 on several cases of cutaneous canine mast cell tumors (MCTs), cutaneous mastocytosis, and atopic dermatitis. The results obtained show how this double staining technique, although limited to non-cytoplasmic markers and of little use in poorly differentiated MCTs in which MC metachromasia is hard to see, can be used during the evaluation of nuclear and/or membranous immunohistochemical markers in all canine cutaneous disorders, especially if characterized by the presence of a low number of MCs. It can help to evaluate those MCTs in which neoplastic MCs must be clearly distinguished from inflammatory cells that can infiltrate the tumor itself, in facilitating the calculation of the Ki67 index. Moreover, it can be used to study the expression of new markers in both animal and human tissues containing MCs and in MC disorders.

Careful morphological investigations, coupled with experimental hematology studies in animal models and in in vitro human cultures, have identified that platelets are released in the circulation by mature megakaryocytes generated by hematopoietic stem cells by giving rise to lineage-restricted progenitor cells and then to morphologically recognizable megakaryocyte precursors, which undergo a process of terminal maturation. Advances in single cell profilings are revolutionizing the process of megakaryocytopoiesis as we have known it up to now. They identify that, in addition to megakaryocytes responsible for producing platelets, hematopoietic stem cells may generate megakaryocytes, which exert either immune functions in the lung or niche functions in organs that undergo tissue repair. Furthermore, it has been discovered that, in addition to hematopoietic stem cells, during ontogeny, and possibly in adult life, megakaryocytes may be generated by a subclass of specialized endothelial precursors. These concepts shed new light on the etiology of myelofibrosis, the most severe of the Philadelphia negative myeloproliferative neoplasms, and possibly other disorders. This perspective will summarize these novel concepts in thrombopoiesis and discuss how they provide a framework to reconciliate some of the puzzling data published so far on the etiology of myelofibrosis and their implications for the therapy of this disease.

Abstract The murine white (W) spotting locus is the site of the c‐kit gene and encodes a tyrosine kinase receptor while the complementary Steel (Sl) iocus encodes its ligand. Mutations at either locus have profound effects on hematopoiesis, particularly erythroid and mast cell proliferation. We added c‐kit antisense oligonucleotides to long‐term suspension cultures of enriched human umbilical cord progenitor cells. This resulted in the suppression of c‐kit gene expression and the preferential suppression of the generation of erythroid burst‐forming cells (BFU‐E) which extended over the life of the culture (3 weeks). The results provide an in vitro model of the “W phenotype” in human hematopoiesis and confirm the importance of c‐kit gene function in early erythropoiesis. Because the generation of BFU‐E was suppressed even after c‐kit gene expression had recovered, this gene product may be critical to the erythroid commitment process. © 1993 Wiley‐Liss, Inc.

Summarized here, and in forthcoming issues of, are the concepts that emerged at a recent international workshop on cell therapy organized by The Istituto Superiore di Sanità in Rome in collaboration with Istituto Dermatopatico dell'Immacolata, Rome; Istituto Nazionale Ricerca Cancro-Centro Biotecnologie Avanzate, Genova; and University G. D'Annunzio, Chieti. The meeting intent was to provide an overview of the most recent developments in cell therapy, the future perspectives for these clinical trials, and the regulatory issues they involve, as well as a progress report on the clinical protocols that have been approved up to now in Italy. The meeting included six scientific sessions (Immunotherapy, Epithelium, Osteoregeneration, Hematopoiesis, Future Perspectives, and Overview of the National and International Regulations) and involved lectures from Italian and foreign scientists.

The recognition of cord blood as a discarded resource rich in high proliferative potential hematopoietic stem/progenitor cells (HSPCs) triggered a flurry of studies in the 1990s exploiting its potential for transplantation. These studies established placental cord blood (PCB) as an excellent source of stem cells for related and unrelated transplants; pediatric, and adult. However, the fact that each PCB is quantitatively a limited resource restricts its efficacy to patients with low body weight, mostly children. Therefore, strategies are continuously being explored to overcome this limitation. Attempts to increase the HSPC content, or to improve their homing and engraftment potential, or the receptivity and/or the immunological status of the recipient are ongoing. Most importantly, apart from HSPCs, PCB contains stem cells for other tissues. This awareness has propelled explorations on the use of PCB for tissue repair and tissue engineering for a variety of clinical applications. In the present communication we will review differences between fetal–neonatal and adult HSPCs, current progress and limitations in the use of PCB for transplantation, and targeted approaches to overcome these limitations in the clinical use of PCB for treatment of hematopoietic and nonhematopoietic disorders.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the canine gastrointestinal tract and are diagnosed by the immunohistochemical expression of the receptor tyrosine kinase (RTK) KIT. Activating mutations of the proto-oncogenes c-KIT and PDGFRA drive GIST oncogenesis and are used to predict the response to RTK-inhibitors in human oncology. Currently, the frequency and significance of these mutations in canine GIST have not been adequately explored. Therefore, we investigated the mutational status of c-KIT (exons 9, 11 and 13) and PDGFRA (exons 12 and 18) genes by PCR followed by fragment analysis for c-KIT deletions and PCR followed by screening with DHPLC and direct sequencing confirmation for single nucleotide variations in 17 formalin-fixed paraffin-embedded canine GISTs confirmed by KIT immunopositivity. c-KIT mutations were detected in 47% of cases, with a mutation detection rate significantly higher (p = 0.0004, Fisher's exact test) and always involving exon 11. A PDGFRA gene mutation (exon 18) was identified in one case. Even if follow-up data were not available for all cases, four cases with documented abdominal metastases displayed c-KIT mutations. These data confirm that c-KIT exon 11 mutations occur frequently in canine GISTs, and identify the presence of a PDGFRA mutation similar to human GISTs. This study also suggests a potential association of c-KIT mutation with more aggressive biological behavior.

