Comparison of efficacy and toxicity according to etoposide and cytarabine dosing in BEAM conditioning followed by autologous stem cell transplantation in Hodgkin lymphoma.

The combination of carmustine, etoposide, cytarabine, and melphalan (BEAM) followed by autologous stem cell transplantation (ASCT) is a commonly used intensification regimen for patients with Hodgkin lymphoma. As etoposide and cytarabine dosing are not defined, we conducted a retrospective, multicenter study, to compare efficacy and toxicity in 130 patients with Hodgkin lymphoma receiving etoposide and cytarabine at either 200 mg/m2/d (n = 50), 400 mg/m2/d (n = 35), or etoposide 200 mg/m2/d and cytarabine 400 mg/m2/d (n = 45). Progression-free survival and overall survival were not associated with the intensity of conditioning. Increased conditioning intensity was associated with longer duration of thrombocytopenia, a higher number of transfused RBC and platelet units and a higher frequency of mucositis, but serious adverse events or infectious complications were not increased. The intensity of BEAM regimen was not associated with survival but with the rate of cytopenia and mucositis advocating for the use of lower dosing in frail patients.

Impact of allogeneic stem cell transplantation comorbidity indexes after haplotransplant using post-transplant cyclophosphamide.

BACKGROUND: Three different scoring systems have been developed to assess pre-transplant comorbidity in allogeneic hematopoietic stem cell transplantation (Allo-HSCT): the Hematopoietic Cell Transplantation-Specific Comorbidity Index, the Comorbidity/Age index, and the Augmented Comorbidity/Age index. All were devised to predict overall survival (OS) and disease-free survival (DFS) survivals and non-relapse mortality (NRM) in patients receiving HLA-matched Allo-HSCT, but their performance has scarcely been studied in the haploidentical Allo-HSCT setting with post-transplant cyclophosphamide, a procedure in constant expansion worldwide. METHODS: To address this issue, their impact on survivals and NRM was examined in a cohort of 223 patients treated with haploidentical Allo-HSCT in four different centers. RESULTS: With a median follow-up of 35.6 months, 3-year OS, DFS, and NRM were 48.1% ± 4%, 46.3% ± 4%, and 30.0% ± 3%, respectively. No impact was found for any of the three comorbidity scores in univariate analysis. In multivariate analyses, the only three factors associated with lower OS were DRI (p < 0.001), an older age of recipients (≥55 years old, p = 0.02) and of donors (≥40 years old, p = 0.005). Older donor age was also associated with lower DFS and higher NRM. CONCLUSION: The comorbidity scores do not predict survivals nor NRM in haploidentical Allo-HSCT with PTCY, suggesting that pre-transplant comorbidities should not be a contra-indication to this procedure.

Early Post-Transplantation Serum Ferritin Level Predicts Survival in Recipients of Haploidentical Stem Cell Transplantation Using Post-Transplantation Cyclophosphamide as Graft-versus-Host Disease Prophylaxis.

