Cefazolin tissue concentrations with a prophylactic dose administered before sleeve gastrectomy in obese patients: a single centre study in 116 patients.

BACKGROUND: In obese patients undergoing sleeve gastrectomy, the blood and fatty-tissue concentrations of cefazolin required for adequate antibiotic prophylaxis are uncertain. METHODS: This was a single centre prospective study in obese (Group A: 40≤ BMI ≤50 kg m-2) and severely obese (Group B: 50< BMI ≤65 kg m-2) patients undergoing bariatric surgery. Blood and fatty-tissue samples were collected after a cefazolin 4 g i.v. injection. The primary aim was to compare cefazolin concentrations in subcutaneous fatty tissue with a targeted tissue concentration of 4 µg g-1 according to Staphylococcus aureus resistance breakpoint. RESULTS: One hundred and sixteen patients were included: 79 in Group A and 37 in Group B. At the beginning of the surgery, cefazolin concentration in subcutaneous fatty tissue was 12.2 (5.4) µg g-1 in Group A and 12 (6.1) µg g-1 in Group B (P=0.7). At the end, cefazolin concentrations in subcutaneous fatty tissue were 9.0 (4.9) and 7.8 (4.2) µg g-1 in Groups A and B, respectively (P=0.2). The plasma concentration of free cefazolin during surgery was higher in Group A than in Group B (P<0.0001). Fatty-tissue concentrations of 95% and 83% patients in Groups A and B, respectively, were above S. aureus resistance breakpoint. CONCLUSIONS: After a 4 g dose, the concentrations of cefazolin in fatty tissue were above the 4 µg g-1 tissue concentration target, providing adequate antibiotic tissue concentrations during bariatric surgery. As cefazolin concentration in fatty tissue is a surrogate endpoint, the results should be considered in conjunction with the results on free cefazolin concentrations in subcutaneous tissue. CLINICAL TRIAL REGISTRATION: NCT01537380.

Leaf wounding or simulated herbivory in young N. attenuata plants reduces carbon delivery to roots and root tips.

MAIN CONCLUSION: In Nicotiana attenuata seedlings, simulated herbivo ry by the specialist Manduca sexta decreases root growth and partitioning of recent photoassimilates to roots in contrast to increased partitioning reported for older plants. Root elongation rate in Nicotiana attenuata has been shown to decrease after leaf herbivory, despite reports of an increased proportion of recently mobilized photoassimilate being delivered towards the root system in many species after similar treatments. To study this apparent contradiction, we measured the distribution of recent photoassimilate within root tissues after wounding or simulated herbivory of N. attenuata leaves. We found no contradiction: herbivory reduced carbon delivery to root tips. However, the speed of phloem transport in both shoot and root, and the delivery of recently assimilated carbon to the entire root system, declined after wounding or simulated herbivory, in contrast with the often-reported increase in root partitioning. We conclude that the herbivory response in N. attenuata seedlings is to favor the shoot and not bunker carbon in the root system.

Increased Th17/Treg ratio in chronic liver GVHD.

The contribution of Th17 cells to chronic GVHD (cGVHD) has been demonstrated in cGVHD mouse models. However, their contribution to human liver cGVHD remains unclear. We evaluated Th17 cells in biopsies from a cohort of 17 patients with liver cGVHD. We observed a significant increase in Th17 cells in the liver of patients with cGVHD, as demonstrated by an increase in CCR6+, CD161+ and RORγt+ T cells (P=0.03, P=0.0001 and P=0.03, respectively). We also assessed the presence of Th1 and regulatory (Treg) T cells: the numbers of Th1 and Treg cells were very low, with no difference between the two groups (P=0.88 and P=0.12, respectively). Furthermore, Th17/Th1 and Th17/Treg ratios were significantly increased in the liver of patients with liver cGVHD (P=0.005 and P=0.002, respectively). This study provides evidence for an infiltration by Th17 cells in the liver of patients with cGVHD and an increased Th17/Treg ratio, suggesting a defect in the regulatory mechanism driven by Treg cells or an inappropriate activation of effectors cells, especially Th17 cells, or both mechanisms, in human liver cGVHD.

