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Background Hyperglycemia-induced oxidative stress and inflammation are potent drivers of atherosclerotic cardiovascular disease (ASCVD). Gestational diabetes (GDM) is characterized by chronic hyperglycemia during pregnancy and may represent a clinical model to study the mechanisms of oxidative stress and inflammation induced by hyperglycemia. GDM is associated with a range of adverse perinatal and long-term outcomes for both mother and offspring. In this perspective, it is emerging a putative association between maternal GDM and offspring's epigenetic trait. Purpose To investigate the link between histone modifications, oxidative stress and inflammatory phenotype as well as the transmission of epigenetic signatures to the offspring. Methods We analyzed peripheral blood mononuclear cells (PBMC) from GDM and control mothers as well as human umbilical vein endothelial cells (HUVEC) and cord blood mononuclear cells (CBMC) isolated from newborn umbilical cords obtained at delivery from both groups. Histone methyltransferase MLL1-dependent trimethylation of histone 3 at lysine 4 amino residue (H3K4me3) on NF-kB p65 subunit promoter region was assessed by chromatin immunoprecipitation (ChIP) and real-time qPCR in HUVEC, PBMC and CBMC, respectively. MLL1 and downstream inflammatory and redox genes were determined by real-time qPCR and immunocytochemistry in the presence and in the absence of MLL1 inhibitor MM-102. Measurement of reactive oxygen species (ROS) was performed by electron spin resonance spectroscopy. Results For the first time, we demonstrated a significant increase of MLL1 expression with subsequent MLL1-induced upregulation of NF-kB p65 gene via H3K4me3 in GDM as compared to control cells. MLL1-driven epigenetic remodeling of NF-kB p65 promoter is upstream to the activation of inflammatory pathway. Indeed, treatment with MM-102 decreased H3K4me3 and blunted expression of NF-kB p65 as well as VCAM-1, MCP-1 and IL-6 genes. We also found that expression of ROS scavenger aldehyde dehydrogenase 2 is reduced, whereas pro-oxidant nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit NOX4 is upregulated. Interestingly, the increased ROS generation observed in GDM is involved in the upregulation of MLL1 as shown by the restoring effect of antioxidant vitamin C on MLL1 expression levels. Conclusions Our results suggest that a complex interplay between oxidative stress and histone modifications are responsible for the GDM maternal inflammatory and oxidative phenotypes and its transmission to the offspring. The deciphering of epigenetic-induced chromatin remodelling opens the perspective for pharmacological reprogramming of adverse chromatin changes to reduce the burden of early development of metabolic phenotypes and ASCVD. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University G. d'Annunzio MIUR fundings Schematic figure

In the setting of T cell-depleted, full-haplotype mismatched transplantation, adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) can prevent graft-versus-host disease (GVHD) and improve post-transplantation immunologic reconstitution and is associated with a powerful graft-versus-leukemia effect. To improve the purity and the quantity of the infused Tregs, good manufacturing practices (GMP)-compatible expansion protocols are needed. Here we expanded Tregs using an automated, clinical-grade protocol. Cells were extensively characterized in vitro, and their efficiency was tested in vivo in a mouse model. Tregs were selected by CliniMacs (CD4+CD25+, 94.5 ± 6.3%; FoxP3+, 63.7 ± 11.5%; CD127+, 20 ± 3%; suppressive activity, 60 ± 7%), and an aliquot of 100 × 106 was expanded for 14 days using the CliniMACS Prodigy System, obtaining 684 ± 279 × 106 cells (CD4+CD25+, 99.6 ± 0.2%; FoxP3+, 82 ± 8%; CD127+, 1.1 ± 0.8%; suppressive activity, 75 ± 12%). CD39 and CTLA4 expression levels increased from 22.4 ± 12% to 58.1 ± 13.3% (P 95%). When sorted populations were analyzed, TIM3+ cells showed significant increases in IL-10 and granzyme B (P © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.

