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This study reports the pharmacologic effects of isatuximab, a CD38 mAb, on T- and B-cell acute lymphoblastic leukemia (ALL). We analyzed CD38 expression in 50-T-ALL and 50 B-ALL clinical samples, and 16 T-ALL and 11 B-ALL cell lines. We primarily focused on in vitro assessments of isatuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In vivo assessment of isatuximab activity was performed in several ALL xenograft models, including disseminated and subcutaneous tumor models in female C.B-17 severe combined immunodeficiency mice. Our study reveals that most patients (90%–100%) carried CD38+ blasts independent of disease burden. The median CD38 receptor density on abnormal lymphoblasts is 41,026 copies/cell on T-ALL and 28,137 copies/cell on B-ALL, respectively. In patients with T-ALL, there is a significant increase of CD38 expression in abnormal blasts compared with normal T cells. High-level CD38 receptor density (RD) is critical to trigger effective isatuximab-mediated ADCC against target ALL cells. In addition, a correlation between CD38 RD and isatuximab-mediated ADCP is demonstrated. In the disseminated CD38+, T-ALL, and B-ALL xenograft models, isatuximab is able to induce robust antitumor activity, even at low doses. This study shows that isatuximab has significant in vitro and in vivo activity against ALL cells with robust ADCC and ADCP effects that are associated with CD38 expression levels in both T-ALL and B-ALL.

Resume Introduction En France, la decision de pratiquer une autopsie, lorsqu’un obstacle medico-legal est etabli, incombe au magistrat enqueteur. Il est demontre que la realisation de l’autopsie reduit le risque d’erreur dans la determination du mode et de la cause du deces. Malgre la suppression, en 2011 de la tarification a l’acte des autopsies, des considerations budgetaires peuvent entrer en jeu, notamment en fonction de la distance entre le lieu du deces et le lieu de la realisation de l’autopsie. L’objectif de cette etude etait de comparer les circonstances de deces selon leur survenue dans des zones eloignees ou des zones proches du lieu d’autopsies. Methodes L’ensemble des rapports d’autopsie des instituts medico-legaux de Grenoble, Nancy et Toulouse pour l’annee 2012 ont ete recueillis et anonymises. Le tribunal demandeur, le mode de deces, l’âge et le sexe du defunt et la cause pathologique de la mort ont ete renseignes. Les codes de la classification internationale des maladies ont ete utilises pour les causes de deces. L’hypothese principale etait que la repartition entre modes de deces violents et non violents etaient differentes entre les tribunaux proches des instituts medico-legaux et les tribunaux eloignes. Les comparaisons entre les donnees observees et les donnees attendues ont ete effectuees a l’aide du test du Chi 2 . Resultats Sept cent quarante-quatre rapports d’autopsie rediges en 2012 ont pu etre analyses. Il n’a pas ete observe de difference significative permettant de valider l’hypothese principale : les autopsies pour morts violentes demandees par les tribunaux proches representaient 278 autopsies contre 243 en peripherie, et les circonstances non violentes respectivement 134 et 84 autopsies ( p [Chi 2 ] = 0,09). Seuls les resultats en sous-groupes de la region grenobloise etaient significatifs. Il a ete mis en evidence que, rapportees au nombre de deces, davantage d’autopsies etaient realisees pour les tribunaux proches du lieu d’autopsie, soit 16,2 autopsies pour 1000 deces, contre 8,3 pour les autopsies realisees pour les tribunaux eloignes. Le materiel etudie, en termes de sex-ratio et d’âge moyen, etait conforme aux autres travaux realises dans ce domaine. Conclusion Cette etude n’a pas permis de montrer une difference significative entre les zones eloignees et proches des instituts medico-legaux quant aux circonstances de deces, cependant le volume d’autopsies rapporte au nombre de deces est plus important pour les zones ou se trouvent le lieu d’autopsie.

Resume Une serie de 9 deces intervenus dans un contexte d’usage ou de mesusage de dispositifs transdermiques (TD) a base de fentanyl (Durogesic ® ou generiques) est rapportee. Les observations concernent 5 hommes et 4 femmes âges de 26 a 73 ans, avec un nombre de patches impliques compris entre 1 et 16 (doses de 48 a 1500 μg/h). Huit cas correspondaient a des applications multiples de patches au niveau cutane (avec dans un cas une possible injection intraveineuse associee du fentanyl recueilli par deconditionnement d’un ou plusieurs patches), et un cas a l’ingestion d’un patch. Les concentrations sanguines en fentanyl variaient de 2,2 a 234 ng/mL. Au vu des donnees de la litterature, les auteurs insistent sur les difficultes d’interpretation posees par ces situations medico-legales, s’agissant notamment d’apprecier la responsabilite du ou des dispositif(s) TD dans la survenue du deces ; plusieurs facteurs (proximite voire recouvrement entre taux sanguins therapeutiques et toxiques, frequence des polyintoxications, redistribution post-mortem) rendent compte de ces interpretations delicates, et imposent de considerer chaque situation au cas par cas, non seulement au vu des taux sanguins mais aussi de l’ensemble des donnees disponibles (anamnese, autopsie, histologie).