Pharmacological characterization of histone deacetylase inhibitor and tumor cell-growth inhibition properties of new benzofuranone compounds.

Epigenetic modifications, such as DNA methylation or histone deacetylation, are early events in cell tumorigenesis. The consequences of these modifications are repression of gene transcription and, notably, of tumor suppressor gene transcription. New therapeutic strategies aim to 'normalize' the epigenetic status of cancer cells. Histone deacetylase inhibitors (HDACi) have shown promising effects against proliferation and resistance to apoptosis of a large number of cancer cells. Vorinostat (SAHA), a hydroxamate HDACi, has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of refractory cutaneous T-cell lymphoma (CTCL). However, HDACi are poorly specific, present toxicities and many have very low half-lives in the plasma. Thus, the development of new compounds is necessary in order to increase the potential of HDACi in cancer treatment. We designed an assay, based on bioluminescence resonance energy transfer (BRET) technology, to screen and characterize HDACi activity in living cells. Using our specific and reproducible BRET assay, we characterized the pharmacological properties of benzofuranone HDACi compounds for the induction of histone acetylation and performed a comparison with the properties of suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA). We defined a benzofuranone HDACi compound that induced histone acetylation at nanomolar concentrations and showed an increased duration of histone acetylation. These properties correlated with the pharmacological properties of this HDACi for the growth inhibition of cancer cells. We, thus, demonstrated the applicability of BRET technology for the screening and characterization of new HDACi compounds in living cells, and identified an interesting benzofuranone HDACi.

Recognition of pleural mesothelioma by mucin-1(950-958)/human leukocyte antigen A*0201-specific CD8+ T-cells.

Recent clinical investigations have demonstrated that T-cell-based immunotherapy of malignant pleural mesothelioma (MPM) could represent an alternative to the other therapeutic strategies. However, its development suffers from the lack of identified tumour antigenic targets. Mucin (MUC)1, which is expressed and recognised by cytotoxic T-cells in numerous cancer types, has not been investigated as a potential immune target in MPM. Thus, the objective of this study was to analyse MUC1 expression by MPM cells and to determine whether this antigen can be the target of cytotoxic CD8+ T-cells (cytotoxic T-lymphocytes (CTLs)). We first evaluated the expression and glycosylation of MUC1 by MPM cell lines using different MUC1-specific monoclonal antibodies. We then obtained a CTL clone specific for a MUC1 peptide (residues 950-958) presented by human leukocyte antigen (HLA)-A*0201 and studied its interferon-γ and cytotoxic response to MPM cell lines. We found that all MPM cell lines expressed MUC1 protein at the cell surface with different glycosylation profiles. We also observed that HLA-A*0201+ MPM cell lines are recognised and lysed by a HLA-A*0201/MUC1(950-958)-specific CTL clone independently of the MUC1 glycosylation profile. Thus, MUC1 expression and antigen presentation by MPM cells may represent an attractive target for immunotherapeutic treatment of MPM despite its hyperglycosylated profile.

A 5-aza-2'-deoxycytidine/valproate combination induces cytotoxic T-cell response against mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive tumour with a limited response to conventional therapy. The aim of this study was to evaluate the anticancer effect of a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-azaCdR), and two histone deacetylase inhibitors, valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA). Human mesothelioma cells were treated with each epigenetic drug, either alone or in combinations. The cytotoxic effects on treated cells and the expression of specific tumour antigens were evaluated. The recognition of treated cells by a specific CD8+ T-cell clone was also measured. Additionally, the effect of combined treatments was tested in a murine model of mesothelioma. We showed that VPA and SAHA synergised with 5-azaCdR to kill MPM cells and induce tumour antigen expression in the remaining living tumour cells. As a consequence, tumour cells expressing these antigens were recognised and lysed by specific CD8+ cytotoxic T-cells. In vivo, treatment with 5-azaCdR/VPA inhibited tumour growth, and promoted lymphocyte infiltration and an immune response against tumour cells. Appropriate epigenetic drug combinations, in addition to inducing mesothelioma cell death, also affect the immunogenic status of these cells. This property could be exploited in clinical investigations to develop MPM treatments combining chemotherapeutic and immunotherapeutic approaches.