EDITORIAL article Front. Oncol., 25 November 2022Sec. Cancer Molecular Targets and Therapeutics Volume 12 - 2022 | https://doi.org/10.3389/fonc.2022.1090658

Abstract We have investigated the phenotypic and functional characteristics of murine pre‐B cells obtained in semisolid and liquid culture with stem cell factor (SCF) and interleukin 7 (IL‐7). Both serum‐supplemented and serum‐deprived culture conditions were used. The source of bone marrow cells was either normal mice (CD1 and C3H) or the lupus strain of mice MRL/Ipr and its congenic strain MRL/+. SCF (100 ng/ml) and IL‐7 (250 ng/ml) supported murine B cell proliferation in vitro from all the murine strains analyzed both in serum‐supplemented and serum‐deprived conditions. Maximal colony growth was observed in both cases when the factors were used in combination. The growth factors alone induced some colony growth in serum‐supplemented cultures but were either ineffective or had modest activity in serum‐deprived cultures. Cells harvested from the colonies or generated in liquid cultures and stimulated with SCF + IL‐7 in the absence of serum had almost exclusively a pre‐B cell phenotype (BP‐1+, B220+, slg‐, CD4‐, CD8‐, Mac‐1, RB‐6‐). Both the maximal colony growth in semisolid culture and the maximal number of cells in liquid culture were observed at day 12–14. At this time, the pre‐B cells failed to differentiate further and started to die. Pre‐B cells generated in vitro were, however, capable of differentiating in vivo. SCID mice injected with 2 × 10 6 pre‐B cells had readily detectable serum levels of IgM (54 ± 26 m̈g/ml) and IgG (60 ± 95 m̈g/ml) at 4 weeks and 6 weeks posttransplantation, respectively. Mature B and T cells of the donor major histocompatibility complex type were detected in the SCID mice at sacrifice 14 weeks posttransplantation. These data indicate that purified (&gt;80% BP‐1+) populations of functional pre‐B cells can be grown from murine bone marrow of normal mice as well as of lupus mice in serum‐deprived cultures stimulated with SCF and IL‐7. These cultures, therefore, provide a highly enriched source of pre‐B cells but also contain T cell precursors that differentiate upon adoptive transfer into SCID mice. © 1995 Wiley‐Liss, Inc.

Extract Essentials Introduction Mature circulating red cells are specialized cellular elements of the blood responsible for both the delivery of oxygen to and for the removal of CO2 from all tissues of the body. In the adult, their number is constantly maintained by the balance of two ongoing processes: the destruction of old red cells, mainly in the spleen; and the generation of new red cells within the bone marrow by a process referred to as erythropoiesis. The generation of new red cells, like other cellular elements, is accomplished through a complex interplay between haemopoietic cells, stromal cells, and the extracellular matrix within the bone marrow microenvironment. Unique to the erythropoietic process is its regulation not only by growth factors produced in situ in the bone marrow, but also by circulating erythropoietin (EPO), a true ‘hormone’ produced by the kidneys in the adult. Positive or negative alterations of EPO production, whether acquired or congenital, and/or of its signalling pathway, result in quantitative changes in red cell production—i.e. anaemia or erythrocytosis.

In this issue of Blood, by generating a novel dual oxidase 2 (DUOX2) mutation responsible for congenital hypothyroidism (CH) in pigs, Zhang et al identify Kr üppel-like factor 9 (KLF9) as the mediator for the regulation exerted by the thyroid gland on hematopoiesis. 1

The engraftment potential of murine stem cells (HSC) is greatly reduced when these cells are expanded in vitro with stem cell factor and interleukin-3. We have evaluated if the addition of MIP-1 alpha or LIF to these cultures would protect the ability of murine wild type HSC to engraft the stem cell defective W/Wv recipient. In this transplantation model red and white blood cell reconstitution is assessed by hemoglobin electrophoresis and c-kit PCR genotyping, respectively. The results obtained indicate that both MIP-1 alpha and LIF protect, at least transiently, the HSC repopulating ability in vivo in spite of the modest expansion in the number of nucleated and progenitor cells observed.

