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We previously demonstrated that natural product‐inspired 3,4‐dihydropyrrolo[1,2‐a]pyrazin‐1(2H)‐ones derivatives delivered potent and selective PIM kinases inhibitors however with non‐optimal ADME/PK properties and modest oral bioavailability. Herein, we describe a structure‐based scaffold decoration and a stereoselective approach to this chemical class. The synthesis, structure–activity relationship studies, chiral analysis, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Compound 20c demonstrated excellent potency on PIM1 and PIM2 with exquisite kinases selectivity and PK properties that efficiently and dose‐dependently promoted c‐Myc degradation and appear to be promising lead compounds for further development. [ABSTRACT FROM AUTHOR]

The availability of genomic datasets in association with clinical, phenotypic, and drug sensitivity information represents an invaluable source for potential therapeutic applications, supporting the identification of new drug sensitivity biomarkers and pharmacological targets. Drug discovery and precision oncology can largely benefit from the integration of treatment molecular discriminants obtained from cell line models and clinical tumor samples; however this task demands comprehensive analysis approaches for the discovery of underlying data connections. Here we introduce PATRI (Platform for the Analysis of TRanslational Integrated data), a standalone tool accessible through a user-friendly graphical interface, conceived for the identification of treatment sensitivity biomarkers from user-provided genomics data, associated with information on sample characteristics. PATRI streamlines a translational analysis workflow: first, baseline genomics signatures are statistically identified, differentiating treatment sensitive from resistant preclinical models; then, these signatures are used for the prediction of treatment sensitivity in clinical samples, via random forest categorization of clinical genomics datasets and statistical evaluation of the relative phenotypic features. The same workflow can also be applied across distinct clinical datasets. The ease of use of the PATRI tool is illustrated with validation analysis examples, performed with sensitivity data for drug treatments with known molecular discriminants. [ABSTRACT FROM AUTHOR]

Background In our Center, the cell viability, the integrity of the bag, and the clonogenic assay were evaluated before the reinfusion of hematopoietic progenitor cells-apheresis ( HPC-A). This quality control (QC) should be made 14 days before the reinfusion to the patient to have the result of the functional test on the proliferative capacity of hematopoietic progenitors. Study Design and Methods This study was designed to assess the potential of an automatic cell counting system ( Nucleo Counter NC-3000, Chemo Metec) in our clinical routine as a support of the clonogenic assay and the cytofluorimetric analysis for the QC of the cryopreserved HPC-A. The cell viability was evaluated by flow cytometry using the modified International Society of Hematotherapy and Graft Engineering protocol. The proliferative potential was assessed by specific clonogenic tests using a commercial medium. Furthermore, we evaluated the cellular functionality with Nucleo Counter NC-3000, by using two protocols: 'vitality assay' and 'mitochondrial potential assay.' Results The evaluation of the total nucleated cells in preapoptosis measured by 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazol-carbocyanine iodide ( JC-1) assay showed a negative correlation (r = −0.43) with the total number of colonies (colony-forming unit [CFU]-granulocyte-macrophage progenitors plus burst-forming unit-erythroid progenitors plus CFU-granulocyte, erythroid, macrophage, megakaryocyte progenitors) obtained after seeding of 50 × 106/ L viable total nucleated cells. We observed a significant difference (p < 0.0001) comparing the median number of colonies (166.70; SD, ±136.36) obtained with a value of JC-1 less than 30% to the number of colonies (61.75; SD, ±59.76) obtained with a value of JC-1 more than 30%. Conclusion The evaluation of cell functionality by the use of the Nucleo Counter NC-3000 is in agreement with results from clonogenic assay and can be considered an effective alternative in the routine laboratory. [ABSTRACT FROM AUTHOR]