Jasmonic acid does not mediate root growth responses to wounding in Arabidopsis thaliana.

Jasmonic acid (JA) is a crucial plant defence signalling substance that has recently been shown to mediate herbivory-induced root growth reduction in the ecological model species Nicotiana attenuata. To clarify whether JA-induced reduction of root growth might be a general response increasing plant fitness under biotic stress, a suite of experiments was performed with the model plant Arabidopsis thaliana. JA bursts were elicited in leaves of A. thaliana in different ways. Root growth reduction was neither induced by foliar application of herbivore oral secretions nor by direct application of methyl jasmonate to leaves. Root growth reduction was observed when leaves were infected with the pathogen Pseudomonas syringae pv. tomato, which persistently induces the JA signalling pathway. Yet, high resolution growth analyses of this effect in wild type and JA biosynthesis knock-out mutants showed that it was elicited by the bacterial toxin coronatine that suggests ethylene- but not JA-induced root growth reduction in A. thaliana. Overall, the results demonstrate that the reaction of root growth to herbivore-induced JA signalling differs among species, which is discussed in the context of different ecological defence strategies among species.

Herbivore-induced jasmonic acid bursts in leaves of Nicotiana attenuata mediate short-term reductions in root growth.

Root growth in Nicotiana attenuata is transiently reduced after application of oral secretions (OS) of Manduca sexta larvae to wounds in leaves. Feeding of M. sexta or OS elicitation is known to result in jasmonic acid (JA) and ethylene bursts, and activates a suite of defence responses. Because both plant hormones are known to strongly reduce root growth, their activation might account for the observed reduction of root growth following herbivory. To test this hypothesis, we measured primary root growth with digital image sequence processing at high temporal resolution in antisense-lipoxygenase 3 (asLOX3) and inverted repeat-coronatin-insensitive 1 (irCOI1) seedlings which are impaired in JA biosynthesis and perception, respectively, and wild-type (WT) seedlings. Higher root growth rates in irCOI1 compared with WT were observed after OS elicitation. The dynamics of wound-induced root growth reduction coincide with the dynamics of root growth reduction induced by external application of methyl JA. In an experiment with 1-methylcyclopropen (1-MCP), a potent ethylene receptor blocker, no wounding-specific difference between growth of 1-MCP-treated plants and non-treated plants was observed, suggesting that wound-induced endogenous JA and not ethylene mediates the wounding-specific reduction in root growth. Yet, inhibiting the ethylene response by applying 1-MCP led to markedly increased root growth compared with that of control plants, indicating that ethylene normally suppresses plant growth in N. attenuata seedlings.

Heterogeneous patterns of amplification of the NUP214-ABL1 fusion gene in T-cell acute lymphoblastic leukemia.

Episomes with the NUP214-ABL1 fusion gene have been observed in 6% of T-ALL. In this multicentric study we collected 27 cases of NUP214-ABL1-positive T-ALL. Median age was 15 years with male predominance. Outcome was poor in 12 patients. An associated abnormality involving TLX1 or TLX3 was found in all investigated cases. Fluorescent in situ hybridization revealed a heterogeneous pattern of NUP214-ABL1 amplification. Multiple episomes carrying the fusion were detected in 24 patients. Episomes were observed in a significant number of nuclei in 18 cases, but in only 1-5% of nuclei in 6. In addition, intrachromosomal amplification (small hsr) was identified either as the only change or in association with episomes in four cases and two T-ALL cell lines (PEER and ALL-SIL). One case showed insertion of apparently non-amplified NUP214-ABL1 sequences at 14q12. The amplified sequences were analyzed using array-based CGH.These findings confirm that the NUP214-ABL1 gene requires amplification for oncogenicity; it is part of a multistep process of leukemogenesis; and it can be a late event present only in subpopulations. Data also provide in vivo evidence for a model of episome formation, amplification and optional reintegration into the genome. Implications for the use of kinase inhibitors are discussed.