The negative impact of high serum ferritin level (SFL) before and after allogeneic hematopoietic cell transplantation (allo-HSCT) on outcomes is well recognized. However, it is poorly documented in adults undergoing haploidentical HSCT (haplo-HSCT) with post-transplantation cyclophosphamide (PTCY) for hematologic malignancies. The main objective was to assess the impact of pretransplantation and post-transplantation SFL on overall survival (OS), disease-free survival (DFS), and nonrelapse mortality (NRM) in patients undergoing haplo-HSCT with PTCY. The secondary objective was to identify factors associated with outcomes after transplantation by comparing SFL with other parameters related to the status of patients or donors. This multicentric retrospective study included 223 consecutive patients who underwent haplo-HSCT with PTCY in 4 French centers (Nantes, Angers, Besançon, and Brest) between October 2013 and January 2020. The impact of SFL on OS, DFS, and NRM at different time points was assessed based on receiver operating characteristic curves. With a median follow-up of 37.6 months (interquartile range, 23.5 to 51.0 months), 3-year OS, DFS, and NRM were 48.1 ± 4%, 46.3 ± 4%, and 30.0 ± 3%, respectively. Pretransplantation SFL had no impact on outcomes irrespective of the cutoff tested. Considering patients alive at 3 months post-transplantation, an SFL ≥3500 µg/L at 3 months was statistically significantly associated with worse 3-year OS (32.7 ± 8.7% versus 53.4 ± 7.2%; P = .01) and DFS (30.1 ± 8.2% versus 53.1 ± 7.1%; P = .008), with a trend toward higher NRM (33.2 ± 8.6% versus 17.6 ± 5.4%; P = .10). Similarly, high SFL (≥2700 µg/L) at 6 months post-transplantation was associated with worse 3-year OS (56.1 ± 9.1% versus 79.2 ± 6.0%; P = .02) and DFS (53.6 ± 8.7% versus 74.9 ± 6.2%; P = .01), with a trend toward higher NRM (21.4 ± 7.4% versus 8.2 ± 4.0%; P = .10). In multivariate analysis, high 3-month and 6-month FL remained associated with lower OS and DFS, with a trend toward higher NRM. Pretransplantation SFL appears to have no impact on outcomes in haplo-HSCT with PTCY, in contrast to what is documented in the matched allo-HSCT setting. In contrast, in the haplo-HSCT setting, high SFL early post-transplantation is associated with lower survival and a trend toward higher NRM.

Adhesion, proliferation and osteogenic differentiation of human MSCs cultured under perfusion with a marine oxygen carrier on an allogenic bone substitute.

Tissue engineering strategies have been developed to optimize osseointegration in dental implant surgery. One of the major problems is the non-homogeneous spatial cell distribution in the scaffold, as well as subsequent matrix production. Insufficient nutrient and oxygen supplies inside the scaffold are factors in this phenomenon. To mediate this gradient formation, we have implemented a perfusion culture method to seed human bone marrow mesenchymal stem cells (MSCs) into three-dimensional (3-D)-allogenic bone scaffolds in combination with a marine haemoglobin, HEMOXCell®, for oxygen delivery. Cell culture was performed under static and perfusion conditions, with standard and osteogenic media, with and without HEMOXCell®. The cell seeding efficiency, as well as MSC/scaffold cytocompatibly were assessed using viability and proliferation assays. Scaffolds' cellularization and extracellular matrix (ECM) formation were analyzed using scanning electron microscopy and histological staining. Cell differentiation was investigated with osteogenic biomarkers gene expression analysis. The perfusion culture was observed to significantly promote MSC proliferation and differentiation throughout the scaffolds, especially when using the induction medium w/HEMOXCell®. Our data suggest that perfusion culture of MSC into allogenic bone substitute with HEMOXCell® as a natural oxygen carrier is promising for tissue engineering applications to oxygenate hypoxic areas and to promote cellular proliferation.

HEMOXCell, a New Oxygen Carrier Usable as an Additive for Mesenchymal Stem Cell Culture in Platelet Lysate-Supplemented Media.

Human mesenchymal stem cells (MSCs) are promising candidates for therapeutic applications such as tissue engineering. However, one of the main challenges is to improve oxygen supply to hypoxic areas to reduce oxygen gradient formation while preserving MSC differentiation potential and viability. For this purpose, a marine hemoglobin, HEMOXCell, was evaluated as an oxygen carrier for culturing human bone marrow MSCs in vitro for future three-dimensional culture applications. Impact of HEMOXCell on cell growth and viability was assessed in human platelet lysate (hPL)-supplemented media. Maintenance of MSC features, such as multipotency and expression of MSC specific markers, was further investigated by biochemical assays and flow cytometry analysis. Our experimental results highlight its oxygenator potential and indicate that an optimal concentration of 0.025 g/L HEMOXCell induces a 25%-increase of the cell growth rate, preserves MSC phenotype, and maintains MSC differentiation properties; a two-fold higher concentration induces cell detachment without altering cell viability. Our data suggest the potential interest of HEMOXCell as a natural oxygen carrier for tissue engineering applications to oxygenate hypoxic areas and to maintain cell viability, functions and "stemness." These features will be further tested within three-dimensional scaffolds.