Background: High-dose chemotherapy followed by reinfusion of autologous stem cells harvested from peripheral blood has been increasingly applied for a variety of disorders. The critical importance of cell dose in the clinical outcome, after transplant, has motivated the need to develop techniques aimed at reducing cell losses and increasing reproducibility. Objectives: The aim of this study is to evaluate the efficacy of the Sepax S-100 device to process thawed HPC-A products in comparison with manual procedure. Methods/Materials: We have analysed viability, total nucleated cells (TNC), haematopoietic progenitors and CD34+ cells recovery. Results: The TNC and CD34+ cells recovery in the automatic procedure was of 91·9% (73-100; SD ± 12·60) and 86·7% (69-100; SD ± 10·21), respectively. Instead the recovery of TNC and CD34+ cells using the manual method was of 84·7% (47-100; SD ± 22·9) and 80·29% (23-100; SD ± 25·96). The results, obtained from the assessment of viability of CD34+ both 7-AAD)+ and AnnV+ showed a high percentage of necrosis and apoptosis in this cell subset by using the manual procedure in respect to the Sepax automated system. Conclusion: Overall, our data suggest that the automated washing procedure is safe and suitable for processing of thawed HPC-A products and can be daily used in clinical routine. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented about a 3.5-year-old patient diagnosed with non-Hodgkin lymphoma treated with immunomagnetic selection (ICS) of CD34+ stem cells collected by means of leukaphaeresis (LKF).

Background Despite improvements in hepatitis B surface antigen (HBsAg) test sensitivity, post-transfusion hepatitis B virus (HBV) infection still occurs because HBsAg is undetectable during the early window phase (WP) of the infection, in the convalescence core window phase of the infection, or in serologically silent chronic hepatitis or in mutant forms of HBV. HBV-DNA screening using high sensitivity nucleic amplification technology (NAT) assays has recently been introduced to reduce the residual risk of transmission of HBV by transfusion of blood components. Materials Over 1 year 75 063 donations were individually screened for HBV-DNA by the Ultrio Procleix assay on the Tigris platform. The donations were collected in the Latium region, an area of the central Italy, and they accounted for the 40% of the total blood units collected in this area per year. The initial reactive samples were re-tested and confirmed by the discriminatory HBV assay. Additional HBV serological markers were also performed. Suspected WP infections were followed-up to monitor the development of the immune response. All HBV-DNA-positive donors were called back to check up their infectious status. Results The results of testing the 75 063 donations are: 33 donations HBsAg positive, 31 out of them HBV-DNA-positive and two HBV-DNA negative; 22 donations HBsAg-negative but HBV-DNA positive with low viral load. Six of the 22 were found to be consistently HBV-DNA reactive whereas the remaining 16 donations showed inconsistent results on multiple NAT retesting. One WP infection was confirmed by the follow-up of the donor for 3 months following the index blood donation. Conclusions In the donor population of the Latium region, NAT screening has revealed a higher than expected number of donors who were HBsAg non-reactive but HBV-DNA-positive with three donors showing HBV-DNA as the only marker of infection. The adoption of genome screening has increased the safety of the blood supply and has also contributed to the protection of donor health by identifying either WP or clinically silent infections. [ABSTRACT FROM AUTHOR]

In this study, the optimisation of biopharmaceutical properties of a dried commercial extract of St John's Wort were evaluated employing the in vivo forced swimming test (FST). Three new dosage forms containing β-cyclodextrin and surfactants (SDS, ASC8) were compared in the FST with the commercial extract. The commercial extract showed antidepressant activity in mice after 60 min at a dosage of 100 mg/kg. The same antidepressant activity appeared in 30 min with a micellar solution of SDS containing the same quantity of extract (100 mg/kg), while with micelles of ASC8 the effect appeared at 15 min and with a dosage of 30 mg/kg. In the case of β-cyclodextrin the best results were obtained at 30 min, administering 60 mg/kg of the extract. Finally, the influence of the formulations on the water solubility of the constituents of the extract is reported. The tensides dramatically enhanced solubility, in particular that of the more lipophilic compounds, in the case of β-cyclodextrin this effect was very pronounced for flavonoids and biapigenin, lower for hypericins and practically insignificant for hyperforins. Copyright © 2008 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Examines the functional characterization and mechanism of action of recombinant human kynurenin 3-hydroxylase (rec-K3H). Involvement of NADPH-dependent flavin mono-oxygenase in the tryptophan pathway; Specific activity of the pure recombinant protein; Mechanism of rec-K3H-catalyzed reaction by overall initial rate measurement.

