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Summary High‐temperature bioconversion of lignocellulose into fermentable sugars has drawn attention for efficient production of renewable chemicals and biofuels, because competing microbial activities are inhibited at elevated temperatures and thermostable cell wall degrading enzymes are superior to mesophilic enzymes. Here, we report on the development of a platform to produce four different thermostable cell wall degrading enzymes in the chloroplast of Chlamydomonas reinhardtii. The enzyme blend was composed of the cellobiohydrolase CBM3GH5 from C. saccharolyticus, the β‐glucosidase celB from P. furiosus, the endoglucanase B and the endoxylanase XynA from T. neapolitana. In addition, transplastomic microalgae were engineered for the expression of phosphite dehydrogenase D from Pseudomonas stutzeri, allowing for growth in non‐axenic media by selective phosphite nutrition. The cellulolytic blend composed of the glycoside hydrolase (GH) domain GH12/GH5/GH1 allowed the conversion of alkaline‐treated lignocellulose into glucose with efficiencies ranging from 14% to 17% upon 48h of reaction and an enzyme loading of 0.05% (w/w). Hydrolysates from treated cellulosic materials with extracts of transgenic microalgae boosted both the biogas production by methanogenic bacteria and the mixotrophic growth of the oleaginous microalga Chlorella vulgaris. Notably, microalgal treatment suppressed the detrimental effect of inhibitory by‐products released from the alkaline treatment of biomass, thus allowing for efficient assimilation of lignocellulose‐derived sugars by C. vulgaris under mixotrophic growth.

Summary In Arabidopsis mature seeds, the onset of the embryo-to-seedling transition is nonautonomously controlled, being blocked by endospermic abscisic acid (ABA) release under unfavorable conditions. Whether the mature endosperm governs additional nonautonomous developmental processes during this transition is unknown. Mature embryos have a more permeable cuticle than seedlings, consistent with their endospermic ABA uptake capability. Seedlings acquire their well-sealing cuticles adapted to aerial lifestyle during germination. Endosperm removal prevents seedling cuticle formation, and seed reconstitution by endosperm grafting onto embryos shows that the endosperm promotes seedling cuticle development. Grafting different endosperm and embryo mutant combinations, together with biochemical, microscopy, and mass spectrometry approaches, reveal that the release of tyrosylprotein sulfotransferase (TPST)-sulfated CIF2 and PSY1 peptides from the endosperm promotes seedling cuticle development. Endosperm-deprived embryos produced nonviable seedlings bearing numerous developmental defects, not related to embryo malnutrition, all restored by exogenously provided endosperm. Hence, seedling establishment is nonautonomous, requiring the mature endosperm.

Because of their abundance and extensive phosphorylation, numerous thylakoid proteins stand out amongst the phosphoproteins of plants and algae. In particular, subunits of light-harvesting complex II (LHCII) and of photosystem II (PSII) are dynamically phosphorylated and dephosphorylated in response to light conditions and metabolic demands. These phosphorylations are controlled by evolutionarily conserved thylakoid protein kinases and counteracting protein phosphatases, which have distinct but partially overlapping substrate specificities. The best characterized are the kinases STATE TRANSITION 7 (STN7/STT7) and STATE TRANSITION 8 (STN8), and the antagonistic phosphatases PROTEIN PHOSPHATASE 1/THYLAKOID-ASSOCIATED PHOSPHATASE 38 (PPH1/TAP38) and PHOTOSYSTEM II CORE PHOSPHATASE (PBCP). The phosphorylation of LHCII is mainly governed by STN7 and PPH1/TAP38 in plants. LHCII phosphorylation is essential for state transitions, a regulatory feedback mechanism that controls the allocation of this antenna to either PSII or PSI, and thus maintains the redox balance of the electron transfer chain. Phosphorylation of several core subunits of PSII, regulated mainly by STN8 and PBCP, correlates with changes in thylakoid architecture, the repair cycle of PSII after photodamage as well as regulation of light harvesting and of alternative routes of photosynthetic electron transfer. Other kinases, such as the PLASTID CASEIN KINASE II (pCKII), also intervene in thylakoid protein phosphorylation and take part in the chloroplast kinase network. While some features of thylakoid phosphorylation were conserved through the evolution of photosynthetic eukaryotes, others have diverged in different lineages possibly as a result of their adaptation to varied environments.

In Arabidopsis mature seeds, the onset of the embryo-to-seedling transition is nonautonomously controlled, being blocked by endospermic abscisic acid (ABA) release under unfavorable conditions. Whether the mature endosperm governs additional nonautonomous developmental processes during this transition is unknown. Mature embryos have a more permeable cuticle than seedlings consistent with their endospermic ABA uptake capability. Seedlings acquire their well-sealing cuticles adapted to aerial lifestyle during germination. Endosperm removal prevents seedling cuticle formation and seed reconstitution by endosperm grafting onto embryos shows that the endosperm promotes seedling cuticle development. Grafting different endosperm and embryo mutant combinations, together with biochemical, microscopy and mass spectrometry approaches, reveals that release of tyrosylprotein sulfotransferase (TPST)-sulfated CIF2 and PSY1 peptides from the endosperm promotes seedling cuticle development. Endosperm-deprived embryos produced nonviable seedlings bearing numerous developmental defects, not related to embryo malnutrition, all restored by exogenously provided endosperm. Hence, seedling establishment is nonautonomous, requiring the mature endosperm.

International audience; Protein phosphorylation plays important roles in short-term regulation of photosynthetic electron transfer, and during state transitions, the kinase STATE TRANSITION7 (STT7) ofChlamydomonas reinhardtiiphosphorylates components of light-harvesting antenna complex II (LHCII). This reversible phosphorylation governs the dynamic allocation of a part of LHCII to PSI or PSII, depending on light conditions and metabolic demands, but counteracting phosphatase(s) remain unknown inC. reinhardtii. Here we analyzed state transitions inC. reinhardtiimutants of two phosphatases, PROTEIN PHOSPHATASE1 and PHOTOSYSTEM II PHOSPHATASE, which are homologous to proteins that antagonize the state transition kinases (STN7 and STN8) in Arabidopsis (Arabidopsis thaliana). The transition from state 2 to state 1 was retarded inpph1, and surprisingly also inpbcp. However, both mutants eventually returned to state 1. In contrast, the double mutantpph1;pbcpappeared strongly locked in state 2. The complex phosphorylation patterns of the LHCII trimers and of the monomeric subunits were affected in the phosphatase mutants. Their analysis indicated that the two phosphatases have different yet overlapping sets of protein targets. The dual control of thylakoid protein dephosphorylation and the more complex antenna phosphorylation patterns inC. reinhardtiicompared to Arabidopsis are discussed in the context of the stronger amplitude of state transitions and the more diverse LHCII isoforms in the alga.