Advancement in recombinant protein production using a marine oxygen carrier to enhance oxygen transfer in a CHO-S cell line.

Recombinant proteins, particularly proteins used as therapeutics, are widely expressed for bioprocessing manufacturing processes. Mammalian cell lines represent the major host cells for bioproduction, according to their capacities of post-translational modifications and folding of secreted proteins. Many parameters can affect cell productivity, especially the rate of oxygen transfer. Dissolved oxygen, in high or low proportions, is a crucial parameter which can affect cell viability and thus productivity. HEMARINA has developed a new technology, commercially proposed as HEMOXCell(®), to improve cell culture at a large production scale. HEMOXCell(®) is a marine oxygen carrier having properties of high oxygen sensitivity, to be used as an oxygen additive during cell culture manufacturing. In this study, we investigated the effects of HEMOXCell(®) on the culture of the commonly used CHO-S cell line. Two main objectives were pursued: 1) cell growth rate and viability during a batch mode process, and 2) the determination of the effect of this oxygen carrier on recombinant protein production from a CHO-transfected cell line. Our results show an increase of CHO-S cellular growth at a rate of more than four-fold in culture with HEMOXCell(®). Moreover, an extension of the growth exponential phase and high cell viability were observed. All of these benefits seem to contribute to the improvement of recombinant protein production. This work underlines several applications using this marine-type oxygen carrier for large biomanufacturing. It is a promising cell culture additive according to the increasing demand for therapeutic products such as monoclonal antibodies.

Biosimilars of filgrastim in autologous stem cell transplant: reduction in granulocyte-colony stimulating factor costs, but similar effects on bone marrow recovery.

Granulocyte-colony stimulating factors (G-CSFs) enhance bone marrow (BM) recovery after autologous stem cell transplant (ASCT) in patients with lymphoma and myeloma. Few publications exist that discuss the use of filgrastim biosimilars after ASCT. We conducted a single-center retrospective study in patients with lymphoma and myeloma treated at Brest Hospital to assess the cost reductions related to and the efficiency and safety of filgrastim biosimilars. We identified 65 patients with lymphoma or myeloma treated with filgrastim biosimilars for ASCT and compared 19 parameters of these patients, including BM recovery, side effects, infectious complications and treatment costs, with published historical data on a cohort of 50 patients treated with classic filgrastim. We observed a significant reduction of G-CSF costs in both groups but did not observe a change in total hospitalization costs (representing less than 2% of the costs) between groups. Additionally, we did not observe differences between the two groups in BM recovery, infectious complications, side effects or the other studied parameters. In this retrospective study, the absence of differences between groups after ASCT in lymphoma and myeloma led us to believe that these drugs could be safely and effectively used for such indications without a significant impact on hospitalization costs. A prospective study should be conducted to confirm our results.

Delayed G-CSF stimulation after PBSCT does not seem to modify the biological parameters of bone marrow recovery.

There are currently no recommendations indicating when stimulation should begin after autologous peripheral blood stem cell transplantation (PBSCT). We compared the outcome following between two treatment groups, in which daily granulocyte colony stimulating factor (G-CSF) administration began on either the fifth or the eighth day after PBSCT in lymphoma and myeloma patients. We studied eight clinical parameters: number of G-CSF injections, number of days of hospitalization, of red blood cell or platelet transfusions; days when body temperature exceeds 38°C; days of parenteral nutrition; weight loss and hospitalization costs. We studied also four biological parameters: number of CD34+ cells, days with leucocytes less than 1 × 10(9) /L, days with hemoglobin less than 90 g/L or with less than 50 × 10(9) /L of platelets. There were no statistical significant differences between the study arms. It seems that delayed stimulation by G-CSF after PBSCT is safety and does not seem to modify bone marrow recovery timing.

Preclinical evidence that use of TRAIL in Ewing's sarcoma and osteosarcoma therapy inhibits tumor growth, prevents osteolysis, and increases animal survival.