Hematopoietic cell transplant (HCT) recipients have a substantial risk of developing secondary solid cancers (SSCs). The aim of this retrospective study was to compare the incidence of SSC in a monocentric cohort of thalassemia major (TM) patients (n=122) who received HCT versus an hematopoietic cell donor monocentric cohort (n=122) and versus a large multicenter cohort of age- and sex-matched TM patients (n=244) who received conventional therapy. With a median follow-up of 24 years, 8 transplanted patients were diagnosed with SSC at a median of 18 years after HCT and at a median age of 33 years. Three patients died of cancer progression and 5 are living after a follow-up ranging from 10 months to 16 years after SSC diagnosis. The 30-year cumulative incidence of developing SSC was 13.24%. The occurrence of solid cancers in the hematopoietic cell donor cohort was limited to only one case for a significantly lower cumulative incidence (3.23%, P=0.02) and to 3 cases in the cohort of nontransplant patients for a significantly lower cumulative incidence (1.32%, P=0.005). This study shows that the magnitude of increased risk of SST is fourfold to sixfold for patients treated with HCT as compared with hematopoietic cell donors and nontransplant patients.

In high-risk acute leukemia patients undergoing HLA haploidentical T cell-depleted tranplantation, we demonstrated that adoptive immunotherapy with donor T regulatory cells (Tregs; 2x106/kg) co-infused with conventional T cells (Tcon; 1x106/kg ) provided significant protection from acute graft-versus-host disease (aGvHD) and was associated with an almost complete control of leukemia relapse (graft versus leukemia effect, GvL) (Di Ianni et al., Blood 2011; Martelli et al., Blood 2014; Ruggeri et al., ASH 2018). In the present study we investigated whether Tregs interact with bone marrow (BM) and peripheral blood (PB) dendritic cells (DCs) and whether such interaction is responsible for GvHD protection and GvL effect. Twenty six patients (median age 54 ; 20 AML; 4 ALL; 2 MDS) transplanted between July 2016 and April 2019 were evaluated up to one year after the transplant. BM and PB DCs (using CD123 for plasmocitoid DC-pDC; CD11c for myeloid DC-mDC; CD80/CD86 for costimulatory molecules) and T cells (CD3/CD4/CD8; CD4/CD25/CD127; CD28/PD-1/TIM3) were analysed by flow-cytometry. DCs were also sorted and analysed by RT-PCR for a panel of genes involved in activation (IL-6; TNF-a; IL-12; CCR7; NOTCH ligands) vs tolerigenic (TGF-beta; PD-1/PDL1; IDO; IL-10; ICOS) pathways. To study the effects of DCs on T cell proliferation, pre-activated (with GM-CSF at 50 ng/ml, IL-4 at 800 U/ml and TNF-a at 50 ng/ml for 18 hrs) BM and PB CD1c+ DCs were co-cultured for 96 hrs with autologous CFSE labelled BM and PB CD3+ cells at a DC:CD3 ratio of 1:10. mDC numbers were significantly higher in BM than PB during the first 6 months after transplant. BM-derived mDCs expressed higher levels of the co-stimulatory receptor CD86. No differences emerged in pDCs. RT-PCR showed an activation signature in BM-DCs (significantly higher IL-6 level) and a tolerigenic signature in PB-DCs (significantly higher TGF-beta and PDL-1 levels). BM-derived CD8+ T cells displayed a higher expression of the co-stimulatory receptor CD28 than PB-derived CD8+ T cells (30.3±18.8 vs 9.2±4.9; p Disclosures No relevant conflicts of interest to declare.

