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Pediatric adrenocortical tumors (PACTs) represent rare causes of malignancies. However, the south/southeast regions of Brazil are known to have a high incidence of PACTs because of the founder effect associated with a germline pathogenic variant of tumor suppressor gene TP53. We aimed to retrospectively analyze the types of variables among hormone production, radiological imaging, tumor staging, histological and genetic features that were associated with the occurrence of malignancy in 95 patients (71% females) with PACTs from a unique center. The worst prognosis was associated with those aged > 3 years (p < 0.05), high serum levels of 11-desoxicortisol (p < 0.001), tumor weight ≥ 200 g (p < 0.001), tumor size ≥ 5 cm (p < 0.05), Weiss score ≥ 5 (p < 0.05), Wieneke index ≥ 3 (p < 0.001) and Ki67 ≥ 15% (p < 0.05). Furthermore, patients with MacFarlane stage IV had an overall survival rate almost two times shorter than patients with other stages (p < 0.001). Additionally, the subtractions of BUB1B-PINK1 (

Resume Introduction Dans le cadre des lesions traumatiques du plexus brachial, l’obtention d’une epaule mobile, stable et forte fait partie des objectifs prioritaires. Les arthrodeses scapulo-humerales (ASH) restent principalement utilisees en complement ou apres echec d’une chirurgie nerveuse initiale. Notre objectif etait de comparer les resultats obtenus au niveau de l’epaule apres chirurgie nerveuse directe (CND) ou ASH dans les paralysies supra-claviculaires traumatiques du plexus brachial. Hypothese Les ASH, consideree comme un traitement palliatif, peuvent etre considerees comme une alternative therapeutique aux CND. Materiel et methodes Cinquante-huit patients, d’âge moyen 24 ans, ont ete retrospectivement inclus avec un recul minimum de 2 ans. Vingt patients ont fait l’objet d’une neurotisation du nerf spinal accessoire partiel sur le nerf supra-scapulaire (groupe CND) et 38 patients d’une ASH. Nous avons evalue les mobilites de l’epaule, la force musculaire isometrique et le score DASH. Resultats Le recul moyen etait de 46 mois (24–156). Il n’existait pas de difference statistiquement significative entre les deux groupes concernant les mobilites de l’epaule. La dispersion des donnees etait statistiquement plus importante dans le groupe CND que dans le groupe ASH en antepulsion (p = 0,0011), abduction (p Conclusions Nos resultats contrastent avec ceux classiquement admis selon lesquels la neurotisation du nerf supra-scapulaire permettrait l’obtention de meilleurs resultats cliniques que les ASH. Nous n’avons pas retrouve cette superiorite dans la presente etude. Les differences de dispersion des donnees indiquent que la chirurgie nerveuse procurerait des resultats imprevisibles et heterogenes en opposition avec l’ASH qui procurerait des resultats modestes, mais homogenes et predictibles. Niveau de preuve IV, etude retrospective, serie clinique.

The UN Convention on the Rights of the Child confers on children a number of rights, not the least of which is the guarantee that their best interests shall be a primary consideration in all actions that concern them. This article applies the Best Interests of the Child [BIC] principle—a widespread concept in family law—to the context of criminal policy. It calls for a rebalancing of Canadian youth justice so as to prioritize children’s best interests. As marginalized societal actors, children are at risk of seeing their interests increasingly overlooked if they are not brought to the fore. Once a defining feature of Canada’s response to juvenile delinquency, the BIC principle has waned in the face of sweeping penal populism. Examples from Scotland, Scandinavia and Washington reveal means by which child-friendly justice systems can withstand the influence of punitive public sentiment. Mirroring such approaches in Canada would require repurposing the deeply engrained concept of proportionality and viewing children’s interests as inherent to advancing public safety., La Convention relative aux droits de l’enfant (convention des Nations Unies) confère aux enfants plusieurs droits, l’un des plus importants étant la garantie que l’intérêt supérieur de l’enfant doit être une considération primordiale dans toutes les décisions qui le concernent. Dans cet essai, l’auteur applique le principe de l’intérêt supérieur de l’enfant — concept largement répandu en droit de la famille — au contexte des politiques en matière criminelle. Il évoque la nécessité de rééquilibrer le système canadien de justice pour les jeunes afin de prioriser l’intérêt supérieur des enfants. En raison du rôle marginal qu’ils jouent dans la société, les enfants sont de plus en plus exposés au risque que leurs intérêts soient ignorés s’ils ne sont pas mis de l’avant. Dans la foulée de la progression du populisme pénal, le principe de l’intérêt supérieur de l’enfant, qui constituait jadis un élément fondateur de l’intervention du Canada en matière de délinquance juvénile, a peu à peu été ébranlé. À l’aide d’exemples de situations vécues en Écosse, en Scandinavie et à Washington, l’auteur explique qu’un système de justice adapté aux enfants peut se prémunir contre l’influence les sentiments du public quant une démarche punitive. Cependant, pour utiliser des approches semblables au Canada, il serait nécessaire de redéfinir le concept profondément enraciné de la proportionnalité en reconnaissant que les intérêts des enfants sont intrinsèquement liés à la sécurité publique.