Protein phosphorylation plays important roles in short-term regulation of photosynthetic electron transfer. In a mechanism known as state transitions, the kinase STATE TRANSITION 7 (STT7) ofChlamydomonas reinhardtiiphosphorylates components of light-harvesting antenna complex II (LHCII). This reversible phosphorylation governs the dynamic allocation of a part of LHCII to photosystem I or photosystem II, depending on light conditions and metabolic demands. Little is however known in the green alga on the counteracting phosphatase(s). In Arabidopsis, the homologous kinase STN7 is specifically antagonized by PROTEIN PHOSPHATASE 1/THYLAKOID-ASSOCIATED PHOSPHATASE 38 (PPH1/TAP38). Furthermore, the paralogous kinase STN8 and the countering phosphatase PHOTOSYSTEM II PHOSPHATASE (PBCP), which count subunits of PSII amongst their major targets, influence thylakoid architecture and high-light tolerance. Here we analyze state transitions inC. reinhardtiimutants of the two homologous phosphatases, CrPPH1 and CrPBCP. The transition from state 2 to state 1 is retarded inpph1, and surprisingly also inpbcp. However both mutants can eventually return to state 1. In contrast, the double mutantpph1;pbcpappears strongly locked in state 2. The complex phosphorylation patterns of the LHCII trimers and of the monomeric subunits are affected in the phosphatase mutants. Their analysis indicates that the two phosphatases have different yet overlapping sets of protein targets. The dual control of thylakoid protein de-phosphorylation and the more complex antenna phosphorylation patterns in Chlamydomonas compared to Arabidopsis are discussed in the context of the stronger amplitude of state transitions and the more diverse LHCII isoforms in the alga.

Biofuels from renewable plant biomass are gaining momentum due to climate change related to atmospheric CO2increase. However, the production cost of enzymes required for cellulosic biomass saccharification is a major limiting step in this process. Low-cost production of large amounts of recombinant enzymes by transgenic plants was proposed as an alternative to the conventional microbial based fermentation. A number of studies have shown that chloroplast-based gene expression offers several advantages over nuclear transformation due to efficient transcription and translation systems and high copy number of the transgene. In this study, we expressed in tobacco chloroplasts microbial genes encoding five cellulases and a polygalacturonase. Leaf extracts containing the recombinant enzymes showed the ability to degrade various cell-wall components under different conditions, singly and in combinations. In addition, our group also tested a previously described thermostable xylanase in combination with a cellulase and a polygalacturonase to study the cumulative effect on the depolymerization of a complex plant substrate. Our results demonstrate the feasibility of using transplastomic tobacco leaf extracts to convert cell-wall polysaccharides into reducing sugars, fulfilling a major prerequisite of large scale availability of a variety of cell-wall degrading enzymes for biofuel industry.

Myeloid-derived suppressor cells (MDSCs) are powerful immunomodulatory cells that in mice play a role in infectious and inflammatory disorders, including acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. Their relevance in clinical acute GVHD is poorly known. We analyzed whether granulocyte colony-stimulating factor (G-CSF) administration, used to mobilize hematopoietic stem cells, affected the frequency of MDSCs in the peripheral blood stem cell grafts of 60 unrelated donors. In addition, we evaluated whether the MDSC content in the peripheral blood stem cell grafts affected the occurrence of acute GVHD in patients undergoing unrelated donor allogeneic stem cell transplantation. Systemic treatment with G-CSF induces an expansion of myeloid cells displaying the phenotype of monocytic MDSCs (Linlow/negHLA-DR−CD11b+CD33+CD14+) with the ability to suppress alloreactive T cells in vitro, therefore meeting the definition of MDSCs. Monocytic MDSC dose was the only graft parameter to predict acute GVHD. The cumulative incidence of acute GVHD at 180 days after transplantation for recipients receiving monocytic MDSC doses below and above the median was 63% and 22%, respectively (P = .02). The number of monocytic MDSCs infused did not impact the relapse rate or the transplant-related mortality rate (P > .05). Although further prospective studies involving larger sample size are needed to validate the exact monocytic MDSC graft dose that protects from acute GVHD, our results strongly suggest the modulation of G-CSF might be used to affect monocytic MDSCs graft cell doses for prevention of acute GVHD.

The high cost of recombinant enzymes for the production of biofuel from ligno-cellulosic biomass is a crucial factor affecting the economic sustainability of the process. The use of plants as biofactories for the production of the suitable recombinant enzymes might be an alternative to microbial fermentation. In the case of enzyme accumulation in chloroplasts, it is fundamental to focus on the issue of full photosynthetic efficiency of transplastomic plants in the field where they might be exposed to abiotic stress such as high light intensity and high temperature. Xylanases (EC 3.2.1.8), a group of enzymes that hydrolyse linear polysaccharides of beta-1,4-xylan into xylose, find an application in the biofuel industry favouring biomass saccharification along with other cell-wall degrading enzymes. In the present study, we analysed how a high level of accumulation of a thermostable xylanase in tobacco chloroplasts does not impact on photosynthetic performance of transplastomic plants grown outdoors. The recombinant enzyme was found to be stable during plant development, ex planta and after long-term storage.

Plerixafor "on demand" after chemotherapy plus granulocyte-colony-stimulating factor (G-CSF) is efficient in peripheral stem cell mobilization, but the timing of administration and criteria for patient selection are under investigation. To devise an algorithm for the "on demand" use of plerixafor at the first mobilization attempt, we analyzed the kinetics of hematopoietic recovery and peripheral blood CD34+ cells in 107 patients treated with high-dose cyclophosphamide plus G-CSF. Fifty-one patients with myeloma were treated with cyclophosphamide 3-4 g/m(2) on day 0 followed by G-CSF 10 μg/kg from day + 6, and 56 patients with lymphoma received cyclophosphamide 6-7 g/m(2) followed by G-CSF 5 μg/kg from day + 1. Peripheral blood CD34+ cell monitoring was started on day + 8 in patients with myeloma and day + 10 in patients with lymphoma. The outcome of interest was a collection of ≤ 2 × 10(6) CD34+/kg. By a multivariate logistic regression model, CD34+ cell count10/μL at leukocyte recovery (1000/μL) or leukocyte count1000/μL after day + 12 in myeloma and day + 14 in lymphoma predicted the failure of mobilization by 2.7 and 2.8 times (p = 0.001 and p = 0.02) with a sensitivity of 89% and specificity of 88%, respectively. Plerixafor "on demand" may be considered in patients with myeloma and lymphoma with delayed hematopoietic recovery and10/μL CD34+ cells, as a first-line mobilization strategy.

The bulk production of recombinant enzymes by either prokaryotic or eukaryotic organisms might contribute to replace environmentally non-friendly chemistry-based industrial processes with enzyme-based biocatalysis, provided the cost of enzyme production is low. In this context, it is worth noting that the production of recombinant proteins by photosynthetic organisms offer both eukaryotic (nuclear) and prokaryotic (chloroplast) alternatives, along with the advantage of an autotrophic nutrition. Compared to nuclear transformation, chloroplast transformation generally allows a higher level of accumulation of the recombinant protein of interest. Furthermore, among the photosynthetic organisms, there is a choice of using either multicellular or unicellular ones. Tobacco, being a non-food and non-feed plant, has been considered as a good choice for producing enzymes with applications in technical industry, using a transplastomic approach. Also unicellular green algae, in particular Chlamydomonas reinhardtii, have been proposed as candidate organisms for the production of recombinant proteins. In the light of the different features of these two transplastomic systems, we decided to make a direct comparison of the efficiency of production of a bacterial endoglucanase. With respect to the amount obtained, 14 mg g-1 of biomass fresh weight equivalent to 8-10% of the total protein content, and estimated production cost, 1.5-2 € Kg-1, tobacco proved to be far more favourable for bulk enzyme production when compared to Chlamydomonas reinhardtii which accumulated this endoglucanase at 0.003% of the total protein.