Arsenic Trioxide (ATO) is widely acknowledged as the treatment of choice for Acute Promyelocytic Leukemia (APL). It is a “two-sided” drug since it can induce differentiation or kill APL and other tumor cells according to the dosage. Part of the cytotoxic effects of ATO on APL cells is due to its pro-oxidant activity, a characteristic which ATO shares with a number of other compounds, including high doses of ascorbate (ASC). In a comparative investigation on the cytotoxic effects of both ATO and ASC on HL60 (APL) cell lines, in Vitro, we have been able to confirm the known cytotoxic effects of ATO, but, more importantly, we have demonstrated that ASC is significantly more effective than ATO, in killing these cancer cells in Vitro, when the concentrations are maintained within the millimolar (mM) range, i.e. the range of plasma concentrations at which ASC induces oxidative damage to tumor cells. Since these plasma levels can be reached only by the intravenous administration of high doses of ASC, we propose that intravenous high doses of ASC may represent a potentially revolutionary new approach in the management of APL.

The final quality control of cryopreserved progenitor cells is a successful and persistent three lineage engraftment after transplantation. Of course, the stem cell providing institution is obliged to have a program for controlling and monitoring the manufacturing of cellular therapy products before the patients’ conditioning therapy is started. The FACT-JACIE Standards [1] and the Netcord ⁄ FACT Stan- dards prescribe that the director of the institute shall define tests and procedures for measuring and assaying cellular therapy products to ensure their safety, viability and integ- rity and shall also ensure that products meet predetermined release specifications. This requires specifications of assays and the definition of thresholds to allow release.

PKCalpha 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 gamma/gamma + beta globin expression ratio represented the major difference between neonatal and adult erythroblasts (58 +/- 12 vs. 7 +/- 3, respectively), we tested the hypothesis that PKCalpha might affect gamma-globin expression by measuring the levels of (A)gamma- or beta-promoter-driven reporter activity in erythroid cells stably (GM979) or transiently (K562, primary adult and neonatal erythroblasts) transfected with a dual microLCRbetaprRluc(A)gammaprFluc reporter in the presence of transient expression of either the constitutively active (sPKCalpha) or catalytically inactive (iPKCalpha) PKCalpha. As further control, GM979 cells were incubated with the PKC inhibitor rottlerin (30 microM). In all the cells analyzed, sPKCalpha significantly increased (by two- to sixfold) the levels of luciferase activity driven by the (A)gamma-promoter and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. In GM979 cells, rottlerin inhibited (by 50%) the (A)gamma-driven luciferase activity and the (A)gamma-F/((A)gamma-F + 2beta-R) expression ratio. These results suggest that different PKC isoforms may exert ontogenetic-specific functions in erythropoiesis and that modulation of PKCalpha might affect the activity of (A)gamma-promoter-driven reporters.

The present invention relates to a method of ex vivo amplification of neonatal T cells from umbilical cord blood which comprises obtaining light density mononuclear cells from a sample of umbilical cord blood and then culturing said light density mononuclear cells in a serum-deprived culture medium supplemented with various cytokine combinations. Particularly, there are reported the effects exerted by cytokine combinations including stem cell factor (SCF), interleukin-7, interleukin-4 and interleukin-2, on the amplification of T cells from cord blood mononuclear cells cultured for 10-11 days under serum-deprived conditions. Of all the combinations investigated, SCF plus interleukin-7 sustained the best fold increase (FI) of total nucleated cells (FI=6.4±1.17), amplifying preferentially CD4 + over CD8 + T cell subsets (FI=4.72±0.79 vs 2.73±1.2, respectively, p