Resume Introduction La reforme francaise de 2011 a cree l’observatoire national de la medecine legale. En thanatologie, les donnees recueillies sont succinctes, notamment par rapport aux donnees du bureau du coroner au Quebec. L’objectif de ce travail etait de recenser les methodes statistiques employees dans les instituts medicolegaux francais. Methodes Un entretien telephonique a ete realise aupres des instituts medicolegaux francais. Les questions portaient sur le type de donnees recueillies, le personnel responsable du recueil, le logiciel informatique utilise et le moyen utilise pour le codage des resultats d’autopsie. Resultats Vingt-neuf des 31 instituts medicolegaux ont ete contactes entre mars et mai 2014. Dix-neuf centres (66 %) codaient les resultats d’autopsie a l’aide d’une liste courte de termes, six centres (20 %) utilisaient la classification internationale des maladies, et quatre centres (14 %) ne codifiaient pas leurs resultats d’autopsie. Il n’y avait pas de travail statistique joint entre plusieurs instituts medicolegaux. Conclusion L’utilisation de la classification internationale des maladies pour coder les resultats d’autopsie est effective dans plusieurs instituts medicolegaux francais ; elle peut servir de base methodologique a un travail commun des donnees thanatologiques.

Isatuximab (Isa) is an IgG1 monoclonal antibody (Ab) that specifically recognizes human CD38. Once Isa engages multiple myeloma (MM) cells expressing a high level of CD38, it can induce tumor cell killing via Fc-dependent mechanisms including Ab-dependent cell-mediated cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). To better understand the mechanism of Isa-mediated cytotoxicity, we studied CD38 levels in 16 established MM lines and measured Isa-mediated ADCC, ADCP, and CDC in tumor cell killing. The cytotoxic functions of Isa were dependent on CD38 receptor density (RD) in most cell lines. Isa-mediated ADCC was observed in a subset (7/16) of MM cell lines that displayed CD38 RD >100,000 molecules/cell. Similarly, the same subset of MM cells, with 1 exception, were sensitive to Isa-mediated ADCP, indicating a similar CD38 RD is also required for this killing effect. A much higher CD38 RD (>250,000 molecules/cell) alone was insufficient for Isa-mediated CDC. Cell lines MOLP-8 and MOLP-2 have CD38 RD >250,000 molecules/cell, but are resistant to Isa-mediated CDC. Overexpression of CD38 in cell lines with low endogenous CD38 expression did not always sensitize the cells to Isa-mediated CDC. These results suggest that additional mechanisms are involved in the regulation of such cytotoxic effects. We further investigated the expression of complement-cascade inhibitors CD46, CD55, and CD59. High-level expression of at least one of these molecules was associated with resistance to Isa-mediated CDC, even when cells were CD38 high (hi). By comparing 4 selected MM cell lines, we found that a minimal RD level of 50,000 molecules/cell for CD46, CD55 or CD59 appears to be an important threshold to suppress Isa-mediated CDC. When CD59 function was neutralized by an anti-CD59 antibody, we were able to re-sensitize the cells to Isa-mediated CDC in CD38hi MOLP-8 cells. Neutralizing CD59 function alone did not rescue Isa-mediated CDC in CD38-low expressing NCI-H929 cells. However, overexpressing CD38 and inhibiting CD59 rendered NCI-H929 cells sensitive to CDC. Taken together, the high-level of CD38 expression and low-level of CD59 (and perhaps other inhibitors) expression are important for Isa-mediated CDC in killing of target tumor cells. In conclusion, the main immune effector mechanisms involved in Isa-mediated killing of MM cells include ADCC and ADCP. These effects are dependent on high levels of CD38 RD in MM cell lines in vitro. Further confirmation is under way using samples from MM patients. Citation Format: Zhili Song, Guang Yang, Anlai Wang, Rita Greco, Joachim Theilhaber, Elvis Shehu, Daniel Ajona, Bruno Paiva, Chen Zhu, Dmitri Wiederschain, Marielle F. Chiron Blondel, Francisco Adrian. Isatuximab-induced multiple myeloma cell killing through effector functions is dependent on CD38 expression and complement inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2966.