PURPOSE: Osteosarcoma and Ewing's sarcoma are high-grade neoplasms typically arising in the bones of children and adolescents. Despite improvement in therapy, the five-year survival rate is only 20% for patients not responding to treatment or presenting with metastases. Among new therapeutic strategies, the efficacy of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily with strong antitumoral activity and minimal toxicity to most normal cells and tissues, was investigated by complementary approaches both in vitro and in preclinical models. EXPERIMENTAL DESIGN: The sensitivity of osteosarcoma and Ewing's sarcoma cell lines to TRAIL was investigated in vitro by determining TRAIL receptor expression together with TRAIL effects on cell viability and apoptosis. Complementary preclinical studies were carried out in respective tumor models by inoculation of osteosarcoma or Ewing's sarcoma tumor cells in paraosseous location. In addition, a model of lung nodule dissemination was developed by i.v. injection of osteosarcoma cells. RESULTS: In vitro, both osteosarcoma and Ewing's sarcoma cells that express the TRAIL death receptors were highly sensitive to TRAIL-induced caspase-8-mediated apoptosis. TRAIL administered in vivo by nonviral gene therapy inhibited primary bone tumor incidence and growth by 87% and prevented tumor-induced osteolysis, leading to a significant 2-fold increase in animal survival 40 days after tumor induction. Furthermore, TRAIL inhibited tumor nodule dissemination in lungs and increased survival in an osteosarcoma model. CONCLUSION: These findings suggest that TRAIL is a promising candidate for the development of new therapeutic strategies in the most frequent malignant primary bone tumors.

A novel cationic lipophosphoramide with diunsaturated lipid chains: synthesis, physicochemical properties, and transfection activities.

Cationic lipophosphoramidates constitute a class of cationic lipids we have previously reported to be efficient for gene transfection. Here, we synthesized and studied a novel lipophosphoramidate derivative characterized by an arsonium headgroup linked, via a phosphoramidate linker, to an unconventional lipidic moiety consisting of two diunsaturated linoleic chains. Physicochemical studies allowed us to comparatively evaluate the specific fluidity and fusogenicity properties of the liposomes formed. Although corresponding lipoplexes exhibited significant but relatively modest in vitro transfection efficiencies, they showed a remarkably efficient and reproducible ability to transfect mouse lung, with in vivo transfection levels higher than those observed with a monounsaturated analogue previously described. Thus, these results demonstrate that this diunsaturated cationic lipophosphoramidate constitutes an efficient and versatile nonviral vector for gene transfection. They also invite further evaluations of the transfection activity, especially in vivo, of gene delivery systems incorporating the lipid reported herein and/or other lipids bearing polyunsaturated chains.

Activation of tumor-specific T cells by dendritic cells expressing the NY-ESO-1 antigen after transfection with the cationic lipophosphoramide KLN5.

BACKGROUND: Genetic modification of human monocyte-derived dendritic cells (DC) with cDNA sequences encoding tumor-associated antigens (TAA) is a promising strategy for cancer immunotherapy. The present study aimed to develop a nonviral gene transfer method based on the use of the cationic lipophosphoramide reagent, KLN-5, as an alternative to the commonly used viral vectors. METHODS: First, the efficiency of KLN5 for gene transfection into DC was investigated using the green fluorescent protein (GFP) reporter gene. The highest transfection efficiency/cell viability ratio was determined by flow cytometry. Next, DC were transfected with a plasmid encoding NY-ESO-1, a TAA expressed in numerous cancers, according to the transfection protocol previously established with the GFP reporter. Transfected DC were then co-cultured with a CD8+ NY-ESO-1 specific HLA-A*02.01 T cell clone to control their ability to correctly process and present the corresponding epitope in the HLA-A*02.01 context. Finally, T cell activation was assessed via flow cytometry-based detection of interferon-gamma production. RESULTS: An optimal KLN5/plasmid DNA ratio allowing both significant transgene expression and high viability of DC could be determined. Under the established experimental conditions, antigen processing and presentation of the immunodominant (SLLMWITQC(157-165)) epitope in the HLA-A*0201 context was demonstrated by activation of the NY-ESO-1-specific CD8+ T cell clone. CONCLUSIONS: KLN5-based gene transfection into DC allows the efficient induction of TAA presentation and may thus represent a novel attractive nonviral approach for cancer vaccination.