A trial was conducted to investigate whether the sequential administration of recombinant human granulocyte colony-stimulating factor (G-CSF) and recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) could accelerate reconstitution of hematopoiesis, compared with G-CSF alone following high-dose chemotherapy (HDCT). A group of 34 consecutive patients with solid tumors undergoing HDCT and autologous peripheral blood progenitor cell (PBPC) transplantation was studied. Conditioning regimen included carboplatin, etoposide, mitoxantrone, and melphalan for breast cancer and cyclophosphamide or ifosfamide, carboplatin, and etoposide for the other tumors. HDCT was delivered from day -3 to day -1. PBPC were infused on day 0, and on the same day growth factors were administered subcutaneously (s.c.) 5 microg/kg each. Seventeen patients were randomized to receive G-CSF from day 0 to day 13 after HDCT (arm A), and 17 patients received G-CSF from day 0 to day 6 and GM-CSF from day 7 to day 13 (arm B). Patients were stratified, and their characteristics were homogeneous in both arms for age, performance status, and number of previous chemotherapy courses and CD34+ infused. The median time to absolute neutrophil count (ANC) >500/microl was 10 days in arm A and 9 days in arm B (p = 0.96). Days to platelet (PLT) count >20,000 were not different in the two treatment arms (p = 0.1), but patients randomized to arm A had a lower platelet count compared with patients in arm B. One month after PBPC transplantation, a statistically significant difference in PLT count was observed (arm A median 150x10(3)/microl (90-310), arm B median 254x10(3)/microl (117-387),p = 0.0013). The days patients had fever >38 degrees C were 39 in arm A and 26 in arm B (p = 0.18). The difference in the length of hospital stay was not statistically significant between the groups (Mann-Whitney sum rank test). After a median follow-up of 30 months, 21 patients were alive and 20 were disease free. These data show that the two growth factors are associated with different patterns of hematopoietic recovery, and larger randomized trials in groups of more homogeneous patients will be needed to define the effects and benefits of combination growth factor therapies

The efficiency and quality of platelet (PLT) collection were evaluated in a preliminary study using a new cell separator: Baxter-Amicus. The new fully automated blood cell separator combines centrifugation with elutration to obtain higher PLT efficiency with a lower white blood cell (WBC) contamination. We compared procedures performed with the first software version 2.13 and the more recent 2.37 version, then with and without plasma collection. Data from 262 plateletapheresis procedures were analyzed. The mean value of the PLT yield was 4.5±1.0x1011, collection efficiency: 69.4±12%; WBC contamination: 0.8±2.x106; and procedure time: 73± 19 minutes. The use of the new software vs the former permitted the collection of a higher number of platelets: 4.9± 1.1 vs. 4.5± 1.9 x1011 (=ns), with a lower WBC contamination: 0.6± 1.0 vs 0.7± 1.2 x106 (p=ns), in less time: 63± 10 vs 73± 19 minutes (p=0.002). The efficiency of platelet harvesting with simultaneous plasma collection was higher than the standard procedure: 73± 13 vs. 67± 10% (p=0.003).

Over the past few years, hemopoietic transplant has evolved from an investigational phase to routine therapy, thus becoming a potentially curative strategy for a large variety of diseases. Several transplant situations are still outstanding and the need for ex vivo graft manipulation for different transplantation products is growing. To obtain an ideal graft, many different methods, even sophisticated manipulations, may be required. Since transplantation products play an important role in disease outcome, the assessment of graft quality to ensure standard compliance is needed. The development of a regulatory approach to these new manipulated hematopoietic products is very complex and should come under current Good Manufacturing Practices (cGMPs). Manufacturing approach to these new blood products must be urgently introduced to accounting Quality System in Transfusion Medicine. The best way to develop compliance with standards, in agreement with internationally accepted criteria, is, likely, an accreditation system in transplantation programs.

Candida inconspicua was recovered from three patients with hematological malignancies. Two patients had intravenous-catheter-associated fungemia, whereas the third had fungal hepatitis. The three cases of infection occurred over a period of 1 month in patients staying in adjacent single rooms. In vitro susceptibility testing of fungal strains showed all isolates to be resistant to fluconazole, with MICs greater than 32 μg/ml. All of the strains had identical DNA restriction profiles and randomly amplified polymorphic DNA fingerprints. These data suggest a nosocomially acquired infection emanating from a common source within the hospital environment.

Enterococcus hirae is rarely collected from man, while it is a common pathogen in mammals and birds. We describe the first isolation of the organism (strain DSM 27815) from human umbilical cord blood (UCB), thus emphasizing the risk of contamination of UCB units for clinical use. In this context, we also highlight the importance of an extensive training of the collecting personnel as to the observance of the disinfection protocol ensuring UCB units sterility.