Objectif Presenter le premier cas francais de deces imputable a un NPS analogue du fentanyl [1,2] . Description Une femme de 30 ans est decouverte decedee a son domicile avec a proximite un garrot, des seringues et un sachet de poudre de couleur rose. L’autopsie revele de nombreux points d’injection au niveau du pied gauche et du pli du coude droit et conclut a un deces par syndrome asphyxique avec congestion generalisee des visceres. Methodes Nous disposons de prelevements de sang cardiaque et peripherique, d’humeur vitree, de bile, de contenu gastrique, de cheveux et d’ecouvillons nasaux. Une expertise toxicologique de reference est realisee. La poudre est analysee par GC-MS. Des dosages de confirmation de l’ocfentanil sont realises par LC-MS/MS. Au total, 200 μL d’echantillon sont traites avec 200 μL de reactif de precipitation (acide sulfosalicylique, 500 mg/mL) contenant le standard interne deutere (fentanyl-d5). Apres centrifugation, le surnageant est injecte pour subir une premiere phase d’extraction suivie d’une separation en ligne sur colonne Oasis HLB ® (Waters) puis une separation chromatographique sur colonne XSelect ® (Waters). La quantification est realisee en mode d’ionisation positif sur un API4000 Sciex ® . Deux transitions sont enregistrees pour l’ocfentanil ( m/z : 371,3/105,1 et 371,3/188,2) et 1 transition pour le fentanyl-d5 ( m/z : 342,3/188,2). Resultats Les analyses toxicologiques revelent une alcoolemie a 0,20 g/L, des concentrations sanguines de paracetamol a 3 mg/L, de cafeine a 0,7 mg/L et des traces non quantifiables de morphine libre ( Conclusion Les resultats sont en faveur d’une injection de la poudre ayant entraine un deces imputable a l’ocfentanil, opioide de synthese 3 fois plus puissant que le fentanyl [3] . Les screenings toxicologiques realises en GC-MS, y compris dans la bile, n’ont pas permis de reveler l’ocfentanil a cause de tres faibles concentrations. L’interet majeur pour le diagnostic toxicologique de disposer des produits retrouves pres du corps ou dans l’entourage d’un patient hospitalise, est a rappeler aux enqueteurs, aux cliniciens et aux magistrats.

The rose Bengal photosensitized oxidation of the (+) and (−) stereoisomers of α-pinene, β-pinene and limonene has been optimized to produce sufficient quantities of hydroperoxides for antimicrobial screening. Experimental conditions allowed the production of highly concentrated solutions of hydroperoxides (approximately 2 M, approximately 30 w%). The antimicrobial activity of each hydroperoxide solution was evaluated by measuring their minimum inhibitory concentration (MIC) (1,2). All the hydroperoxides behave as powerful antimicrobial agents (similar to hydrogen peroxide). The MIC values were almost independent of the nature of the parent terpene but depended of the nature of the bacterial colony (five conventional families, met in human pathologies, were tested).

The photohydroperoxidation of the stereoisomers of three terpenes ((+) and (−) α-pinene, (+) and (−) β-pinene and (+) and (−) limonene) has been performed by using photocatalysts, such as zinc oxide, or sensitizers, such as anthracene or rose Bengal, supported on cross-linked polystyrene. Hydroperoxides accumulated alone in the first stages of sensitized oxidation but were always associated with alcoholic and carbonyl products in the case of ZnO. Secondary products obtained for longer exposure times in sensitized oxidations were identified by gas chromatography/mass spectrometry and mechanisms for their formation, deriving from the photolysis of parent hydroperoxides, were suggested.