There is evidence that the total cellular content of placental cord blood (PCB) grafts is related to the speed of engraftment, though the total nucleated cell (TNC) dose is not a precise predictor of the time of neutrophil or platelet engraftment. It is important to understand the reasons for the quantitative association and to improve the criteria for selecting PCB grafts by using indices more precisely predictive of engraftment. The posttransplant course of 204 patients who received grafts evaluated for hematopoietic colony-forming cell (CFC) content among 562 patients reported previously were analyzed using univariate and multivariate life-table techniques to determine whether CFC doses predicted hematopoietic engraftment speed and risk for transplant-related events more accurately than the TNC dose. Actuarial times to neutrophil and platelet engraftment were shown to correlate with the cell dose, whether estimated as TNC or CFC per kilogram of recipient's weight. CFC association with the day of recovery of 500 neutrophils/μL, measured as the coefficient of correlation, was stronger than that of the TNC (R = −0.46 and −0.413, respectively). In multivariate tests of speed of platelet and neutrophil engraftment and of probability of posttransplantation events, the inclusion of CFC in the model displaced the significance of the high relative risks associated with TNC. The CFC content of PCB units is associated more rigorously with the major covariates of posttransplantation survival than is the TNC and is, therefore, a better index of the hematopoietic content of PCB grafts.

Abstract Abstract 3156 Poster Board III-93 Hematopoietic progenitor cells cultured with stem cell factor (SCF), interleukin-3 (IL-3) and erythropoietin (EPO) generate erythroid cells that mature in 2 weeks. Addition of dexamethasone and estradiol retards maturation providing the basis for the development of HEMA, a system that generates ex vivo human erythroblasts (EBs) in numbers sufficient for transfusion. In HEMA, EBs at different stages of maturation coexist for &gt; 21 days. FACS analyses for thrombospondin receptor (CD36) and Glycophorin A (CD235a) divides EBs into 4 maturation classes: CD36highCD235aneg (CFU-E) and CD36highCD235alow (pro-EBs) (immature EBs, iEBs); CD36highCD235ahigh (basophilic-polychromatic EBs) (mature EBs) and CD36lowCD235ahigh (orthochromatic EBs) cells. At day 10, 5-19% of the cells are non-erythroid CD36negCD235aneg cells while orthochromatic EBs are barely detectable at any time. The aim of this study was to identify the relative contribution of different populations to the final EBs output of HEMA. The cells present at day 10 in HEMA established with adult mononuclear cells were separated by sorting, labeled with CSFE and cultured for 4 additional days for fate determination (proliferation, maturation and/or death). To identify which population had the ability to generate more EBs, unfractionated cells and cells isolated by sorting (non-erythroid, iEBs and mEBs) were cultured either in colony assay or HEMA. Ninety-three percent of the colony forming ability (all CFU-GM) of unfractionated cells was recovered in the CD36negCD235aneg fraction which did not grow in HEMA. However, iEBs did not contain colony forming cells but generated twice as many EBs as unfractionated cells [3.9±1.0 vs 2.38±0.32 fold increase (FI), respectively). In cultures maintained for 16 days, iEBs sequentially sorted every 2 days generated 5-fold more EBs than the corresponding unfractionated EBs (FI=25 vs 5, respectively). mEBs did not generate colonies or proliferate but died in HEMA. Calculations of hemi CSFE staining decrements indicated that the division index of iEBs and mEBs in the first 24 hrs of HEMA was 1.32±0.08 and 0.4±0.08 (p&lt;0.01), respectively. The higher number of cells generated by iEBs with respect to unfractionated cells suggested that mEBs may inhibit iEBs proliferation. To characterize this effect, iEBs and mEBs were co-cultured for 48 hrs at ratios of 100/0, 5/1, 5/3 and 1/2. One of the two populations was labelled by CSFE staining. To take into account a possible toxic effect of CSFE-labeling, experiments were repeated with either CSFE-labelled iEBs or CSFE-labelled mEBs measuring proliferation/maturation/apoptosis of labeled and unlabelled cells, respectively. iEBs alone increased in numbers by 2-fold over 4 days. By contrast, iEB-derived cells did not increase in number in co-cultures at 5/1 and 5/3 mEB ratios and were undetectable in those at 1/2 mEB ratio. Although cell number did not increase in the co-cultures, the proliferation index (1.3), levels of maturation (50% newly generated mEBs) and apoptosis (barely detectable Annexin Vpos cells) of iEBs in co-cultures remained similar to those observed when these cells were cultured alone. The failure to detect cells after 4 days in iEB/mEB co-cultures at 1/2 ratio suggests that mEBs induce non-apoptotic iEB death. In conclusion, three cells populations were present at day 10 in HEMA of adult mononuclear cells: a CD36negCD235aneg population that contains myeloid progenitor cells and does not generate EBs; iEBs, that generate additional iEBs as well as mEBs; and mEBs that have low proliferation activity, limited life-span and induce non-apototic iEB death. Therefore, the final cellular output of HEMA is determined by a delicate balance between proliferation, maturation and cell death of iEBs and mEBs. Disclosures No relevant conflicts of interest to declare.