A "quasi-experimental" trial was carried out to investigate the effect of three antimicrobial regimens on oral and fecal yeast colonization in patients with hematologic malignancies. Fifty-four patients received ciprofloxacin and oral amphotericin B (group 1); 45 received ceftazidime, amikacin, vancomycin, and oral amphotericin B (group 2); and 30 received ceftazidime, amikacin, vancomycin, and intravenous amphotericin B (group 3). The oral yeast isolation rate showed a decrease in group 1 (from 59.3% to 40.7%) and group 3 (from 56.7% to 46.7%), and a marked increase in group 2 (from 51.1% to 84. 4%). All the groups showed a reduction in their fecal yeast isolation rate. An overgrowth of Candida parapsilosis, C. krusei, and C. tropicalis was observed in all the groups, but it was much higher in group 2. Our findings provide evidence that ceftazidime, amikacin, and vancomycin, given with oral amphotericin B, induce an overgrowth/persistence of Candida species in the mouth and gut, which might be attributable to inclusion of vancomycin. Treatment with intravenous amphotericin B has at least the capacity of counterbalancing yeast proliferation induced by that antibacterial regimen.

Recovery phase infection patterns in 55 patients who had undergone autologous blood progenitor cell transplantation (ABPCT) were evaluated retrospectively. The results were compared to those obtained in a group of 41 patients who received autologous bone marrow transplantation (ABMT). Fever related to documented or suspected infection developed in 38 of 55 patients in the ABPCT group and in 37 of 41 in the ABMT group (p 0.05). However, fewer acquired systemic fungal infections (1/55 vs. 5/41, p < 0.05) as well as fewer days of antibiotic usage were observed in the ABPCT group.

Attempts to identify an early and discriminating marker of acute graft-versus-host disease (aGvHD) have been unsuccessful. The levels of soluble CD4 and soluble CD8 in serum correlate with T cell subset activation and may be important in monitoring and characterizing immunological processes. We determined serum soluble CD4 (sCD4) and sCD8 levels with a two-site sandwich enzyme immunoassay on patients' serum samples collected prior to bone marrow transplantation and weekly after transplantation until day +28. No significant increment of sCD4 was documented in each determination. sCD8 rose significantly before diagnosis or development of maximal clinical symptoms in patients with grade II-III aGvHD than grade 0-I aGvHD [at day +21--median value 447 IU/ml; range 94-713; versus 1136 IU/ml, range 790-1416 (P = 0.002); at day +28--median value 443 IU/ml, range 73-992, versus 1164 IU/ml, range 625-1960 (P = 0.005)]. On the day of marrow infusion the sCD8 levels were significantly higher in patients who subsequently developed grade II-III than in patients with grade 0-I aGvHD (median value 155 IU/ml, range 10-332, versus 350 IU/ml, range 283-830; P = 0.003). Careful monitoring of sCD8 is a useful tool for a prompt aGvHD diagnosis and may be used in a clinical bone marrow transplantation setting.

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Preliminary BM processing to produce an enriched MNC fraction from large BM volumes improves subsequent pharmacological and/or immunological “ex vivo” treatment and cryopreservation. We detail on a multicenter study (6 Transplant Centers) performed to establish an effective and reliable protocol using a CS 3000 continuous flow separator on a large series of BM processed for autologous (96) and allogeneic (12) transplantation. The reduction in volume was 78.6+7.2% while 28.9+12.4% of the original nucleated cells were found in the final product. A mean of 84.3+13.2% of the starting MNC was yielded in a fraction containing over 81% MNC. Cloning efficiency indicated than the final graft was highly enriched in progenitor cells committed to the granulocyte/macrophage pathway (> 100%) as assessed in vitro (CFU-GM). Removal of RBC and PLT was 98.3+1.1 and 37.7+14.6%, respectively. The mean dose of MNC and CFU-GM was 0.6+0.37 x 108 and 0.96+1 x 108 recipient weight. The entire process was accomplished in 87.5+20 min. We concluded that this automated device is a simple and reproducible method for BM processing suitable as first step for further “ex vivo” automated negative and/or positive cell selections.