Pseudomonas exotoxin (PE) is cleaved within mammalian cells between Arg279 and Gly280 to generate an enzymatically active COOH-terminal fragment of 37 kDa which translocates to the cytosol and ADP-ribosylates elongation factor 2. A protease, with toxin cleaving activity, was prepared from beef liver and subsequently characterized. After achieving a 500-fold enrichment in several chromatographic steps, a soluble form of this protease was identified as a furin-like enzyme. It cleaved PE on the COOH-terminal side of the sequence of RQPR (amino acids 276-279) producing the same fragments as those generated within cells. Cleavage had a pH optimum of 5.0-5.5, was inhibited by EDTA or p-hydroxymercuribenzoate but not by O-phenanthroline,N-ethylmaleimide, trans-epoxysuccinyl-L-leukcylamido-(4-guanidino)-butane, or PMSF (or other well known inhibitors of serine proteases). The beef protease cleaved PE with an apparent Km of 7 microM. A mutant form of PE, PEala281, was cleaved at the same site, with the same pH optimum, a similar Km (9 microM) but with a Vmax 150 times faster than was seen with the native toxin. Mutational analysis of the amino acids located just before the site of cleavage, confirmed the importance of arginines at P-1 and P-4. It was also noted that the introduction of a dibasic pair at 278-279 did not increase toxicity or appreciably improve the rate of cleavage. Unnicked diphtheria toxin (DT) was also cleaved by the beef protease; cleavage was on the COOH-terminal side of the sequence RVRR (amino acids 190-193), was seen at pH values ranging from 5.5 to 8.5 and had an optimum at pH 8.0. Recombinant furin cleaved PE, PEala281, and DT with the same characteristics as the beef protease. In addition, Western blot analysis revealed that anti-furin antibodies reacted specifically with components in the beef protease preparation. Immunodepletion experiments showed that all toxin-cleavage activity could be removed from the beef protease using anti-furin antibodies. The relevance of furin-mediated cleavage was further assessed by adding nicked toxins to intact cells. Nicked PE and DT both killed cells at a faster rate than their unnicked counterparts.

Pseudomonas exotoxin (PE) enters cells by receptor-mediated endocytosis and is cleaved by a cellular protease between Arg279 and Gly280 to produce an NH2-terminal fragment of 28 kDa which contains the toxin's binding domain and a COOH-terminal fragment of 37 kDa which has translocating and ADP-ribosylating activity. After proteolysis, the COOH-terminal fragment reaches the endoplasmic reticulum by retrograde transport where it translocates to the cytosol and inhibits protein synthesis by ADP-ribosylating elongation factor 2. To understand how the 37-kDa fragment functions, we focused on the role of specific amino acids located near its NH2 terminus. We found that there was a 4-250-fold loss in toxic activity when tryptophan 281, leucine 284, or tyrosine 289 were changed to other residues. Mutations at these three positions did not interfere with the receptor binding, cell-mediated proteolytic cleavage, or ADP-ribosylating activity. To determine the role of these amino acids, a competition assay was devised in which the addition of excess PE delta 553, a mutant form of PE that lacks ADP-ribosylation activity, competed efficiently for the toxicity of PE. Excess PE with mutations near the NH2 terminus of the 37-kDa fragment competed poorly. This competition occurred after proteolysis since PEGly276, a mutant form of PE that is not cleaved, did not complete. We conclude that specific amino acids at the NH2 terminus of the 37-kDa fragment interact in a saturable manner with an unknown intracellular component.

UML is almost inevitable when dealing with software designing and intends to be extended to cover many different disciplines. At the same time, control and automation designers integrate further object oriented concepts in their traditional programming behaviors in particular with function blocks. Whereas UML was not adapted to model these IEC 61131 compliant items, an important extension called SysML has been proposed. This paper deals with evaluating the new modeling abilities of this language according to programmable logical controllers specific programming rules.

Pseudomonas exotoxin (PE) requires proteolytic cleavage to generate a 37-kDa C-terminal fragment that translocates to the cytosol and ADP-ribosylates elongation factor 2. Cleavage within cells is mediated by furin, occurs between arginine 279 and glycine 280, and requires an arginine at both P1 and P4 residues. To study the proteolytic processing of PE-derived chimeric toxins, TGFalpha-PE38 (transforming growth factor fused to the domains II and III of PE) and a mutant form, TGFalpha-PE38gly279, were each produced in Escherichia coli. When assessed on various epidermal growth factor (EGF) receptor-positive cell lines, TGFalpha-PE38 was 100-500-fold more toxic than TGFalpha-PE38gly279. In contrast to PE, where cleavage by furin is only evident at pH 5.5, furin cleaved TGFalpha-PE38 over a broad pH range, while TGFalpha-PE38gly279 was resistant to cleavage. TGFalpha-PE38 was poorly toxic for furin-deficient LoVo cells, unless it was first pretreated in vitro with furin. Furin treatment produced a nicked protein that was 30-fold more toxic than its unnicked counterpart. Using the single chain immunotoxin HB21scFv-PE40 as a substrate, furin-mediated processing of an antibody-based immunotoxin was also evaluated. HB21scFv-PE40, which targets cells expressing the transferrin receptor, was cleaved in a similar fashion to that of TGFalpha-PE38 and nicked HB21scFv-PE40 exhibited increased toxicity for LoVo cells. In short-term experiments, the rate of reduction in protein synthesis by furin-nicked immunotoxins was increased compared with unnicked protein, indicating that cleavage by furin can be a rate-limiting step. We conclude that furin-mediated cleavage of PE-derived immunotoxins is important for their cytotoxic activity.