Examines the role of protein tyrosine kinase in ZAP-70 phosphorylation and enzymatic activity. Activation of enzyme depends on the binding; Effects of enzymatic activity to tyrosine phosphorylation producing subtle conformational changes; Discussion on the N-terminal sequence analysis of the two bonds for automated protein sequencer using standard techniques.

Purified human T lymphocytes, completely depleted of accessory cells [i.e. monocytes, large granular lymphocytes (LGL) and B lymphocytes], have been grown in serum-free culture in presence of a mitogenic lectin (phytohaemagglutinin, PHA) and different recombinant cytokines. Only 1L2 and IL-4 induced a marked stimulation of [³H] thymidine ([³TdR) uptake, cell proliferation and expression of activation markers (transferrin receptor (TrfR), IL-2R]. The other cytokines (IL-1α,IL-1β,IFN-γ, GM-CSF, TNF-α) had no significant effect, except for a moderate, but significant, stimulation of [³H]TdR uptake induced by IL-3. Simultaneous addition of IL-4 and anti-IL-2 neutralizing monoclonal antibodies (mAb) did not modify the effects induced by IL-4 alone. Furthermore, IL-2 was not detected in the supernatant of T cells grown in the presence of PHA and IL-4. Thus, our results indicate that IL-4 acts on T lymphocytes independently of IL-2. We also observed that IL-6 moderately activates DNA synthesis in PHA-stimulated T lymphocytes, but markedly potentiates the proliferative effect of suboptimal amounts of IL-2. In conclusion, the present study suggests that B-Cell growth factors, in addition to IL-2, control the proliferation of normal circulating T lymphocytes. [ABSTRACT FROM AUTHOR]

Peripheral blood mononuclear cells (PBM) pulsed with lectin (PHA or Con A for 0.25-3 hr) show a low expression of interleukin-2 and transferrin receptors (IL-2Rs, TfRs) and a mild decline of intracellular ferritin level, compared to control cultures grown in continuous presence of mitogen. Interestingly, lectin-pulsed PBM do not release detectable amounts of IL-2 in the medium. Furthermore, expression of TfRs in these lymphocytes is not inhibited by addition of excess anti- IL-2 neutralizing monoclonal antibody, but is significantly inhibited by treatment with iron salts. These observations suggest that mitogen triggers an IL-2-independent expression of TfRs, at least in part via a decrease of intracellular iron level. Addition of either recombinant IL-2 (rILs2) or an iron chelator (picolinic acid) to lectin-pulsed PBM induces both a marked enhancement of TfR synthesis and a sharp decline of intracellular ferritin level, which are comparable to the corresponding pattern observed in control cultures. Conversely, addition of iron salts fully inhibits the increase of Tilt expression induced by rIL-2. These observations strongly suggest that the enhanced TfR synthesis elicited by rIL-2 is mediated by depletion of a regulatory intracellular iron pool. In line with these studies, >99% purified T lymphocytes stimulated by lectin show a low expression of TfPs, which is markedly enhanced by addition of exogenous rIL-2. Altogether, we postulate that: (i) in resting T lymphocytes the gene encoding TfR is apparently in a 'closed' configuration; (ii) even in the absence of IL-2 activity, a mitogen pulse is sufficient to initiate the expression of TfRs, at lent in part via a decline of intracellular iron level; and (iii) TfR synthesis is then largely amplified by IL-2, again via a decrease of the size of a regulatory intracellular iron pool. [ABSTRACT FROM AUTHOR]