Background and Objectives The Dideco ‘Pluricell System’ is a CE-marked medical device allowing haematopoietic stem cell (HSC) expansion. It comprises a kit of cGMP cytokines and reagents, a closed-cell expansion chamber and a cell-washing set. We tested the system in a multicentric study by expanding CD34+ cells from eight fresh umbilical cord blood (UCB) samples. Materials and Methods During culture, the mean nucleated cell (NC) count, the mean CD34+ cell count, fold expansion, viability and apoptosis were measured. Clonogenic assays and immunophenotypical characterization were performed on days 0, 7 and 12. On the expanded cellular product, in three cases cell genotyping, endotoxin level and mycoplasma detection (by polymerase chain reaction) were performed. Results The mean CD34+ cell expansion on days 7 and 12 was sevenfold and 12-fold respectively and the mean NC expansion was 69-fold and 180-fold. The mean NC viability on day 12 was 96·9% (94·4–99·1). After 12 days, granulocyte–macrophage colony-forming units (GM-CFU) showed a 20-fold increase: a slight increase in CD34+ cell apoptosis was observed during culture. In all of three cases neither chromosomal alterations nor mycoplasma contamination was detected. No significant endotoxin levels were detected after expansion. Conclusions The device allows the ex vivo expansion of NC and CD34+ cells in a closed system. The expanded cellular product is a mixture of progenitors (CD34+ cells) and differentiated (mainly myeloid and megakaryocytic) cells. To reduce cell apoptosis, more frequent cell feeding during culture should be tested.

Recovery phase infection patterns in 55 patients who had undergone autologous blood progenitor cell transplantation (ABPCT) were evaluated retrospectively. The results were compared to those obtained in a group of 41 patients who received autologous bone marrow transplantation (ABMT). Fever related to documented or suspected infection developed in 38 of 55 patients in the ABPCT group and in 37 of 41 in the ABMT group (p0.05). The percentages of patients with positive blood cultures did not differ significantly (ABPCT, 8/55 vs. ABMT, 8/41, p0.05). However, fewer acquired systemic fungal infections (1/55 vs. 5/41, p0.05) as well as fewer days of antibiotic usage were observed in the ABPCT group.

Preliminary BM processing to produce an enriched MNC fraction from large BM volumes improves subsequent pharmacological and/or immunological "ex vivo" treatment and cryopreservation. We detail on a multicenter study (6 Transplant Centers) performed to establish an effective and reliable protocol using a CS 3000 continuous flow separator on a large series of BM processed for autologous (96) and allogeneic (12) transplantation. The reduction in volume was 78.6 + 7.2% while 28.9 + 12.4% of the original nucleated cells were found in the final product. A mean of 84.3 + 13.2% of the staring MNC was yielded in a fraction containing over 81% MNC. Cloning efficiency indicated than the final graft was highly enriched in progenitor cells committed to the granulocyte/macrophage pathway (100%) as assessed in vitro (CFU-GM). Removal of RBC and PLT was 98.3 + 1.1 and 37.7 + 14.6%, respectively. The mean dose of MNC and CFU-GM was 0.6 + 0.37 x 10(8) and 0.96 + 1 x 10(5) recipient weight. The entire process was accomplished in 87.5 + 20 min. We concluded that this automated device is a simple and reproducible method for BM processing suitable as first step for further "ex vivo" automated negative and/or positive cell selections.