Several studies indicate that abnormal megakaryocytes (MK) drive bone marrow fibrosis in primary myelofibrosis (PMF). Since the lung is a site where MK lodge to release platelets (Lefrançais, Nature 2017; 544:105), we investigated whether the MK abnormalities observed in PMF also lead to fibrosis in lung. To this aim, we used Gata1low mice that express MK abnormalities similar to those observed in PMF patients and develop myelofibrosis with age (Zingariello, Blood 2013;121:3345) and compared number/morphology of MK and extent of fibrosis in lungs from Gata1low mice or from their wild-type (WT) littermates. Two age groups were considered: adult, A (5-7 months) and old, O (&gt;12 months). Pulmonary fibrosis was assessed by H&amp;E and Masson's staining and graded from 0 (absent) to 3 (severe). MK were evaluated by immunohistochemistry and immunofluorescent analysis with CD42 and their number expressed per 0.144 mm2. Statistical analysis was performed with Shapiro test for normality while chi-square test and Spearman's correlation test for comparisons. Since the mutation is carried in the CD1 background that is known to develop multisite inflammation with age (Brayton, Vet Pathol 2012; 49:85), preliminary experiments compared the pro-inflammatory signature of the mice groups by ELISA determinations of the plasma levels of CXCL1 (murine equivalent of IL-8) and its inducer lipocalin2 (LCN2). WT mice expressed a pro-inflammatory signature with high levels of LCN2 (90±45ng/mL in A; 50±49 in O) and CXCL1 (44.5±11.4pg/mL in A;31.6±4.6 in O compared to 17.0 ±5.1 in O P-Selnull/Gata1low mice which do not express inflammation, p&lt;0.01). These levels were not affected by age. Gata1low mice expressed high levels of LCN2 that increased with age (100±18 in A and 150±45 in O, p&lt;0.05) and levels of CXCL1 significantly lower than those expressed by WT (27.9±1.3 in A and 28.8 ±9.7 in O, p&lt;0.05) but still greater than those in negative controls. Mild and localized interstitial pneumonia characterized by infiltration with lymphocytes, plasma cells and macrophages in the alveolar septae and peribronchial-vascular spaces was observed in 66.6% of WT mice. However, mild interstitial pneumonia was detected in 84.6% of Gata1low mice where it was multi-focal to coalescent and extended from alveolar septae to subpleural interstitium. Lungs from 33% of WT mice had mild fibrosis with accumulation of collagen in alveolar septae (grade 1) while those from 70.8% Gata1low mice presented severe fibrosis (37.5% grade 1, 29.4% grade 2 and 17.6% grade 3) (Fig. 1A, 1B). The mild interstitial pneumonia associated to grade 1 fibrosis present both in Gata1low and WT littermates is considered a background lesion due to the pro-inflammatory cytokine profile of the strain investigated. By contrast, despite the severity of pneumonia was similar in Gata1low and WT littermates, grade 2 and 3 fibrosis was only present in Gata1low mice, indicating that it was not due to the baseline inflammatory milieu of their CD1 background. MK were observed intravascularly and within the perivascular interstitium of lungs from both WT and Gata1low mice, the same location where fibrosis was detected. The number of MK was significantly lower in Gata1low than in WT littermates (median=1, range 0.6-2.57 vs 1.8, range 0.4-8.3, respectively, p=0.02) (Fig 1C). The morphology of MK found in the two groups was significantly dissimilar (p&lt;0.01): most MK (88.8%) in WT lungs contained either a round nucleus with small amount of cytoplasm or a polylobated nucleus with well developed platelet territories (Fig.1D). The morphology of most (70%) of MK in Gata1low lungs was instead characterized by large size, polylobated nuclei but poorly developed platelet territories (Fig.1E). Therefore, the significantly higher number of Gata1low mice that exhibited moderate to severe pulmonary fibrosis compared to WT controls was associated with abnormal MK suggesting that factors released by Gata1low MK in addition to CXCL1/LPN2 are involved in establishing lung fibrosis in this model. Further studies will clarify whether these factors are similar to those responsible for fibrosis in bone marrow. In conclusion, the significantly higher number of Gata1low mice that exhibited moderate to severe pulmonary fibrosis compared to WT controls establishes Gata1low mice as a potential new animal model to study human idiopathic pulmonary fibrosis and to identify possible drug targets to treat this disease. Disclosures No relevant conflicts of interest to declare.

Chronic idiopathic myelofibrosis (IM) is a chronic myeloproliferative disorder characterized by splenomegaly, a leukoerythroblastic blood picture, teardrop poikilocytosis, marrow fibrosis, osteosclerosis, marrow neo-vascularization, abnormal stem/progenitor cell trafficking and extramedullary hematopoiesis. The disease may eventually evolve into acute leukemia. This Philadelphia chromosome negative disorder is thought to originate from a somatic mutation at the level of the multipotent hematopoietic stem cell, the most visible consequence of which is a profound hyperplasia associated with increased proliferation but abnormal differentiation of the megakaryocytes (MKs). The pathobiology of the disease, however, involves not only abnormal hematopoietic stem/progenitor cells functions, but also a defective marrow microenvironment. The molecular nature of the genetic defect in IM and how this defect might induce so many pleiotropic consequences remains unknown. Many of the features of the human disease can be reproduced in mice by genetic alterations that induce MK abnormalities similar to those found in patients. Unfortunately, none of the mutations causing the disease in mice has been detected in the human disease. These animal models, however, allow one to dissect the patho-biological pathway that establishes the complex features of IM. Furthermore, these models also shed light on the cross-talk between stem/progenitor cells and microenvironment in normal hematopoiesis. Keywords: Idiopathic myelofibrosis, animal models, GATA-1, thrombopoietin, megakaryocytes