Root growth of Nicotiana attenuata is decreased immediately after simulated leaf herbivore attack.

Image-based non-destructive methods were used to quantify root growth reactions happening within hours following simulated leaf herbivore attack.1 The induction of wound reactions in leaves of seedlings of Nicotiana attenuata led to transiently decreased root growth rates: Upon application of the oral secretions and regurgitants of the specialist herbivore Manduca sexta, a transient decrease in root growth was observed that was more pronounced than if a mere mechanical wounding was imposed. Root growth reduction was more severe than leaf growth reduction and the timing of the transient growth reduction coincided with endogenous bursts of jasmonate (JA) and ethylene emissions reported in literature. The reaction of root growth was superimposed by a strong diel variation of root growth, which was caused by the fluctuating temperature to which the plants were exposed. Apart from the observed root growth reaction, other defense-related traits such as increased nicotine concentration, trichome length and density were activated within 72 h after wounding. Further experiments indicated that the response was elicited by fatty acid-amino acid conjugates that are contained in the oral secretions and that JA signalling is crucial for root-shoot communication here.

[Translational research and Cancer Plan]. / Recherche translationnelle et plan Cancer.

The French Cancer Plan 2003-2007 has made translational research central to its research programme, to ensure the care-research continuum and the quickest application possible for the most recent discoveries, for the patients' benefit. This is a new field of research, still little-known or ill-understood. A working group, composed of physicians and researchers from academic research and industrial research, sought to define translational research in cancerology and define the issues at stake in it. Translational research needs to develop in close connection with the patients in order to enable a bi-directional flow of knowledge from cognitive research toward medical applications and from observations made on patients toward cognitive research. Placed under the aegis of the French National Cancer Institute and Leem Research, the group has put forth a strategy for implementing translational research in cancerology in France to make it attractive, competitive and efficient and to foster the development of public-private partnerships.

NaRALF, a peptide signal essential for the regulation of root hair tip apoplastic pH in Nicotiana attenuata, is required for root hair development and plant growth in native soils.

Rapid alkalinization factor (RALF) is a 49-amino-acid peptide that rapidly alkalinizes cultivated tobacco cell cultures. In the native tobacco Nicotiana attenuata, NaRALF occurs as a single-copy gene and is highly expressed in roots and petioles. Silencing the NaRALF transcript by transforming N. attenuata with an inverted-repeat construct generated plants (irRALF) with normal wild-type (WT) above-ground parts, but with roots that grew longer and produced trichoblasts that developed into abnormal root hairs. Most trichoblasts produced a localized 'bulge' without commencing root hair tip growth; fewer trichoblasts grew, but were only 10% as long as those of WT plants. The root hair phenotype was associated with slowed apoplastic pH oscillations, increased pH at the tips of trichoblasts and decreased accumulation of reactive oxygen species in the root hair initiation zone. The root hair growth phenotype was partially restored when irRALF lines were grown in a low-pH-buffered medium, and reproduced in WT plants grown in a high-pH-buffered medium. When irRALF plants were grown in pH 5.6, 6.7 and 8.1 soils together with WT plants in glasshouse experiments, they were out-competed by WT plants in basic, but not acidic, soils. When WT and irRALF lines were planted into the basic soils of the native habitat of N. attenuata in the Great Basin Desert, irRALF plants had smaller leaves, shorter stalks, and produced fewer flowers and seed capsules than did WT plants. We conclude that NaRALF is required for regulating root hair extracellular pH, the transition from root hair initiation to tip growth and plant growth in basic soils.

Root growth dynamics of Nicotiana attenuata seedlings are affected by simulated herbivore attack.