This work focuses on the development of a molecular tool for purification of Photosystem II (PSII) from Nicotiana tabacum (L.). To this end, the chloroplast psbB gene encoding the CP47 PSII subunit was replaced with an engineered version of the same gene containing a C-terminal His-tag. Molecular analyses assessed the effective integration of the recombinant gene and its expression. Despite not exhibiting any obvious phenotype, the transplastomic plants remained heteroplasmic even after three rounds of regeneration under antibiotic selection. However, the recombinant His-tagged CP47 protein associated in vivo to the other PSII subunits allowing the isolation of a functional PSII core complex, although with low yield of extraction. These results will open up possible perspectives for further spectroscopic and structural studies.

The nucleo-cytoplasmic compartment exerts anterograde control on chloroplast gene expression through numerous proteins that intervene at posttranscriptional steps. Here, we show that the maturation of psaC mutant (mac1) of Chlamydomonas reinhardtii is defective in photosystem I and fails to accumulate psaC mRNA. The MAC1 locus encodes a member of the Half-A-Tetratricopeptide (HAT) family of super-helical repeat proteins, some of which are involved in RNA transactions. The Mac1 protein localizes to the chloroplast in the soluble fraction. MAC1 acts through the 5' untranslated region of psaC transcripts and is required for their stability. Small RNAs that map to the 5'end of psaC RNA in the wild type but not in the mac1 mutant are inferred to represent footprints of MAC1-dependent protein binding, and Mac1 expressed in bacteria binds RNA in vitro. A coordinate response to iron deficiency, which leads to dismantling of the photosynthetic electron transfer chain and in particular of photosystem I, also causes a decrease of Mac1. Overexpression of Mac1 leads to a parallel increase in psaC mRNA but not in PsaC protein, suggesting that Mac1 may be limiting for psaC mRNA accumulation but that other processes regulate protein accumulation. Furthermore, Mac 1 is differentially phosphorylated in response to iron availability and to conditions that alter the redox balance of the electron transfer chain.

Green microalgae, due to their short growth cycle and to their ability to photosynthetically fix carbon dioxide producing an oil-rich biomass, have been proposed as an attractive alternative feedstock for the production of “second generation” biofuels. However, it has been anticipated that owing to their ability to colonize very different environments characterized by high levels of nitrogen, they can also be good candidates for bioremediation, thus integrating environmental protection with sustainable biomass production. We have isolated a strain belonging to Scenedesmus genus from urban wastewater. This isolate, Scenedesmus acutus PVUW12, was tested for its ability to grow and actively deplete eutrophicating inorganic molecules present in wastewater. In order to test its biomass productivity, the PVUW12 strain was grown in a vertical-column photobioreactor using standard growth medium obtaining a maximal productivity of 0.3 g dry weight L−1 d. When the same strain was grown in the photobioreactor filled with wastewater collected from the final step of the local urban purifier plant containing 18.8 mg L−1 nitrate, we observed complete nitrogen removal coupled with a biomass production of about 0.74 g dry weight L−1 within 3 days. After 10 days, the recovered biomass was analyzed for triglyceride content which was found to be 9.3% of the dry biomass. However, when algal cells were left for additional 10 days in static conditions the triglyceride content increased to 28.8%. These data show that this Scenedesmus strain can be used for wastewater bioremediation producing a biomass suitable for energy production.

Haploidentical hematopoietic stem cell transplantation provides an option for patients with advanced hematologic malignancies lacking a compatible donor. In this prospective phase 1/2 trial, we evaluated the role of reduced-intensity conditioning (RIC) followed by early add-backs of CD8-depleted donor lymphocyte infusions (DLIs). The RIC regimen consisted of thiotepa, fludarabine, cyclophosphamide, and 2 Gy total body irradiation. Twenty-eight patients with advanced lymphoproliferative diseases (n = 24) or acute myeloid leukemia (n = 4) were enrolled. Ex vivo and in vivo T-cell depletion was carried out by CD34+ cell selection and alemtuzumab treatment. The 2-year cumulative incidence of nonrelapse mortality was 26% and the 2-year overall survival (OS) was 44%, with a better outcome for patients with chemosensitive disease (OS, 75%). Overall, 54 CD8-depleted DLIs were administered to 23 patients (82%) at 3 different dose levels without loss of engraftment or acute toxicities. Overall, 6 of 23 patients (26%) developed grade II-IV graft-versus-host disease, mainly at dose level 2. In conclusion, our RIC regimen allowed a stable engraftment with a rather low nonrelapse mortality in poor-risk patients; OS is encouraging with some long-term remissions in lymphoid malignancies. CD8-depleted DLIs are feasible and promote the immune reconstitution.

Objective To optimize transduction efficiency of mobilized CD34 + cells with serotype-5 adenoviruses (Ad5s), we investigated the activity of the chemical cocktail BoosterExpress Reagent in enhancing Ad5-mediated gene transfer into CD34 + cells. Methods Enriched CD34 + cells were transduced with three different Ad5s at increasing multiplicity of infections (MOIs) in the presence and absence of BoosterExpress Reagent. Percentages of transduced cells and levels of transgene expression were quantified by flow cytometry. Propidium iodide staining and colony growth were used to assess the toxicity of the transduction protocol. Expression of adenovirus receptors was investigated by flow cytometry. Results Irrespective of the Ad5 used, transduction with BoosterExpress Reagent using an MOI of 500 resulted in an average six- to seven-fold increase of transduction efficiencies and 1.5- to 2-fold increase of the levels of transgene expression, which could be detected up to 7 days post-transduction. Although BoosterExpress Reagent alone did not affect the plating efficiency of CD34 + cells, adenovector transduction plus BoosterExpress Reagent significantly reduced the plating efficiency due to the marked increase of transduced cells. However, adenoviral transduction in the presence of BoosterExpress Reagent failed to significantly reduce the recovery of CD34 + cells as compared with transduction in the absence of the compound. Coxsackievirus and adenovirus receptor as well as α v β 3 , α v β 5 , α 5 , and β 1 integrins were upregulated by BoosterExpress Reagent. Conclusions BoosterExpress Reagent allows high-levels of durable transgene expression, thus making CD34 + cells a suitable target for Ad5-mediated gene transfer.