Abstract Real-time, in vivo imaging provides an essential window into the spatiotemporal cellular and molecular events contributing to tissue development and pathology. By coupling longitudinal intravital imaging with genetic lineage tracing, here we captured the earliest cellular events underlying the impact of active Wnt/β-catenin signaling on the organization and differentiation of the mammary epithelium. This enabled us to interrogate how Wnt/β-catenin regulates the dynamics of distinct subpopulations of mammary epithelial cells in vivo and in real time. We show that β-catenin stabilization, when targeted to either of the mammary luminal or basal epithelial lineages, invariably leads to cellular rearrangements that precipitate the formation of hyperplastic lesions that undergo squamous transdifferentiation. These results enhance our understanding of the earliest stages of hyperplastic lesion formation in vivo , and reveal that in mammary neoplastic development, β-catenin activation dictates a hair-follicle/epidermal differentiation program independently of the targeted cell of origin.

Abstract A mayor pathobiological role for interleukin 8 in the etiology of myelofibrosis has been suggested by observations indicating that megakaryocytes expanded in culture from these patients express great levels of interleukin 8 1 and that the plasma levels of this cytokine are predictive of poor prognosis 2. In preliminary experiments we demonstrated that the megakaryocytes from the bone marrow of the Gata1 low model of myelofibrosis express not only high levels of TGF-β, but also levels greater than normal of lipokalin-2, a known inducer of IL-8 production, and of CXCL1, the murine equivalent of IL-8. In addition, these megakaryocytes express also high levels of the CXCL1 receptors CXCLR1 and CXCR2 and the bone marrow from these mice express an CXCR1/CXCR2 activated signature. Using these data as a foundation, we tested here the effects of treatment of Gata1 low mice with the CXCR1/R2 inhibitor reparixin on the myelofibrosis phenotype expressed by this models. To these aim, Gata1 low mice (8-month old) were treated either with vehicle (3 males and 3 females) or with reparixin (formerly referred to as repertaxin) 3 (5 males and 5 females) for either 20 or 37 days. The drug was administered by minipumps implanted subcutaneously in the dorsal region set to deliver 7.5mg of drug/hr/Kg of body weight. The mice receiving the drug for 37 days had the minipumps replaced by day 17. The efficiency of drug delivery decreased over time since the plasma levels of reparixin were 13.90±4.18 and 6.71±4.18ug/mL at day 20 and 37, respectively (p&amp;lt;0.05).The drug was well tolerated with no death or change in body weight recorded over the period of observation. Since the results observed in males and females were similar, the data were pooled for statistical analyses. The treatment did not affect blood values (hematocrit (%): 34.32±3.87 vs 35.63±3.45 and 30.92±3.58, platelets: (x10 3/uL) 187.80±26.12 vs 181.30±53.30 and 99.83±71.92 and white cell counts (x10 3/uL): 2.78±0.55 vs 3.27±0.72 and 3.57±1.43, respectively, in vehicle and day 20- or day 37-reparixin treated mice). The treatment had also little effects on bone marrow (20.55±5.83 vs 22.24±0.85 and 21.68±6.49) and on spleen 141.40±29.04 vs 99.54±15.55 and 173.00±76.54) cellularity. However, the bones were reddish and their sections contained great numbers of erythroid cells, a sign of increased hematopoiesis. Great reductions in the fibrosis of the bone marrow and spleen was observed in mice that had been treated with reparixin compared to vehicle which were statistically significant by day 20 (day 20 bone marrow fibrosis 28.09±15.69 in vehicle and 4.54±0.45 in reparixin treated mice by Gomori, p&amp;lt;0.05; 19.30±7.86 vs 3.19±1.89 by reticulin, staining, p&amp;lt;0.05, respectively by Anova; day 20 spleen fibrosis 20.51±5.25 in vehicle and 10.85±3.82 in reparixin treated mice by Gomori, p&amp;lt;0.05; and 13.15±3.06 vs 6.13±2.34 by reticulin, staining, p&amp;lt;0.05, respectively). Of note when the levels of Gomori and reticulin fibrosis detected at day 20 and 37 in individual mice were inversely correlated with the plasma levels of reparixin observed in the same mice (Figure 1, p&amp;lt;0.01-0.05 by Pearson). Mechanistic insights on these results were provided by Immunostaining of marrow and spleen sections of vehicle and reparixin-treated mice indicating that the megakaryocytes from the reparixin-treated group express levels of TGF-β significantly lower than those expressed by the corresponding cells from vehicle while the levels of LCN-2, CXCL1, CXCR1 and CXCR2 expressed by the reparixin treated megakaryocytes are similar to that of the vehicle treated cells. These results indicate that inhibition of CXCL1 by reparixin, probably by reducing the abnormally high TGF-β content of the megakaryocytes, reduces fibrosis in Gata1 low mice and provide a preclinical rational to test this drug in patients with myelofibrosis. References: 1) Emadi S et al. Blood. 2005;105:464; 2) Tefferi et al, J Clin Oncol. 2011;29:1356; 3) Bertini R et al, PNAS 2004; 101:11791 Figure 1 Figure 1. Disclosures Crispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy. Massucci: Dompe Farmaceutici Spa R&amp;D: Current Employment. Brandolini: Dompe farmaceutici Spa R&amp;D: Current Employment. Giorgio: Dompe farmaceutici Spa R&amp;D: Current Employment. Allegretti: Dompe farmaceutici Spa R&amp;D: Current Employment. Migliaccio: Dompe farmaceutici Spa R&amp;D: Other: received funding for reserach .