In the present study we have attempted a characterization of the biochemical bases of the interaction of human basic fibroblast growth factor (bFGF) with glycosaminoglycans (GAGs) in solution. This interaction has been evidenced as the capacity of different GAGs and various sulfated compounds to protect bFGF and different bFGF mutants from tryptic cleavage. Heparin protects bFGF from trypsin digestion in a dose-dependent fashion, Substitution by sitedirected mutagenesis of two or more basic residues with neutral glutamine residues in the amino, terminal region bFGF(27-32) or in the carboxyl4erminai region bFGF(118-129) does not significantly affect the protective effect exerted by heparin. In contrast, heparin protection is abolished when the full region bFGF(27-32) is deleted. The capacity of different GAGs to protect bFGF from proteolytic cleavage decreases in the following order: heparin > heparan sulfate > dermatan sulfate = chondroitin sulfates A and C > hyaluronic acid = K5 polysaccharide, indicating that both the degree of sulfation and the backbone structure of GAG modulate its interaction with bFGE This is confirmed by the different capacity of various sulfated compounds (including dextran sulfates, suramin, trypan blue, and sulfate ion) to protect bFGF from tryptic digestion. Moreover, tryptic digestion studies performed with various heparin molecules and dextran sulfates of different size, ranging from 2.0 kDa to 500 kDa, indicate that the number of bFGF molecules which interact with a single molecule of polysaccharide is related to the molecular mass of the GAG and that six hexose residues are sufficient to protect 1 2 molecules bFGE In conclusion, our findings indicate that the capacity of GAGs to protect bFGF from tryptic cleavage depends upon their size, sulfation, distribution of the anionic sites along the chain, and structural requirements of the bFGF molecule, These studies will help to design synthetic oligosaccharides endowed with different bFGF agonist and/or antagonist activities. [ABSTRACT FROM AUTHOR]

The functional features of a recombinant fibroblast growth factor (FGF) receptor (FGF-R) were investigated by expressing at high level in Escherichia coli a soluble non-glycosylated form of FGF-R1. The extracellular domain of the mature protein (XC-FGF-R), comprising the first 356 amino acids, was purified from a large-scale fermentation. After cell lysis, the protein was quantitatively found in the pellet. XC-FGF-R was solubilized using guanidine/HC1 and allowed to refold using two dialysis steps. The refolded protein was obtained in a homogeneous form after ammonium sulphate precipitation and gel-filtration chromatography. The soluble receptor had the ability to form a complex with recombinant human basic FGF (rhbFGF) in solution, as demonstrated by immunoprecipitation with anti-(FGF-R) serum. Formation of a rhbFGF/XC-FGF-R complex was visualized by cross-linking experiments. Quantitative binding experiments with the XC-FGF-R immobilized on Affi-Gel resin showed high binding affinity for 125I-bFGF (Kd = 5–10 nM). Purified XC-FGF-R inhibited binding of 125I-bFGF to its high-affinity receptors on baby hamster kidney cells. These data suggest that glycosylation of the FGF-R is not necessary for its ligand-binding activity. The use of an E. coli expression system resulted in the efficient production of a soluble receptor in a form suitable for ligand/receptor structural studies and screening of new potential agonists and antagonists of angiogenesis. These results indicate that E. coli can be used for the production of complex molecules such as Ig-like receptors. [ABSTRACT FROM AUTHOR]

BACKGROUND: Immunomagnetic selection of peripheral blood progenitor cells (PBPCs) in patients with tumoral infiltration in marrow makes it possible to reduce contamination of cellular concentrates, but this procedure cannot always be used, mainly because of the low cellular count in apheresis concentrates. STUDY DESIGN AND METHODS: In this case two cellular concentrates taken separately at two different times were selected and cryopreserved; they were thawed with an automatic instrument. RESULTS: After manipulation, a selected concentrate containing 24.16 × 106 CD34+ cells with a purity of 90.15 percent was obtained; vitality after thawing and selection was 88 and 96 percent, respectively. The engraftment was achieved on Day +17 from the infusion of the previously selected PBPCs, as the literature also shows us. CONCLUSION: The time passed between the infusion and the engraftment gives us evidence of the efficacy of immunomagnetic selection carried out after thawing 2 cell units that were collected at different times from the same patient. In this way, it has been possible to perform an autologous transplant in a patient in which CD34+ cells transplant is recommended, but from whom the number of collected cells after a single mobilization cycle would not have been sufficient for the engraftment. [ABSTRACT FROM AUTHOR]