Recombinant RFB4 single-chain immunotoxin that is cytotoxic towards CD22-positive cells E. Mansfield*, M. F. Chiron*, P. Arnlott, I. Pastan* and D. J. FitzGeraldV *Laboratory of Molecular Biology, DBS, National Cancer Institute, National Institutes of Health, 37/4E I 6, 37 Convent Drive MSC 4255, Bethesda, MS 20892, U.S.A., and tDepartment of Immunology, Royal Free Hospital, Rowland Hill Street, London NW3 2PF, U.K.

To be toxic for mammalian cells, Pseudomonas exotoxin (PE) requires proteolytic cleavage between Arg-279 and Gly-280. Cleavage, which is mediated by the cellular protease furin, generates an active C-terminal fragment which translocates to the cytosol and inhibits protein synthesis. In vitro, furin-mediated cleavage is optimal at pH 5.5 with a relatively slow turnover rate. Within cells, only 5-10% of cell-associated PE is cleaved. To investigate the reasons for this inefficient cleavage, the amino acid composition near the cleavage site was altered to resemble more closely the arginine-rich sequence from the functionally similar region of diphtheria toxin (DT). Four PE-DT mutants were generated, whereby 1, 5, 6 or 8 amino acids at the PE-cleavage site were changed to amino acids found at the DT-cleavage site. Mutant proteins were expressed in Escherichia coli, purified and then analysed for their susceptibility to cleavage by furin and trypsin, susceptibility to cell-mediated cleavage, and cytotoxic activity relative to wild-type PE. At pH 5.5, the rate of both furin-mediated cleavage and trypsin-mediated cleavage increased dramatically when amino acids in PE were altered to resemble the DT sequence. This increase did not alter the pH optimum for furin-mediated cleavage of PE toxins, which remained at pH 5.0-5.5. When radioactive versions of selected PE-DT proteins were added to intact cells, an increase in the percentage of molecules that were cleaved relative to wild-type PE was also seen. However, changes that favoured increased proteolysis apparently interfered with other important toxin functions because none of the PE-DT proteins exhibited enhanced toxicity for cells when compared with the activity of wild-type PE.

Pseudomonas exotoxin (PE) is cleaved within mammalian cells between Arg279 and Gly280 to generate an enzymatically active COOH-terminal fragment of 37 kDa which translocates to the cytosol and ADP-ribosylates elongation factor 2. A protease, with toxin cleaving activity, was prepared from beef liver and subsequently characterized. After achieving a 500-fold enrichment in several chromatographic steps, a soluble form of this protease was identified as a furin-like enzyme. It cleaved PE on the COOH-terminal side of the sequence of RQPR (amino acids 276-279) producing the same fragments as those generated within cells. Cleavage had a pH optimum of 5.0-5.5, was inhibited by EDTA or p-hydroxymercuribenzoate but not by O-phenanthroline,N-ethylmaleimide, trans-epoxysuccinyl-L-leukcylamido-(4-guanidino)-butane, or PMSF (or other well known inhibitors of serine proteases). The beef protease cleaved PE with an apparent Km of 7 microM. A mutant form of PE, PEala281, was cleaved at the same site, with the same pH optimum, a similar Km (9 microM) but with a Vmax 150 times faster than was seen with the native toxin. Mutational analysis of the amino acids located just before the site of cleavage, confirmed the importance of arginines at P-1 and P-4. It was also noted that the introduction of a dibasic pair at 278-279 did not increase toxicity or appreciably improve the rate of cleavage. Unnicked diphtheria toxin (DT) was also cleaved by the beef protease; cleavage was on the COOH-terminal side of the sequence RVRR (amino acids 190-193), was seen at pH values ranging from 5.5 to 8.5 and had an optimum at pH 8.0. Recombinant furin cleaved PE, PEala281, and DT with the same characteristics as the beef protease. In addition, Western blot analysis revealed that anti-furin antibodies reacted specifically with components in the beef protease preparation. Immunodepletion experiments showed that all toxin-cleavage activity could be removed from the beef protease using anti-furin antibodies. The relevance of furin-mediated cleavage was further assessed by adding nicked toxins to intact cells. Nicked PE and DT both killed cells at a faster rate than their unnicked counterparts.