Calreticulin (CALR) is a multifunctional protein normally found within the lumen of the endoplasmic reticulum that mediates the cellular response to Ca2+ by chaperoning other proteins to their acting sites. Somatic loss-of-function mutations in the CALR gene were recently discovered in 70% of patients with the Philadelphia-negative myeloproliferative neoplasm (MPN) primary myelofibrosis (PMF) who did not harbor gain-of-function mutations of JAK21,2. Nevertheless, the JAK2 pathway is constitutively activated also in patients carrying CALR mutation and treatments with JAK2 inhibitors are effective not only in MPN patients (PMF and polycythemia vera, PV) harboring JAK2 mutations but also in PMF patients harboring mutations in CALR3. We have previously reported that erythroid cells from PV and PMF patients express abnormal activity of the glucocorticoid receptor (GR), a nuclear receptor whose transcriptional activity plays an important role in the regulation of stress erythropoiesis4,5. Since GR is one of the numerous proteins regulated by CALR6, we hypothesized that in human erythroid cells CALR regulates GR functions and that this regulation is disrupted both by CALR and JAK2 mutations in MPN. In this study we tested this hypothesis by determining whether GR and CALR are associated in normal erythroid cells and whether this association is impaired in those from MPN patients. First, biochemical studies determined that human erythroblasts (Erys) expanded ex-vivo from normal stem cell sources [cord blood (CB) and adult blood (AB)] and from MPN patients contain similar levels of CALR and GR. Analyses of cell fractions indicated that in normal Erys, CALR was constitutively localized in the cytoplasm while GR was detected either in the cytoplasm or in the nucleus, depending on the growth factor (the glucocorticoid receptor agonist dexamethasone, erythropoietin or stem cell factor) to which they had been exposed. Second, robust levels of CALR and GR expression were also detected by confocal microscopy. In addition, this analyses revealed that in Erys expanded from normal sources CALR and GR are co-localized in the cytoplasm and that the cytoplasmic association between the two proteins is increased by growth factor deprivation and further enhanced by stimulation with growth factors that activate the JAK2/STAT5 signaling (dexamethasone and/or erythropoietin) while it is inhibited by stimulation with factors that do not use this pathway (stem cell factor). By contrast, in Erys expanded from MPN carrying either CALR or JAK2 mutations, CALR and GR are not associated and remain not associated when the cells are exposed to dexamethasone or erythropoietin. However, in Erys from JAK2V17F-positive MPN patients, association between CALR and GR in the cytoplasm is restored by exposing the cells to the JAK2 inhibitor ruxolitinib. These results suggest that CALR/GR association is a downstream event induced by the JAK2/STAT5 pathway and identify for the first time that CALR functions are impaired in erythroid cells from MPN patients carrying JAK2 mutations.

Splenomegaly is a major manifestation of primary myelofibrosis (PMF) contrib- uting to clinical symptoms and hematologic abnormalities. The spleen from PMF patients contains increased numbers of hematopoietic stem cells (HSC) and megakar- yocytes. These megakaryocytes express high levels of P-selectin (P-sel) that, by trig- gering neutrophil emperipolesis, may cause TGF-β release and disease progression. This hypothesis was tested by deleting the P-sel gene in the myelofibrosis mouse model carrying the hypomorphic Gata1 low mutation that induces megakaryocyte abnormalities that recapitulate those observed in PMF. P-sel null Gata1 low mice survived splenectomy and lived three months longer than P-sel WT Gata1 low littermates and did not express fibrosis and osteosclerosis in the marrow or splenomegaly. Furthermore, deletion of P-sel disrupted megakaryocyte/neutrophil interactions in spleen, reduced TGF-β content and corrected the HSC distribution that in Gata1 low  mice, as in PMF patients, is abnormally expanded in spleen. Conversely, pharmacological inhibition of TGF-β reduced P-sel expression in megakaryocytes and corrected HSC distribution. Spleens, but not marrow, of Gata1 low  mice contained numerous cKITpos activated fibro- cytes, probably of dendritic cell origin, whose membrane protrusions interacted with megakaryocytes establishing niches hosting immature cKITpos hematopoietic cells. These activated fibrocytes were not detected in spleens from P-selnullGata1 low or TGF-β-inhibited Gata1 low  littermates and were observed in spleen, but not in marrow, from PMF patients. Therefore in Gata1low mice, and possibly in PMF, abnormal P-sel expres- sion in megakaryocytes may mediate the pathological cell interactions that increase TGF-β content in MK and favor establishment of a microenvironment that supports myelofibrosis-related HSC in spleen.