To investigate the therapeutic activity of the fully human anti–HLA-DR antibody 1D09C3 in multiple myeloma (MM), we reevaluated HLA-DR expression on CD138+ cells, analyzed the capacity of IFN-γ to up-regulate HLA-DR expression on MM cell lines, and tested the in vitro and in vivo activity of 1D09C3 alone or in combination with IFN-γ. CD138+HLA-DR+ cells were detected in 31 of 60 patients, with 15 of 60 patients having ≥20% CD138+HLA-DR+ cells (median, 50%; range, 23–100). Because primary plasma cells cannot be efficiently cultured in vitro, we used a panel of MM cell lines with a dim/negative to bright HLA-DR expression to evaluate 1D09C3-induced cell death. Annexin V/propidium iodide (PI) staining showed that 1D09C3-induced cell death correlated with constitutive HLA-DR expression. Induction of HLA-DR by IFN-γ restored the sensitivity of HLA-DR dim cell lines to 1D09C3. In vivo, the combined IFN-γ/1D09C3 treatment significantly increased the median survival of nonobese diabetic/severe combined immunodeficient mice xenografted with KMS-11 cell line, compared with controls (147 versus 48 days, P ≤ 0.0001) or mice receiving 1D09C3 alone (147 versus 92 days, P ≤ 0.03). The better therapeutic activity of IFN-γ/1D09C3 treatment over 1D09C3 alone was further shown by a 2-fold increase of mice being disease-free at 150 days after xenograft (47% versus 25%). No mice experienced any apparent treatment-related toxicity. Our data show that (a) one fourth of MM patients express HLA-DR on CD138+ cells and (b) IFN-γ–induced up-regulation of HLA-DR results in a potent enhancement of the in vivo antimyeloma activity of 1D09C3. [Cancer Res 2007;67(7):3269–75]

Although high cardiovascular risk patients should be the main target of preventive strategies, modifiable risk factors are often inadequately controlled.To assess feasibility and results of a comprehensive personalized method for cardiovascular prevention in high risk patients followed by their general practitioner.Between 2004 and 2007, 12,513 patients (mean age 64.0 ± 9.5 years; 61.5% males) with multiple cardiovascular risk factors or history of atherosclerotic disease were identified and followed for five years. If control of major modifiable cardiovascular risk factors (hypertension, hypercholesterolaemia, diabetes, obesity, smoking, unhealthy diet, physical inactivity) was sub-optimal, at baseline and yearly thereafter general practitioners planned with patients, with the help of a brief checklist, preventive interventions to improve the global risk profile. Main outcome was the control of the seven major modifiable cardiovascular risk factors during follow-up. Secondary outcome was the incidence of cardiovascular deaths and hospitalization for cardiovascular reasons according to the improvement in global cardiovascular risk profile during the first year.Control of all major modifiable risk factors except physical inactivity improved gradually and significantly (p 0.0001) during follow-up.The improvement in the global cardiovascular risk profile during the first year was independently and significantly associated with a lower rate of major cardiovascular events in the following years (hazard ratio 0.939; 95% confidence interval 0.887-0.994, p = 0.03).Our comprehensive, personalized method for cardiovascular risk prevention in people at high risk appears feasible in general practice. The improvement in the global cardiovascular risk profile was associated with a better prognosis.

Objectives To assess the pharmacological treatment and the control of major modifiable cardiovascular risk factors in everyday practice according to the patients’ cardiovascular risk level. Methods In a cross-sectional study general practitioners (GPs) had to identify a random sample of their patients with cardiovascular risk factors or diseases and collect essential data on the pharmacological treatment and control of hypertension, hyperlipidemia, and diabetes according to the patients’ cardiovascular risk level and history of cardiovascular disease. Participants were subjects of both sexes, aged 40–80 years, with at least one known cardiovascular risk factor or a history of cardiovascular diseases. Results From June to December 2000, 162 Italian GPs enrolled 3120 of their patients (2470 hypertensives, 1373 hyperlipidemics, and 604 diabetics). Despite the positive association between the perceived level of global cardiovascular risk and lipid-lowering drug prescriptions in hyperlipidemic subjects (from 26% for lowest risk to 56% for highest risk p < 0.0001) or the prescription of combination therapy in hypertensives (from 41% to 70%, p < 0.0001) and diabetics (from 24% to 43%, p = 0.057), control was still inadequate in 48% of diabetics, 77% of hypertensives, and 85% of hyperlipidemics, with no increase in patients at highest risk. Trends for treatment and control were similar in patients with cardiovascular diseases. Conclusions Even in high-risk patients, despite a tendency towards more intensive treatment, pharmacological therapy is still under used and the degree of control of blood pressure, cholesterol level and diabetes is largely unsatisfactory.

The complex hematopoietic effects of placental growth factor (PlGF) prompted us to test in mice and nonhuman primates the mobilization of peripheral blood progenitor cells (PBPCs) elicited by recombinant mouse PlGF-2 (rmPlGF-2) and recombinant human PlGF-1 (rhPlGF-1). PBPC mobilization was evaluated by assaying colony-forming cells (CFCs), high-proliferative potential-CFCs (HPP-CFCs), and long-term culture-initiating cells (LTC-ICs). In mice, both rmPlGF-2 and rhPlGF-1 used as single agents failed to mobilize PBPCs, whereas the combination of rhPlGF-1 and granulocyte colony-stimulating factor (rhG-CSF) increased CFCs and LTC-ICs per milliliter of blood by four- and eightfold, respectively, as compared with rhG-CSF alone. rhPlGF-1 plus rhG-CSF significantly increased matrix metalloproteinase-9 plasma levels over rhG-CSF alone, suggesting a mechanistic explanation for rhPlGF-1/rhG-CSF synergism. In rhesus monkeys, rhPlGF-1 alone had no mobilization effect, whereas rhPlGF-1 (260 μg/kg per day) plus rhG-CSF (100 μg/kg per day) increased rhG-CSF-elicited mobilization of CFCs, HPP-CFCs, and LTC-ICs per milliliter of blood by 5-, 7-, and 15-fold, respectively. No specific toxicity was associated with the administration of rhPlGF-1 alone or in combination. In conclusion, our data demonstrate that rhPlGF-1 significantly increases rhG-CSF-elicited hematopoietic mobilization and provide a preclinical rationale for evaluating rhPlGF-1 in the clinical setting.

Objective To explore new treatments specifically targeting malignant plasma cells (PCs), we examined CD52 antigen expression on primary PCs as well as multiple myeloma (MM) cell lines, and investigated in vivo the antimyeloma activity of alemtuzumab. Materials and Methods PCs were enriched from the marrow of MM patients (n = 39) according to CD138 expression and then analyzed by 3-color flow cytometry and quantitative PCR. The in vivo activity of alemtuzumab was evaluated in a xenotransplant model of MM in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Results CD52 expression revealed a substantial heterogeneity in terms of both percentage of positive cells and fluorescence intensity, with 25/39 (64%) MM patients showing ≥30% CD138+ PCs expressing the CD52 antigen (mean = 79%; range, 33–100%). Similarly to primary cells, cell lines showed heterogeneous CD52 expression. Expression of CD52 mRNA by quantitative PCR analysis strongly correlated with CD52 antigen detection by flow cytometry. In vivo, alemtuzumab treatment significantly increased the median survival of animals with an early- (64 vs 77 days, p ≤ 0.0005) or advanced-stage (66 vs 75 days, p ≤ 0.02) disease. Conclusion We conclude that: 1) CD52 is expressed on PCs of a significant proportion of MM patients; 2) alemtuzumab used as a single agent exerts a good antitumor activity in NOD/SCID mice bearing an early-stage disease; and 3) alemtuzumab might have therapeutic potential in a subset of MM patients.