Aim of our study was to evaluate the intraprocedure dynamic variables during PBSC collection in G-CSF mobilized healthy donors. Eleven donors aged 30 to 53 years were mobilized with Lenograstim 5 μg/kg twice and underwent to a median of 2 LKs/donor (range 1–4) starting from day 4. A total of 21 collections lasting 5.3 hours (4.3–6) were performed with Amicus-Baxter cell separator. Peripheral blood (PB) and separate collection bag samples were taken for each body volume processed (BVP) to analyze intraprocedure dynamic changes and CD34+ cell release. A median of 62% (37–95) of initial CD34/ml values were found at end of procedures. CD34+ yields were 163% (72–309) of initial circulating values. Amount of CD34+ cell release during apheresis was 0.89 × 10 6 (0.19–3.15) per minute of procedure and 87.09 × 10 6 (17.05–292.49) per BVP. Intraprocedure data are shown in table. In an univariate analysis BVP, baseline and precount CD34/ml values showed a significant correlation with intraprocedure CD34+ cell release ( p = 0.002; 0.03 and 0.007 , respectively). This study suggests that continous CD34+ cell release occurs from the bone marrow during LK up to 6 hours of procedure.

PKCs are serine-threonine kinases that play an important role in many cellular functions. Almost all the 12 PKC isoforms described so far are expressed throughout erythroid differentiation of human CD34pos cells. Besides the downmodulation of PKCε expression (Bassini et al, Blood93; 1178, 1999), little is known on the role exerted by each PKC on erythroid differentiation. PKCα and PKCδ are two isoforms expressed at comparable levels (as mRNA and protein) throughout differentiation of CD36highCD235alow pro-erythroblasts into orthochromatic erythroblasts. For both proteins, the ratio between phosphorylated and total form remains constant during the differentiation of adult cells. In contrast, the ratio P-PKCα/total PKCα (but not that of P-PKCδ/total PKCδ) increases by 3-fold (with slight donor-to-donor variability) during the differentiation of CD36highCD235alow cells from cord blood. Furthermore, the protein becomes prominently localized in the nucleus of these cells. Since the most stricking difference in the differentiation of adult vs. neonatal erythroblasts is the γ/γ+β globin expression ratio (0.02–0.08 vs. 0.20–0.40 by Taqman, respectively), we hypothesized that the levels of PKCα activity might affect γ-globin expression in erythroblasts. To test this hypothesis, we used two in vitro models of HbF switching. The first model is represented by GM979 cells stably transfected with a dual luciferase (Renilla, R - Firefly, F) reporter driven by either the human γ- or β-globin promoter. The second model is represented by human erythroblasts obtained in HEMA culture. GM979 cells were transiently transfected with expression constructs encoding either the catalytic subunit (sPKCα) or the catalytic inactive (iPKCα) PKCα. The levels of expression of the γ-driven and β-driven luciferase reporters were then measured 24 hrs after transfection. Alternatively, GM979 cells were incubated with concentrations of rottlerin (30 μM) that specifically inhibit PKCα activity. sPKCα increased by 2–3-fold the expression of the luciferase driven by the γ-promoter but did not affect expression from the β promoter. Therefore, the ratio Aγ-F/(Aγ-F+2β-R) was also increased by 2–3 fold. Conversely, rottlerin inhibited (by 50%) both expression of the γ-driven luciferase and the Aγ-F/(Aγ-F+2β-R) ratio. On the other hand, transfection of the double luciferase reporter gene into adult and neonatal CD36highCD235alow cells (30–50% transfection efficiency) resulted in high levels of expression of both reporters, in a ratio consistent with the ontogenic stage of the cells [high Aγ-F/(Aγ-F+2β-R) in neonatal erythroblasts; low Aγ-F/(Aγ-F+2β-R) in adult erythroblasts]. Co-trasfection of sPKCα with this luciferase reporter in CD36highCD235alow cells (both adult and neonatal) increased by 5–6-fold the expression of the luciferase driven from the γ-promoter. Co-trasfection of iPKCα, sPKCδ and iPKCδ had no effect on the expression of the reporters in these cells. In conclusion, using several models of erythroid differentiation, we observed that increased levels of PKCα activity results in increased activity of the γ-promoter, suggesting that element(s) of the haemoglobin switching machinery may represent a specific PKCα substrate in erythroid cells.