Many studies demonstrate resource-based trade-offs between growth and defence on a large timescale. Yet, the short-term dynamics of this growth reaction are still completely unclear, making it difficult to explain growth-defence trade-offs mechanistically. In this study, image-based non-destructive methods were used to quantify root growth reactions happening within hours following simulated herbivore attack. The induction of wound reactions in Nicotiana attenuata in the seedling stage led to transiently decreased root growth rates. Application of the oral secretion of the specialist herbivore Manduca sexta to the leaves led to a transient decrease in root growth that was more pronounced than if a mere mechanical wounding was imposed. Root growth reduction was more pronounced than leaf growth reduction. When fatty acid-amino acid conjugates (FACs) were applied to wounds, root growth reduction occurred in the same intensity as when oral secretion was applied. Timing of the transient growth reduction coincided with endogenous bursts of jasmonate (JA) and ethylene emissions reported in literature. Simulation of a wound response by applying methyl jasmonate (MeJA) led to more prolonged negative effects on root growth. Increased nicotine concentrations, trichome lengths and densities were observed within 72 h in seedlings that were treated with MeJA or that were mechanically wounded. Overall, these reactions indicate that even in a very early developmental stage, the diversion of plant metabolism from primary (growth-sustaining) to secondary (defence-related) metabolism can cause profound alterations of plant growth performance.

Clinical, cytogenetic and molecular characteristics of 14 T-ALL patients carrying the TCRbeta-HOXA rearrangement: a study of the Groupe Francophone de Cytogénétique Hématologique.

Recently, we and others described a new chromosomal rearrangement, that is, inv(7)(p15q34) and t(7;7)(p15;q34) involving the T-cell receptor beta (TCRbeta) (7q34) and the HOXA gene locus (7p15) in 5% of T-cell acute lymphoblastic leukemia (T-ALL) patients leading to transcriptional activation of especially HOXA10. To further address the clinical, immunophenotypical and molecular genetic findings of this chromosomal aberration, we studied 330 additional T-ALLs. This revealed TCRbeta-HOXA rearrangements in five additional patients, which brings the total to 14 cases in 424 patients (3.3%). Real-time quantitative PCR analysis for HOXA10 gene expression was performed in 170 T-ALL patients and detected HOXA10 overexpression in 25.2% of cases including all the cases with a TCRbeta-HOXA rearrangement (8.2%). In contrast, expression of the short HOXA10 transcript, HOXA10b, was almost exclusively found in the TCRbeta-HOXA rearranged cases, suggesting a specific role for the HOXA10b short transcript in TCRbeta-HOXA-mediated oncogenesis. Other molecular and/or cytogenetic aberrations frequently found in subtypes of T-ALL (SIL-TAL1, CALM-AF10, HOX11, HOX11L2) were not detected in the TCRbeta-HOXA rearranged cases except for deletion 9p21 and NOTCH1 activating mutations, which were present in 64 and 67%, respectively. In conclusion, this study defines TCRbeta-HOXA rearranged T-ALLs as a distinct cytogenetic subgroup by clinical, immunophenotypical and molecular genetic characteristics.

[A case of de novo acute basophilic leukaemia: diagnostic criteria and review of the literature]. / A propos d'un cas de leucémie à basophiles de novo: critères diagnostiques et revue de la littérature.

We report a case of a de novo acute basophilic leukaemia, revealed by an infectious pneumopathy in a 73 year old man. The full blood count revealed an hyperleucocytosis associated with an unregenerative normocytic normochrom anaemia and a thrombocytopenia. The blood and bone marrow smears showed a mixture of undifferentiated blast cells and basophiloblasts (high nucleo-cytoplasmic ratio, coarse basophilic cytoplasmic granules), along with basophilic precursors and basophilic polymorphonuclears. All the blasts were MPO negative but positive for the toluidine blue metachromatic coloration, which is considered as consistent with basophilic lineage. Immunophenotypic studies showed myeloid blasts, without maturity marker, CD 117 negative and CD203 cytoplasmic positive, the latter known to be highly representative of the basophilic lineage. This very clear-cut phenotype, associated with the morphology of cells, were arguments to ascertain the basophilic lineage of the blasts without the need of electron microscopic study. Cytogenetic and RNA analysis revealed the presence of a Philadelphia chromosome and of a BCR-ABL transcript with the unusual junction e6a2. Thus, imatinib was added to the conventional chimiotherapy and the patient is currently in complete remission. This clinical prompted allows us to review the literature on acute basophilic leukaemia and to state on the different diagnostic criteria of this rare disorder.