Lipophosphoramidates as lipidic part of lipospermines for gene delivery.

The DNA compacting properties of polyamines (especially spermine) are well-known, hence the use of spermine as the cationic part in several synthetic DNA carriers. Here, we describe the synthesis of modified spermines, with a "lipophosphoramidate" as the lipidic part, and their use for efficient in vitro transfection. Physicochemical measurements (particle size, zeta potentials, pKa determination) and gel retardation assays were also performed. Theoretical membrane-disrupting ability was established by FRET. Taken together, our results indicate that lipophosphoramidates constitute an interesting alternative to "classical" lipidic parts of cationic lipids used as DNA carriers.

Dicationic lipophosphoramidates as DNA carriers.

Lipophosphoramidates with two different permanent cations as polar heads were synthesized and evaluated for their gene transfer activity. Physicochemical measurements (particle size, zeta potentials) and gel retardation assays were also performed. In vitro biological evaluation was conducted with A542 and HeLa cell lines, and cytotoxicity determined by a chemiluminescent assay. The set of results indicates that, on the whole, dicationic lipophosphoramidates constitute an interesting alternative to their monocationic analogues.

EPI-hNE4, a proteolysis-resistant inhibitor of human neutrophil elastase and potential anti-inflammatory drug for treating cystic fibrosis.

EPI-hNE4 (depelstat) is a potent inhibitor of human neutrophil elastase derived from human inter-alpha-trypsin inhibitor and designed to control the excess proteolytic activity in the sputum of cystic fibrosis patients. We analyzed its resistance to the proteolysis it is likely to encounter at inflammatory sites in vivo. EPI-hNE4 resisted hydrolysis by neutrophil matrix metalloproteases (MMPs) and serine proteases that are released from activated neutrophils in inflammatory lung secretions, including MMP-8 and MMP-9, and the elastase-related protease 3 and cathepsin G. It also resisted degradation by epithelial lung cell MMP-7 but was broken down by the Pseudomonas aeruginosa metalloelastase pseudolysin, when used in a purified system, but not when this protease competed with equimolar amounts of neutrophil elastase. We also investigated the inhibitory properties of EPI-hNE4 at the surface of purified blood neutrophils and in the sputum of cystic fibrosis patients where neutrophil elastase is in both a soluble and a gel phase. The elastase at the neutrophil surface was fully inhibited by EPI-hNE4 and formed soluble complexes. The elastase in cystic fibrosis sputum supernatants was inhibited by stoichiometric amounts of EPI-hNE4, allowing titration of the protease. But the percentage of inhibition in whole sputum homogenates varied from 50 to 100%, depending on the sample tested. EPI-hNE4 was rapidly cleaved by the digestive protease pepsin in vitro. Therefore, EPI-hNE4 seems to be an elastase inhibitor suitable for use in aerosols to treat patients with cystic fibrosis.

Cationic lipophosphoramidates and lipophosphoguanidines are very efficient for in vivo DNA delivery.

Two new families of cationic lipids were designed and synthesized for gene delivery, namely "lipophosphoramidates" and "lipophosphoguanidines", whose efficiency was noteworthy. The most efficient have an arsonium cation as the polar head, and the unsaturated lipidic tails (e.g. oleyl) gave the better in vivo results (mice lungs).

Efficient gene transfer into human epithelial cell lines using glycosylated cationic carriers and neutral glycosylated co-lipids.