Plerixafor ‘on demand’: results of a strategy based on peripheral blood CD34+ cells in lymphoma patients at first or subsequent mobilization with chemotherapy+G-CSF

Although risk assessment charts have been proposed to identify patients at high cardiovascular risk, in everyday practice general practitioners (GPs) often use their knowledge of the patients to estimate the risk subjectively.A cross-sectional study aimed to describe how GPs perceive, qualify and grade cardiovascular risk in everyday practice.General practitioners had to identify in a random sample of 10% of their contacts the first 20 consecutive patients perceived as being at cardiovascular risk. For each patient essential data were collected on clinical history, physical examination and laboratory tests, for the qualification of risk. At the end of the process GPs subjectively estimated the overall patient's level of risk. General practitioners grading was compared with the risk estimate from a reference chart.Over a mean time of 25 days 3120 patients perceived as being at cardiovascular risk were enrolled. According to the inclusion scheme each GP had contact with more than 200 patients at cardiovascular risk every month. Thirty percent of these patients had atherosclerotic diseases. Up to 72% of patients without any history of atherosclerotic diseases but perceived to be at risk could be classified according to a reference chart as being at moderate to very high risk. Comparing GPs' grading of risk with a chart estimate there was agreement in 42% of the cases. Major determinants of GPs' underestimation of risk were age, sex and smoking habits, while obesity and family history were independently associated with overestimation.On the basis of their perception GPs properly identify patients at cardiovascular risk in the majority of cases. General practitioners subjective grading of risk level only partially agreed with that given by a chart.

Objective The aim of this study was to evaluate the capacity of defibrotide in enhancing cytokine-induced hematopoietic mobilization in rhesus monkeys. Materials and methods Animals received recombinant human granulocyte colony-stimulating factor (rhG-CSF, 100 μg/kg/day SC for 5 days) and, after a 4- to 6-week washout period, were remobilized with defibrotide (15 mg/kg/hour continuous intravenous for 5 days) plus rhG-CSF. Hematopoietic mobilization was evaluated by complete blood counts, differential counts, as well as frequency and absolute numbers of colony-forming cells (CFCs), high-proliferative potential CFCs (HPP-CFCs), and long-term culture-initiating cells (LTC-ICs). Results Compared to baseline values, rhG-CSF increased circulating CFCs, HPP-CFCs, and LTC-ICs by 158-, 125-, and 67-fold, respectively; the same figures for defibrotide/rhG-CSF were 299-, 1452-, and 295-fold, respectively. Defibrotide/rhG-CSF treatment compared to rhG-CSF alone increased CFCs, HPP-CFCs, and LTC-ICs by 1.4- (35,089 vs 25,825, p ≤0.02), 6- (4358 vs 748, p ≤0.02), and 5-fold (884 vs 168, p ≤0.04), respectively. We then evaluated the effects of a 2-day defibrotide treatment associated with a 5-day rhG-CSF treatment. Compared to rhG-CSF, defibrotide/rhG-CSF increased the mobilization of CFCs, HPP-CFCs, and LTC-ICs by 2- (31,128 vs 15,527, p ≤0.05), 8- (5361 vs 660, p ≤0.01), and 8-fold (954 vs 119, p ≤0.01), respectively. Conclusions Our data demonstrate that in nonhuman primates: 1) defibrotide enhances rhG-CSF–elicited mobilization of primitive and committed progenitors; and 2) a 2-day defibrotide injection is as effective as a 5-day injection.

CD2+ T lymphocytes obtained from either the donor of bone marrow stromal cells (BMSCs) or a third party were cultured in mixed lymphocyte reactions (MLRs) with either allogeneic dendritic cells (DCs) or peripheral blood lymphocytes (PBLs). When autologous or allogeneic BMSCs were added back to T cells stimulated by DCs or PBLs, a significant and dose-dependent reduction of T-cell proliferation, ranging from 60% ± 5% to 98% ± 1%, was evident. Similarly, addition of BMSCs to T cells stimulated by polyclonal activators resulted in a 65% ± 5% (P = .0001) suppression of proliferation. BMSC- induced T-cell suppression was still evident when BMSCs were added in culture as late as 5 days after starting of MLRs. BMSC-inhibited T lymphocytes were not apoptotic and efficiently proliferated on restimulation. BMSCs significantly suppressed both CD4+ and CD8+ T cells (65% ± 5%, [P = .0005] and 75% ± 15% [P = .0005], respectively). Transwell experiments, in which cell-cell contact between BMSCs and effector cells was prevented, resulted in a significant inhibition of T-lymphocyte proliferation, suggesting that soluble factors were involved in this phenomenon. By using neutralizing monoclonal antibodies, transforming growth factor β1 and hepatocyte growth factor were identified as the mediators of BMSC effects. In conclusion, our data demonstrate that (1) autologous or allogeneic BMSCs strongly suppress T-lymphocyte proliferation, (2) this phenomenon that is triggered by both cellular as well as nonspecific mitogenic stimuli has no immunologic restriction, and (3) T-cell inhibition is not due to induction of apoptosis and is likely due to the production of soluble factors.

Summary Evolution of vascular plants required compromise between photosynthesis and photodamage. We analyzed representative species from two divergent lineages of vascular plants, lycophytes and euphyllophytes, with respect to the response of their photosynthesis and light-harvesting properties to increasing light intensity. In the two analyzed lycophytes, Selaginella martensii and Lycopodium squarrosum, the medium phase of non-photochemical quenching relaxation increased under high light compared to euphyllophytes. This was thought to be associated with the occurrence of a further thylakoid phosphoprotein in both lycophytes, in addition to D2, CP43 and Lhcb1-2. This protein, which showed light intensity-dependent reversible phosphorylation, was identified in S. martensii as Lhcb6, a minor LHCII antenna subunit of PSII. Lhcb6 is known to have evolved in the context of land colonization. In S. martensii, Lhcb6 was detected as a component of the free LHCII assemblies, but also associated with PSI. Most of the light-induced changes affected the amount and phosphorylation of the LHCII assemblies, which possibly mediate PSI–PSII connectivity. We propose that Lhcb6 is involved in light energy management in lycophytes, participating in energy balance between PSI and PSII through a unique reversible phosphorylation, not yet observed in other land plants.

A detailed lipid characterization of Scenedesmus acutus PVUW12, with emphasis on the evaluation of triacylglycerols (TAGs) as a biodiesel feedstock, is presented. When algal cells were grown in nitrogen-free medium (N stress), a lipid increase was detected that was mainly due to TAG accumulation. In situ fluorescence measurements allowed the kinetics and extent of neutral lipid accumulation to be followed. Under N stress, the productivity of total lipids and TAGs increased significantly (80.99 and 63.74 mg L−1 day−1, respectively) compared with controls (29.51 and 16.23 mg L−1 day−1, respectively). Monounsaturated fatty acids were the major fraction and increased further (49.74 %) in stressed cells, with oleic acid as the most abundant compound (46.97 %). The polyunsaturated fatty acid composition of this algal oil appears to meet the European Standard EN 14214. These results indicate that S. acutus oil meets the requirements for its use as a biodiesel feedstock. Since this strain was also proposed for wastewater bioremediation, this opens up the possibility of its use in an integrated system combined with biofuel production. Fil: Damiani, Maria Cecilia. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Popovich, Cecilia Angelines. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida(i); Argentina Fil: Constenla, Diana Teresita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Planta Piloto de Ingeniería Química (i); Argentina Fil: Martinez, Ana María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Instituto de Química del Sur; Argentina Fil: Doria, Enrico. Università Degli Studi Di Pavia; Italia Fil: Longoni, Paolo. Università Degli Studi Di Pavia; Italia Fil: Cella, Rino. Università Degli Studi Di Pavia; Italia Fil: Nielsen, Erik. Università Degli Studi Di Pavia; Italia Fil: Leonardi, Patricia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida(i); Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina

In the recent years, the studies concerning the cultivation of Neochloris oleoabundans for biofuel purposes have increased, in relation to its capability to accumulate lipids when grown under nutrient starvation. Unfortunately, this cultivation mode does not allow to reach high biomass densities, which are required to improve the feasibility of the process. Increasing knowledge of the microalgal physiology is necessary to obtain new useful information for the improvement of culture performance in the perspective of large-scale cultivation. In this work, the mixotrophic cultivation of N. oleoabundans in a brackish medium added with different glucose concentrations has been tested under shaking, with the aim of stimulating growth alongside lipid accumulation inside cells. Cell morphology, glucose consumption, photosynthetic pigment content and photosynthetic efficiency were also investigated. Among all tested glucose concentrations (0-30 g L(-1)), it was observed that 2.5 g L(-1) was the optimal concentration, allowing to obtain the best compromise between glucose supplement, biomass production and lipid accumulation. Growth was highly enhanced in mixotrophic cultures, linked to the release of cells from sporocysts. A unique feature characterising mixotrophy in N. oleoabundans was the promotion of the maximum quantum yield of Photosystem II. Moreover, when mixotrophic cells entered the stationary phase, high lipid accumulation was induced. This study shows that the addition of glucose to N. oleoabundans remarkably increases the production of biomass enriched in lipids and represents an advancement for the cultivation of this microalga for applied purposes.

Successful second autologous engraftment after long duration storage of hematopoietic stem cells

Most photosynthetically fixed carbon is contained in cell wall polymers present in plant biomasses, the largest organic carbon source in the biosphere. The degradation of these polymers for biotechnological purposes requires the combined action of several enzymes. To identify new activities, we examined which enzymes are activated by an endophytic strain of Chaetomium globosum to degrade cellulose-containing substrates. After biochemical analyses of the secretome of the fungus grown on cellulose or woody substrates, we took advantage of the available genomic data to identify potentially involved genes. After in silico identification of putative genes encoding either proteins able to bind to cellulose or glycohydrolases (GHs) of family 7, we investigated their transcript levels by reverse transcription-quantitative PCR (RT-qPCR). Our data suggest that eight genes compose the core of the cellulose-degrading system of C. globosum . Notably, the related enzymes belong structurally to the well-described GH families 5, 6, 7, 16, and 45, which are known to be the core of the cellulose degradation systems of several ascomycetes. The high expression levels of cellobiose dehydrogenase and two GH 61 enzymes suggest the involvement of this oxidoreductive synergic system in C. globosum . Transcript analysis along with relevant coding sequence (CDS) isolation and expression of recombinant proteins proved to be a key strategy for the determination of the features of two endoglucanases used by C. globosum for the first attack of crystalline cellulose. Finally, the possible involvement of transcriptional regulators described for other ascomycetes is discussed.

Adenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor–related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 105 tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell–derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling–stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.

Double autologous stem cell transplantation is the standard treatment in newly diagnosed multiple myeloma (MM) patients younger than 65 years; therefore, optimization of leukapheresis is crucial. We performed a retrospective analysis of 297 leukaphereses comparing semiautomated (V4.7 in 20% of collections) versus automated (V6.0 in 80%) Caridian (COBE) Spectra versions and analyzing the influence of M-protein on the outcome. Both methods gave comparable collection efficiencies (CE%) (53.4% vs. 55.7% in V6.0 and V4.7, respectively) with similar leukapheresis time and processed volume. Harvest volume was higher in V4.7 (P < 0.0001) with similar contamination of red blood cells (RBCs) (P = 0.77) and platelets (P = 0.09) when compared with V6.0. In patients with higher peripheral white blood cells (WBCs), V6.0 with adjusted harvest volume (

To investigate telomere length (TL) and hematopoietic progenitors in long-term survivors after high-dose chemotherapy and peripheral blood stem cell (PBSC) autograft.Peripheral blood (PB) and bone marrow (BM) samples were obtained from 31 subjects in continuous complete remission from a high-risk lymphoma, at a median of 5.8 years (range: 1-11 years) since autograft. Most of them were grafted with large PBSC quantities (median CD34(+ve) cells/kg: 7 x 10(6)). TL was determined by Southern blot analysis, BM progenitors by in vitro long-term culture-initiating cells (LTC-IC) and colony assays.TL of PB granulocytes was significantly shortened in autografted subjects compared with age-matched healthy subjects; a similar finding was observed in BM. The median TL reduction in granulocytes from autografted subjects compared with age-matched controls (Delta(TelShortening)) was then assessed according to time interval since autograft. Three subject subgroups were identified-at 1 to3 years, 3 to6 years, and 6 to 11 years since autograft-and their telomere loss was the same, with Delta(TelShortening) of 1132, 1379, and 1214 bp in the three subgroups, respectively. The longitudinal assessment of TL in five representative patients followed for up to 40 months since autograft confirmed that telomere shortening occurring during exposure to chemotherapy as well as postautograft is persistent at long term. BM LTC-IC and multipotent and committed progenitors were assessed in subjects at3 years after autograft and found to be markedly reduced compared with normal controls.High-dose chemotherapy and PBSC autograft may result in myelopoietic cell abnormalities that appear to be irreversible.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expected to play a key role in cancer therapy due to its high cancer cell-specificity and potent antitumor activity. We have previously demonstrated that CD34+ cells transduced with an adenovirus encoding the full-length human TRAIL gene (CD34-TRAIL+) exert a potent anti-lymphoma effect in NOD/SCID mice. To investigate the mechanism of action of CD34-TRAIL+ cells, in vivo experiments were perfomed to analyze the tumor homing capacity of CD34-TRAIL+ cells and the effects of transduced cells on tumor vasculature. Tumor homing of CD34-TRAIL+ cells was investigated in NOD/SCID mice bearing subcutaneous lymphoid tumors. Following a single intravenous injection of CD34-TRAIL+ cells (5 x 106), nodules were excised at different time-points and immunostained with an anti-human CD45 antibody. Sections were digitally recorded and the total number of CD45+ cells per tissue section was then counted using the imaging software ImageJ (http://rsb.info.nih.gov/ij/). Tumor homing of CD34-TRAIL+ cells peaked 24 hours after cell injection when a mean of 200 CD34-TRAIL+ cells was recorded per 1 x 105 tumor cells (0.2% CD45+ cells per 1 x 105 tumor cells). Mice pretreatment with the anti-VCAM-1 antibody or the CXCR4 antagonist AMD3100 significantly reduced tumor homing of CD34-TRAIL+ cells, with 0.09% and 0.05% CD45+ cells being recorded per 1 x 105 tumor cells, respectively. To analyze the effects of CD34-TRAIL+ cells on tumor vasculature, mice were injected with sulfo-biotin (0.1 mg, IV, 15 min) and tumor endotelial cells (TEC) were then revealed by staining frozen sections with horseradish peroxidase (HRP)-conjugated streptavidin. As compared with CD34-mock- or soluble (s)TRAIL-treated mice, treatment with CD34-TRAIL+ cells induced within 24 hours a significant (P ≤.001) increase of the thickness of the vessell wall (3.7 ± 1 μm vs 3.4 ± 1 μm vs 6 ± 1 μm, respectively) as well as the endothelial surface (10 ± 4% vs 7 ± 4% vs 21 ± 6% of total tissue section, respectively), suggesting that CD34-TRAIL+ cells induce an early vascular disruption. Confocal microscopic imaging of tumor sections double-stained with anti-CD31 and anti-TRAIL-R2 showed that this receptor was expressed by 8–12% of large tumor vessels. Interestingly, upon treatment with CD34-TRAIL+ cells, but not sTRAIL, TUNEL staining revealed an extensive apoptosis of CD31+/TRAIL-R2+ TEC. Forty-eight hours following injection of CD34-TRAIL+ cells, a 21-fold increase of apoptotic index was detected, which was associated with extensive necrotic areas (20% to 25% of tissue section). These data show that: tumor homing of CD34-TRAIL+ cells induces extensive vascular damage, hemorrhagic necrosis and tumor destruction; the antitumor effect of CD34-TRAIL+ cells is mediated by both indirect vascular-disrupting mechanisms and direct tumor cell killing.