Abstract A collection of tributes and remembrances from esteemed colleagues, mentees, and friends on the life and work of “the father of hematopoietic cytokines”

Calreticulin (CALR) shuttles among cellular compartments with proteins bound to its N-CALR domain in response to Ca2+ sensed by its C-CALR domain. Through mechanism(s) poorly defined, this shuttling also regulates the stress-response of human erythroid cells (Erys). Western Blot analyses indicated that Erys generated in-vitro from normal sources and JAK2V617F+ or CALR+ Type2 myeloproliferative neoplasms (MPNs) expressed robust levels of CALR but Ca2+ regulated CALR conformation only in normal cells. Normal proerythroblasts (proErys) expressed mostly N-CALR on their cell-surface (by flow cytometry) and C-CALR in their cytoplasm (by confocal microscopy) and expression of both epitopes decreased with maturation. In the cytoplasm, C-CALR was associated with the glucocorticoid receptor (GR), which initiates the stress-response. Ca2+-deprivation decreased detection of both C-CALR and C-CALR/GR association, increased GR nuclear localization and reduced proliferation in response to dexamethasone (Dex). C-CALR/GR association and Dex response were also decreased by inhibitors of nuclear export. By contrast, CALR+ and JAK2V617F+ proErys expressed increased cell-surface levels of N-CALR and barely detectable cytoplasmic levels of C-CALR. Furthermore, in the cytoplasm, C-CALR was not associated with GR which remained localized in the nucleus, making MPN proErys unresponsive to Dex. Ruxolitinib rescued both cytoplasmic detection of C-CALR and C-CALR/GR association and Dex responsiveness in JAK2V617F+ proErys and its effects were antagonized by inhibitors of nuclear export and Ca2+-fluxes. These results suggest that Ca2+ induces conformational changes of CALR that regulates nuclear export of GR in normal human proErys priming them toward the stress-response. By contrast, the CALR response to Ca2+ is similarly impaired in Erys from MPN patients harboring CALR and JAK2 mutations and this responsiveness is restored by JAK inhibitors.

Abstract Calreticulin (CALR) mutations disrupting domains of the protein that regulate its intracellular trafficking were discovered in patients with myeloproliferative neoplasms (MPN) without JAK2 mutations and are associated with JAK2 activation (Klampt et al, NEJM 2013;369:2379; Nangalia et al, NEJM 2013;369:2391). Whether JAK2 mutations are associated with altered CALR functions is unknown. We previously reported that erythroid cells (Erys) generated in-vitro from JAK2+-polycythemia vera (PV) patients do not respond to dexamethasone (Dex) because in these cells the glucocorticoid receptor (GRα) is constitutively retained in the nucleus (Varricchio et al, Blood 2011;118:425). This was explained by the high frequency found in PV patients of the A3669G GR polymorphism that increases expression of GRβ, the isoform responsible for nuclear retention of GRα. The A3669G frequency is also increased in primary myelofibrosis (PMF) and, when associated with JAK2 mutations, predicts poor survival (Poletto et al, Blood 2012;120:3112). Although the relationship between GR and CALR in Erys has not been defined as yet, studies in animal models indicate that in non-erythroid cells CALR regulates GRα nuclear export. This suggested that CALR may favor nuclear export of GRα, antagonizing GRβ, also, in Erys, and that this function may be impaired both by JAK2 and CALR mutations found in MPN. To test this hypothesis, we compared content, localization and association of CALR and GR in Erys expanded in-vitro from normal sources (NS) or from JAK2 + and CALR + MPNs. By Western Blot (WB), NS and MPN Erys contained comparable levels of CALR and GR. By FACS, in NS Erys cell-surface CALR expression increased from the proerythroblast (proEry) to the polychromatic (polyEry) stage [mean fluorescent intensity (MFI)=215±51 vs 460±125, p&lt;0.05]. Conversely, confocal microscopy of permeabilized cells (to remove cell-surface proteins) detected greater levels of CALR in the cytoplasm of proErys (Fig 1) than in that of polyErys (not shown). CALR was never detected in the nuclei of NS Erys. Thus, maturation of normal Erys is associated with cytoplasm to cell surface trafficking of CALR. By contrast, JAK2+ -PV proErys expressed greater cell-surface levels of CALR than NS proErys (MFI=304±69, p&lt;0.05) and barely detectable levels of CALR in the cytoplasm (Fig 1). Cytoplasmic levels of CALR were also barely detectable in JAK2 + and CALR + PMF proErys (not shown). These results indicate that CALR trafficking is altered in JAK2 + and CALR + proErys alike. By WB and confocal microscopy in NS proErys GRα was prevalently localized in the nuclei or cytoplasm, depending on whether cells had been exposed to Dex (which provides nuclear export signals) or erythropoietin/stem cell factor (that by activating Ca2+ signaling presumably stimulates CALR). In the cytoplasm, GR was co-localized with CALR in proximity of the nuclear membrane. Multi-regression analyses of 410 Pro-Erys compared single CALR and total and cytoplasmic GR signals as independent parameters against the merged signals (co-localization) as dependent parameter. This showed that co-localization correlated significantly with cytoplasmic levels of both CALR and GR but that the greatest p values were observed for CALR, suggesting that CALR is the driving force in determining the interaction between the two proteins. By contrast, in MPN proErys GRα was localized prevalently in nuclei and CALR remained barely detectable in the cytoplasm and their localization was unaffected by treatments with Dex or erythropoietin/stem cell factor. Treatments of JAK2+ Erys with ruxolitinib increased total CALR levels (by 30%, p&lt;0.05) and reduced its cell-surface expression (MFI=304±69 vs 220±48, p&lt;0.05), increasing that in the cytoplasm of proErys (Fig 1). Although this treatment did not alter expression of GRβ, which remained high, it restored the presence of GRα in the cytoplasm where it was associated with CALR in the perinuclear region (Fig. 1) making JAK2+ Erys responsive to Dex (MTT incorporation 0.334±0.081 vs 0.224±0.045 with and without Dex, p&lt;0.05), suggesting that impairment of the nuclear export signal provided by CALR cooperates with GRβ in retaining GRα in the nucleus of proErys from MPNs. These results provide the first indication that cellular distribution, and possibly GRα nuclear export functions, of CALR are altered in erythroid cells from MPN patients irrespective of their mutation status. Disclosures No relevant conflicts of interest to declare.