To date, no clear and constant relationship has been established between the chemical structure and the efficiency of non-viral transfection reagents. Despite the improvement of synthetic transfection systems, the capacity to transfect a target cell in a specific way is still a major challenge that gene therapy needs to overcome to be successful. Consequently, we developed a strategy aimed specifically at improving transfection of targeted human epithelial cells and to examine the possible effects of electrostatic interactions. Our attention therefore focused on the development of novel glycosylated formulations, based upon the introduction of one or two different carbohydrate ligands into (i) cationic lipid structures and (ii) synthetic neutral lipids incorporated into DNA and lipoplexes. Then, these formulations were tested in vitro on two human cell lines [HeLa and 16HBE14o(-)]. We report here that one of those formulations (CG 1/DOPE) is more efficient than DOTAP/DOPE. We determined that this non-viral transfection process is partially due to an endocytotic phenomenon mediated by targeting specific receptors directed toward specific carbohydrate elements. This was shown on 16HBE14o(-) cells where we observed a 43% and a 69% decrease in transfection when we blocked these receptors by the addition of free lactose and mannose, respectively. These results highlight the large adaptability of such monocationic glycolipids in the context of targeting and gene delivery.

CFTR transgene expression in primary DeltaF508 epithelial cell cultures from human nasal polyps following gene transfer with cationic phosphonolipids.

Cystic fibrosis (CF) is the most common autosomal lethal recessive disorder in the Caucasian population. The major cause of mortality is lung disease, owing to the failure of a functional protein from the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Today, even though the knowledge about the CFTR genomic is extensive, no efficient treatment has been developed yet. In this context, gene therapy represents a potential important advance on condition that it could develop efficient and safe transfection agents. Even though viral vectors have been used in most clinical trials owing to their high transfection efficiency, random integration and immunogenicity are still critical side effects. Consequently, all of these drawbacks brought forth the development of nonviral transfection systems. Although they engender few toxicity and immunogenicity problems, their low transfection efficiency is a hurdle that must be overcome. Over the past decade, we have developed an original family of monocationic lipids, cationic phosphonolipids, whose efficiency has been previously demonstrated both in vitro and in vivo. In this report, we observe that a new cationic phosphonolipid (KLN 30) can lead to the restoration of the CFTR protein following the ex vivo transfection of epithelial cells issuing from a F508 homozygous patient. The transgene expression and the cytotoxicity correlate with the charge ratio of the lipoplex. A kinetic study was performed, and a luminescent signal was detected until 35 d after transfection.

Applicability of different antibodies for the immunohistochemical localization of CFTR in respiratory and intestinal tissues of human and murine origin.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein, which has a major role as a chloride (Cl(-)) channel. Although perhaps all functions of CFTR are still not fully characterized, localization studies are necessary to understand the consequences of the more than 1000 mutations thus far identified. Our aim was to determine the histological localization of CFTR on respiratory and colon epithelia of human and murine origin with a panel of several antibodies produced against different CFTR epitopes, using an indirect immunofluorescence method. Our results on human tissues confirm the apical localization of CFTR in ciliated cells of the respiratory mucosa and show that in colon tissue CFTR is observed in both apical and basolateral membranes of epithelial cells from colon crypts. However, poor tissue preservation of colon biopsies after immunohistochemistry (IHC) raises doubts about the latter localization. Contrary to human, mouse colon epithelium (not biopsed) presents good tissue preservation and evidences many cylindrical surface cells with high apical expression of CFTR. For the antibodies' sensitivity, we demonstrate that MATG1061, 24-1, M3A7, and MPCT-1 give good results, allowing the histological localization of CFTR protein of both human and murine origin.

The use of in situ hybridization to study the transgene pathway following cellular transfection with cationic phosphonolipids.

Gene therapy is a promising field of research and biotechnological development. Considering their safety and non-immunogenicity, cationic lipids are widely used for gene transfer in vitro and show promise for in vivo gene transfer applications. However, a better understanding of the mechanisms by which transfection occurs and the limiting steps in cellular transfer of foreign DNA are critical for significant improvements of gene transfer. In this work, we have traced the plasmid DNA into human hematopoietic cell line (K562) using the in situ hybridization method in order to define the main difficulties in transfection and to design new agents better adapted to cellular constraints. In this hematopoietic cell line, after showing the efficiency of our synthetic vectors and optimizing their formulation, we observed that only 5 h after transfection the nucleus to cytoplasm signal ratio was three to one, whereas at 24 h it was one to one. In parallel, the level of the reporter protein strongly increased between these times. Those results emphasize the rapidity of transfection and lead one to imagine chemical modifications adjusted to the environment.