Culture medium and protein supplementation in the generation and maturation of dendritic cells.

Dendritic cells (DC) are powerful antigen-presenting cells that have drawn many attentions due to the recent development of anti-cancer vaccines. Clinical grade production of monocyte-derived DC (Mo-DC) is extensively studied, and many efforts are made to develop and improve clinical standard operating procedures. Most of the parameters involved, such as the cytokines and maturation agents, have been widely assessed. However, very few are investigated about how culture medium and additional protein components affect DC yield, viability and maturation. Thus, our study aimed to compare the impact of standard culture medium on Mo-DC differentiation and maturation. Commercially available media for hematopoietic cell culture as well as different protein supplementations, that is foetal calf serum (FCS), autologous plasma (AP), human serum (HS) and human serum albumin (HSA) were tested. Culture yields, cell viability and DC maturation were investigated. Differentiation yields were similar between the conditions used. However, we evidenced significant differences in terms of cytotoxicity and DC maturation (phenotypic and functional). This underscores the importance of defining culture medium composition in clinical standard operating procedures to insure quality control, and also when preparing DC for experimental uses.

t(5;14)/HOX11L2-positive T-cell acute lymphoblastic leukemia. A collaborative study of the Groupe Français de Cytogénétique Hématologique (GFCH).

To accurately estimate the incidence of HOX11L2 expression, and determine the associated cytogenetic features, in T-cell acute lymphoblastic leukemia (T-ALL), the Groupe Français de Cytogénétique Hématologique (GFCH) carried out a retrospective study of both childhood and adult patients. In total, 364 patients were included (211 children

[Acute myeloblastic leukemia without maturation (AML-M1) with basophilic elements and associated with translocation t(6;9)]. / Leucémie aiguë myéloblastique sans maturation (LAM1) avec éléments basophiles associée à la translocation t(6;9).

The clinical, hematological, and cytogenetic data from a 4 year-old child with acute myeloid (AML-M1) and basophilia is reported. Interestingly, cytogenetic investigations revealed the presence of the translocation t(6;9) (p23;q34). This abnormality is rare and associated with myelodysplastic syndromes or with subtypes of acute myeloid leukemia (M1, M2, M4, M7), usually with preceding or underlying myelodysplasia. The prognosis is poor, without response to chemotherapy regimen alone. Allogeneic bone marrow transplantation appears likely to be a more appropriate treatment.

Anti-cancer therapy using dendritic cells and apoptotic tumour cells: pre-clinical data in human mesothelioma and acute myeloid leukaemia.

We have recently reported in an experimental model, that treatments based on the injections of dendritic cells which had phagocytosed apoptotic bodies derived from tumour cells were particularly effective in the cure of tumour-bearing animals. We proposed that systems using processing and presentation of antigenic molecules from antigen-presenting cells primed with apoptotic bodies can offer new opportunities in anti-cancer treatment. We first established the technical conditions for purification, characterisation and production of tumour cells isolated from fresh pleural liquid or blood. Then we compared efficacy of different apoptotic inducers agents on the cancer cells in culture. The apoptotic tumour cells were purified, characterised and maintained in coculture with monocytes-derived immature dendritic cells. We subsequently investigated the effect of the maturation process on phagocytosis of apoptotic bodies. We have shown that whatever the nature of the apoptotic cells they are phagocytosed by the dendritic cells which were efficiently matured using the combination of TNFalpha+Poly I:C. Furthermore, we demonstrated that the generation of the mature dendritic cells pulsed with apoptotic tumour cells, successfully generated CD4(+) (Th1) and CD8(+) (CTL) cells. All the experimental procedures that we have used were developed with clinical use in mind, using Good Manufacturing Products. We are presently investigating the feasibility of such a "vaccine" for the treatment of asbestos mesothelioma or acute myeloid leukaemia.