Severe combined immunodeficiency (SCID) represents a group of rare, sometimes fatal, congenital disorders in which there is a combined absence of T-lymphocyte and B-lymphocyte function. Children with SCID die within two years of age, if untreated. The effective treatment for SCID is a hematopoietic stem cell transplantation (HSCT). It has been repeatedly described that in peripheral blood of infants with SCID maternal T cells can be found. Here we report a case of blood chimerism in a one-year-old boy with SCID. [ABSTRACT FROM AUTHOR]

Background: Transfusional supporting therapy has steadily increased during the last few years in patients affected by haematologic malignancies and subjected to allogeneic transplantation, specially regarding the paediatric patients. Methods: In our Center we retrospectively studied 8 paediatric patients (median age 7 years - range 1-15) affected by acute myeloid leukemia (2), acute lymphoid leukemia (4), Langherans cells Histiocytosis (1) and Fanconi's anemia (1) and submitted to platelet transfusions from transplantation's day to hematopoietic reconstitution. All patients have been submitted to allogeneic transplantation, 4 using umbilical cord blood, 2 using bone marrow from mismatch unrelated donor and 2 from familiar donor. Results: In our study 2 patients have received exclusively single-donor apheresis platelets and 6 patients a mixture of single donor components and pooled platelet concentrates; all patients were transfused with irradiate and leucodeplete components. The media number Of platelet transfusion received by every patients was 22 (range 5-25); individual response to platelet transfusion was assessed by the evaluation of platelet count before transfusion and after 24 hours and by the calculation of corrected count increment (C.C.I.) after 24 hours only. The platelet number before transfusion was 15.000 mm3 (range 6.000-36.000) while the platelet value after transfusional therapy was 27.000 mm3 (range 8.000-152.000); C.C.I. value was 7.152 mm3 range (6.280-8176) after transfusion (all values are expressed in median), All patients have been submitted to first transfusion one day after stem cells transplantation. We didn't revealed in any patients severe haemorrhagic complications, post-transfusional GVHD and in every case C.C.I. value was appropriate. Conclusion: In our limited experience we report that an accurate transfusional therapy is able to decrease bone marrow and cord blood transplantation haemorrhagic complications, post-transfusional GVHD incidence and platelet refractoriness and offers a better quality of life especially in paediatric patients affected by haematologic malignancies. [ABSTRACT FROM AUTHOR]

BACKGROUND PBPC transplantation has become a widely accepted therapeutic option for patients with chemotherapy-sensitive malignancies. PBPCs facilitate even the transplantation of mismatched allogeneic cells, because of their better engraftment. PBPCs are harvested by leukapheresis after mobilization with G-CSF or chemotherapy and G-CSF. To improve the purity and yield of PBPC grafts, to ensure reproducibility of the procedure and to reduce platelet loss in the patient/donor, different fully automated PBPC collection systems were developed. We report here our experience with the new Amicus blood cell separator. METHODS From July 2000 to March 2001, 46 procedures were performed. Six donors underwent leukaphereses after G-CSF administration (10-12 microgr/Kg/day) beginning on day 4; 14 patients were harvested after chemotherapy and G-CSF or GM-CSF mobilization, when the peripheral CD34+ cell count was >20 cells/micrL. Peripheral venous access was preferred whenever possible, otherwise femoral or subclavian catheters were used. Complete cell counts and CD34+ cell evaluations were done both in patients and donors, before and after each procedure and in all the products of the apheresis. RESULTS A median of 8.959 L of blood (range 3.585-17.021 ) was processed with a duration of 315 minutes (range 215-400) at a blood flow rate of 40 ml/min (range 25-70). ACD was used at a ratio of 1:14. The products of apheresis had a median volume of 143 ml (range 70-306) and a Hct of 12.9% (range 1.6-21). Platelet contamination was low (722 × 10[sup 9]/L, range 55-1,620), according to a low collection efficiency (12%, range 2-34) and a low decrease of platelet level both in donors and patients (21 × 10[sup 9]/L, range 3-83); in contrast, CD34 + cell collection efficiency was high (59.5%, range 15-97). No adverse affects were observed. CONCLUSIONS Amicus blood cell separator has a high collection efficiency for PBPC collections and improves donor safety by reducing platelet loss. [ABSTRACT FROM AUTHOR]