Light-harvesting complex II (LHCII) is a crucial component of the photosynthetic machinery, with central roles in light capture and acclimation to changing light. The association of an LHCII trimer with PSI in the PSI-LHCII supercomplex is strictly dependent on LHCII phosphorylation mediated by the kinase STATE TRANSITION7, and is directly related to the light acclimation process called state transitions. In Arabidopsis (Arabidopsis thaliana), the LHCII trimers contain isoforms that belong to three classes: Lhcb1, Lhcb2, and Lhcb3. Only Lhcb1 and Lhcb2 can be phosphorylated in the N-terminal region. Here, we present an improved Phos-tag-based method to determine the absolute extent of phosphorylation of Lhcb1 and Lhcb2. Both classes show very similar phosphorylation kinetics during state transition. Nevertheless, only Lhcb2 is extensively phosphorylated (>98%) in PSI-LHCII, whereas phosphorylated Lhcb1 is largely excluded from this supercomplex. Both isoforms are phosphorylated to different extents in other photosystem supercomplexes and in different domains of the thylakoid membranes. The data imply that, despite their high sequence similarity, differential phosphorylation of Lhcb1 and Lhcb2 plays contrasting roles in light acclimation of photosynthesis.

The fully human anti-HLA-DR antibody 1D09C3 has been shown to delay lymphoma cell growth in severe combined immunodeficient (SCID) mice. The present study was aimed at (a) investigating the mechanism(s) of 1D09C3-induced cell death and (b) further exploring the therapeutic efficacy of 1D09C3 in nonobese diabetic (NOD)/SCID mice. The chronic lymphocytic leukemia cell line JVM-2 and the mantle cell lymphoma cell line GRANTA-519 were used. Generation of reactive oxygen species (ROS) and mitochondrial membrane depolarization were measured by flow cytometry following cell incubation with dihydroethidium and TMRE, respectively. Western blot analysis was used to detect c-Jun-NH2-kinase (JNK) phosphorylation and apoptosis-inducing factor (AIF). NOD/SCID mice were used to investigate the activity of 1D09C3 in early- or advanced-stage tumor xenografts. In vitro, 1D09C3-induced cell death involves a cascade of events, including ROS increase, JNK activation, mitochondrial membrane depolarization, and AIF release from mitochondria. Inhibition of JNK activity significantly reduced 1D09C3-induced apoptosis, indicating that 1D09C3 activity involves activation of the kinase. In vivo, 1D09C3 induces long-term disease-free survival in a significant proportion of tumor-bearing mice treated at an early stage of disease. Treatment of mice bearing advanced-stage lymphoma results in a highly significant prolongation of survival. These data show that 1D09C3 (a) exerts a potent antitumor effect by activating ROS-dependent, JNK-driven cell death, (b) cures the great majority of mice treated at an early-stage of disease, and (c) significantly prolongs survival of mice with advanced-stage disease. (Cancer Res 2006; 66(3): 1799-808)

Objective In vivo administration of alemtuzumab (an anti-CD52 antibody) is effective to decrease the incidence of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-SCT). However, posttransplant immune reconstitution is impaired, increasing the infection risk. We investigated the effect of in vivo administration of a low-dose alemtuzumab on GVHD prevention and kinetics of immune reconstitution. Patients and Methods Twenty-seven patients entered a pilot study employing reduced-intensity conditioning and low-dose alemtuzumab (15 or 7.5 mg/m 2 ) before peripheral blood allo-SCT from human leukocyte antigen–identical or one antigen–mismatched sibling donors. All lymphoid subsets were longitudinally studied at 1–3, 6, 9, 12 months after transplantation. T-cell receptor (TCR) spectratyping and T-cell receptor excision circles (TRECs) were also analyzed at various time points after allo-SCT. Results All patients but one were engrafted. The probability of nonrelapse mortality at 100 days and 1 year were 7 and 11%, respectively; the overall survival at 2 years was 77%. The cumulative incidence of grade II–IV acute GVHD at day 100 was 11%. The overall incidence of chronic GVHD was 28%. The median time to achieve more than 200 CD4 + /μL and 500 CD8 + /μL were 6 and 9 months, respectively. Natural killer cells remained between the value of 300/μL and 500/μL throughout the period of follow-up whereas the median time to reach CD19 + blood concentrations of >200 cells/μL was 9 months. The normalization of TCR repertoire and increase of TREC counts began at 6 months after allo-SCT. Conclusion We have shown that low-dose alemtuzumab is effective for GVHD prevention, but its use still impairs the immune reconstitution.

To evaluate the appropriate prescription of antiplatelets according to patients’ global cardiovascular risk level in everyday practice. In a cross-sectional study, general practitioners (GPs) identified a random sample of 10% of patients at cardiovascular risk among all subjects coming to the surgery and collected data on cardiovascular risk factors and history of atherosclerotic cardiovascular diseases (CVD). GPs were asked to do a physical examination and record the results of laboratory tests to define the global cardiovascular risk. The use of antiplatelet drugs in patients with established CVD and in healthy subjects at high risk of developing symptomatic atherosclerotic disease was evaluated. A total of 162 GPs from all over Italy recruited 3,120 subjects (51% female, mean age 64 years). Of the 949 with an indication for antiplatelet treatment for secondary prevention of CVD, 442 (47%) were receiving it. Among the 2,071 without CVD, 11% were taking an antiplatelet drug. In this group, antiplatelets were prescribed in 6, 10, 16 and 23%, respectively, of patients perceived by GPs to be at mild, moderate, high and very high cardiovascular risk. Prescription of antiplatelets still seems to be far from what is recommended in virtually all patients with a history of CVD. In subjects with cardiovascular risk factors but without CVD antiplatelet prescription increases in relation to global cardiovascular risk but is still low in patients at high or very high risk of cardiovascular events.