Primary myelofibrosis is the most severe of the Philadelphia-negative myeloproliferative neoplasms and is associated with progressive TGF-β1-dependent scaring of the hematopoietic microenvironment which causes hematopoietic failure in the spleen.Nevertheless, the pathogenetic roleof TGF beta is still unclear because of the modest (2-fold) increases in its plasma levels, both in patients and in animal models. Transmission electron-microscopy (TEM) observations identified that spleen from PMF patients and Gata1low mice contained megakaryocytes with abnormally high levels of TGF-β and collagen fibres embedded in their cytoplasm. Additional immuno-TEM observations of spleen from Gata1low mice revealed the presence of numerous activated fibrocytes establishing with their protrusions a novel cellular interaction, defined as peripolesis, with megakaryocytes. These protrusions infiltrated the megakaryocyte cytoplasm releasing collagen that was eventually detected in its mature polymerized form. Megakaryocytes, engulfed with mature collagen fibres, acquired the morphology of para-apoptotic cells and, in the most advanced cases, were recognized as polylobated heterochromatic nuclei surrounded by collagen fibres strictly associated with TGF-β. These areas contained concentrations of TGF-β-gold particles ~1000-fold greater than normal and numerous myofibroblasts, an indication that TGF-β was bioactive. Loss-of-function studies indicated that peripolesis between megakaryocytes and fibrocytes required both TGF-β, possibly for inducing fibrocyte activation, and P-selectin, possibly for mediating interaction between the two cell types. Loss-of-function of TGF-β and P-selectin also prevented fibrosis. These observations identify that myelofibrosis is associated with pathological increases of TGF-β bioavailability and suggest a novel megakaryocyte-mediated mechanism that may increase TGF-β bioavailability in chronic inflammation.

BACKGROUND: Approximately 50 to 60% of patients with essential thrombocythemia or primary myelofibrosis carry a mutation in the Janus kinase 2 gene (JAK2), and an additional 5 to 10% have activating mutations in the thrombopoietin receptor gene (MPL). So far, no specific molecular marker has been identified in the remaining 30 to 45% of patients.METHODS: We performed whole-exome sequencing to identify somatically acquired mutations in six patients who had primary myelofibrosis without mutations in JAK2 or MPL. Resequencing of CALR, encoding calreticulin, was then performed in cohorts of patients with myeloid neoplasms.RESULTS: Somatic insertions or deletions in exon 9 of CALR were detected in all patients who underwent whole-exome sequencing. Resequencing in 1107 samples from patients with myeloproliferative neoplasms showed that CALR mutations were absent in polycythemia vera. In essential thrombocythemia and primary myelofibrosis, CALR mutations and JAK2 and MPL mutations were mutually exclusive. Among patients with essential thrombocythemia or primary myelofibrosis with nonmutated JAK2 or MPL, CALR mutations were detected in 67% of those with essential thrombocythemia and 88% of those with primary myelofibrosis. A total of 36 types of insertions or deletions were identified that all cause a frameshift to the same alternative reading frame and generate a novel C-terminal peptide in the mutant calreticulin. Overexpression of the most frequent CALR deletion caused cytokine-independent growth in vitro owing to the activation of signal transducer and activator of transcription 5 (STAT5) by means of an unknown mechanism. Patients with mutated CALR had a lower risk of thrombosis and longer overall survival than patients with mutated JAK2.CONCLUSIONS: Most patients with essential thrombocythemia or primary myelofibrosis that was not associated with a JAK2 or MPL alteration carried a somatic mutation in CALR. The clinical course in these patients was more indolent than that in patients with the JAK2 V617F mutation. (Funded by the MPN Research Foundation and Associazione Italiana per la Ricerca sul Cancro.). PMID: 24325356

Ethnographic research is interpretive research in a double sense. One role of ethnographers is to understand the participants’ perspectives: How do they interpret the world? At the same time, ethnographers themselves do interpretive work. These essays address both issues.