The activity of recombinant human growth hormone (rhGH) in enhancing CD34+ cell mobilization elicited by chemotherapy plus recombinant human granulocyte colony-stimulating factor (rhG-CSF) was evaluated in 16 hard-to-mobilize patients, that is, those achieving a peak of circulating CD34+ cells 10/μL or less, or a collection of CD34+ cells equal to or less than 2 × 106/kg. Patients who had failed a first mobilization attempt with chemotherapy plus rhG-CSF (5 μg/kg/d) were remobilized with chemotherapy plus rhG-CSF and rhGH (100 μg/kg/d). As compared with rhG-CSF, the combined rhGH/rhG-CSF treatment induced significantly higher (P ≤ .05) median peak values for CD34+ cells/μL (7 versus 29), colony-forming cells (CFCs)/mL (2154 versus 28 510), and long-term culture-initiating cells (LTC-ICs)/mL (25 versus 511). Following rhG-CSF and rhGH/rhG-CSF, the median yields of CD34+ cells per leukapheresis were 1.1 × 106/kg and 2.3 × 106/kg (P ≤ .008), respectively; the median total collections of CD34+ cells were 1.1 × 106/kg and 6 × 106/kg (P ≤ .008), respectively. No specific side effect could be ascribed to rhGH, except a transient hyperglycemia occurring in 2 patients. Reinfusion of rhGH/rhG-CSF-mobilized cells following myeloablative therapy resulted in prompt hematopoietic recovery. In conclusion, our data demonstrate that in poor mobilizers addition of rhGH to rhG-CSF allows the patients to efficiently mobilize and collect CD34+ cells with maintained functional properties. (Blood. 2004;103: 3287-3295)

Objective The aim of this story was to evaluate the activity of recombinant human (rh) growth hormone (GH) in restoring bone marrow progenitor cell growth as well as cytokine-elicited stem cell mobilization in aged BALB/c mice with impaired marrow hematopoietic function and reduced stem cell mobilizing capacity. Materials and Methods BALB/c mice included in this study were either naturally aged (group I) or aged after having been used for radioprotective assays (group II). Mice were treated for 5 weeks with either rhGH [2.5 mg/kg/day intraperitoneally (IP)] or phosphate-buffered saline (PBS). Subsequently, colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) were evaluated. In addition, progenitor cell mobilization elicited by granulocyte colony-stimulating factor (rhG-CSF) was analyzed. Results Compared with young controls, the growth of marrow CFCs and LTC-ICs was significantly reduced ( P ≤0.05) in group I and II mice. Treatment with rhGH significantly enhanced marrow hematopoiesis in mice of both groups, as demonstrated by a complete restoration of marrow cellularity, and CFC and LTC-IC growth. To further evaluate the hematopoietic potential of rhGH, aged mice treated with rhGH or PBS were mobilized with rhG-CSF (10 μg/day IP for 5 days). Compared with PBS-injected mice, rhGH-treated mice showed a significant improvement of rhG/CSF–elicited stem cell mobilization, with significant increases of white blood cell counts (5633 vs 8133, P ≤0.05), frequency of circulating CFCs per 10 5 mononuclear cells (36 vs 67, P ≤0.009), as well as absolute numbers per mL of blood of circulating CFCs (783 vs 2288, P ≤0.001) and LTC-IC (21 vs 64, P ≤0.001). Conclusion Our data demonstrate in mice that a 5-week treatment with rhGH restores age- and irradiation-associated loss of marrow primitive and committed progenitors.

Defibrotide is a polydeoxyribonucleotide, which significantly reduces the expression of adhesion molecules on endothelial cells. We investigated the activity of Defibrotide alone or in combination with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to mobilize peripheral blood progenitor cells (PBPCs) in BALB/c mice. A 5-day treatment with Defibrotide alone (1-15 mg/mouse/day) had no effect on WBC counts, frequencies and absolute numbers of total circulating colony-forming cells (CFCs), i.e., granulocyte-macrophage colony-forming units, erythroid burst-forming units, and multilineage colony-forming units. As compared with mock-injected mice, administration of rhG-CSF alone (5 micro g/mouse/day) for 5 days significantly (Por = 0.0001) increased WBC counts, CFC frequencies, and CFC absolute numbers by 2-, 13-, and 27-fold, respectively. As compared with control mice, the combined administration of Defibrotide (15 mg/mouse/day) and rhG-CSF significantly (Por = 0.0001) increased WBC counts, frequencies and absolute numbers of CFCs by 4-, 38-, and 119-fold, respectively. As compared with rhG-CSF alone, administration of Defibrotide plus rhG-CSF resulted in a significant increase (Por = 0.001) of the frequency of circulating long-term culture-initiating cells. In addition, transplantation of 2 x 10(5) rhG-CSF- or Defibrotide/rhG-CSF-mobilized mononuclear cells rescued 43% and 71% of recipient mice, respectively. Experiments of CFC homing performed in lethally irradiated or nonirradiated recipients showed that marrow homing of transplanted PBPCs was reduced by 3-fold in Defibrotide-treated animals as compared with mock-injected mice (Por = 0.001), suggesting that the mobilizing effect of Defibrotide might be because of an effect on PBPC trafficking. In conclusion, our data demonstrate that Defibrotide synergizes with rhG-CSF and significantly increases the mobilization of a broad spectrum of PBPCs, including primitive and committed progenitor cells. These data might have relevant implications for autologous and allogeneic anticancer therapy in humans.

With a view to using multiple injections of anti-cancer dendritic cell (DC)-based vaccines, we evaluated the feasibility of the adenoviral transduction of large amounts of human CD34+ cell-derived DCs, and analysed the persistence of the transgene expression and the integrity of DC functional activity after the transduction/cryopreservation procedures. Mature DCs generated from highly enriched human CD34+ cells were transduced by a recombinant adenovirus (rAd-MFG) that carried a modified, membrane-exposed, alkaline phosphatase (AP) sequence as the reporter gene. Cationic lipids such as LipofectAmine or poly-L-lysine were mixed with the viral particles before the transduction of the target cells. The highest transduction efficiency was obtained at a multiplicity of infection (MOI) rate of 500 (AP + DCs: 50 +/- 2%, viability =95%) under both small- and large-scale conditions. The addition of poly-L-lysine or LipofectAmine increased the percentage of transduced cells at an MOI of 500 (CD1a+/AP+ cells = 85 +/- 3% and 80 +/- 2% respectively). Polycations made it possible to reduce the amounts of viral particles, with high efficiency of transduction being achieved at a MOI of 100 with 10 microg/ml poly-L-lysine (CD1a+/AP+: 68 +/- 9%) or 30 microg/ml LipofectAmine (CD1a+/AP+: 60 +/- 7%). Evaluation of the immunophenotype of the transduced DCs showed that the lack of a DC subpopulation was more susceptible to adenoviral transduction. Cryopreservation of transduced DCs did not modify the viability or percentage of AP+ cells that maintain antigen-presenting cell (APC) functions. These findings indicate the efficacy of this method for the transduction of large amounts of CD34+ cell-derived DCs using small quantities of adenoviral vector mixed with polycations. Cryopreservation of transduced DCs did not damage their viability or APC functions, thus making it possible to plan multiple injections of engineered DC-based vaccines.