International audience; BACKGROUND:Restoring shoulder mobility, stability, and strength is a key goal in patients with brachial plexus injuries. Shoulder arthrodesis is chiefly used as an adjunct to, or after failure of, initial direct nerve surgery. The objective of this study was to compare clinical and functional shoulder outcomes after direct nerve transfer vs. shoulder arthrodesis in adults with supra-clavicular brachial plexus injuries.HYPOTHESIS:Shoulder arthrodesis, currently used as a salvage procedure in brachial palsy injuries, deserves to be viewed to a valid alternative to direct nerve transfer.MATERIAL AND METHODS:A retrospective study was conducted in 58 patients with a follow-up of at least 2 years. Among them, 20 were managed by transfer of a spinal accessory nerve fascicle to the supra-scapular nerve and 38 by shoulder arthrodesis. Outcome measures were shoulder range-of-motion, isometric shoulder strength, and the Disabilities of the Arm, Shoulder, and Hand (DASH) score.RESULTS:Mean age at surgery was 24 years and mean follow-up was 46 months (range, 24-156 months). Motion ranges of the shoulder were not significantly different between the two treatment groups. Data variance was significantly greater in the nerve transfer group than in the shoulder arthrodesis group for scapular antepulsion (p=0.0011), abduction (p

The aim of this study was to compare recovery for oral surgery patients given a deep sedation regimen of midazolam, propofol, and remifentanil with a standard control of fentanyl in place of remifentanil.This investigation was designed as a randomized, prospective, single-blinded controlled study. Group 1, the control, received midazolam 0.03 mg/kg, fentanyl 1 microg/kg, and propofol initially at 140 microg/kg/min. Group 2 received midazolam 0.03 mg/kg, remifentanil: propofol (1:500) given at an initial propofol infusion rate of 40 microg/kg/min. Outcome measures included time to response to verbal command, Aldrete score = 9, Postanesthesia Discharge Scoring System = 7, and assessment by the Digit Symbol Substitution Test.Forty-seven subjects were entered in the study. Baseline findings were homogenous between the 2 groups. Subjects in group 2 recovered earlier (P.005) and required less propofol for both the induction (0.8 +/- 0.4 versus 1.2 +/- 0.6 mg/kg; mean +/- SD, P.01) and maintenance of deep sedation (46 +/- 9 versus 131 +/- 17 microg/kg/min; P.005). There were minor differences in vital signs.This study demonstrated that this remifentanil regimen provided significantly more rapid recovery and used significantly less propofol compared with the fentanyl regimen.

Resume L’arthrodese scapulo-humerale trouve sa principale justification dans les sequelles de paralysies du plexus brachial. Elle reste, par contre, une intervention d’indication controversee et limitee dans le cadre des etiologies non neurologiques. Nous rapportons une serie de 8 arthrodeses d’epaule dans des etiologies non neurologiques, afin de determiner et preciser la place de cette intervention de nos jours. Il s’agissait d’une serie de 8 patients, 5 hommes et 3 femmes d’âge moyen 47 ans (23-87). Le cote opere a ete 2 fois le cote dominant et 6 fois le non dominant. 7 des patients avaient eu au moins une intervention chirurgicale au prealable. L’arthrodese a ete realisee une fois pour une reaction a corps etranger sur tendon prothetique de coiffe, 2 fois pour osteonecrose cephalique post-traumatique, 3 fois pour omarthrose excentree avec rupture massive de coiffe, et 2 fois pour omarthrose avec instabilite multidirectionnelle. Dans tous les cas, sauf un, l’arthrodese a ete realisee par voie posterieure avec pour osteosynthese un vissage interne associe a un fixateur externe laisse en place en moyenne 2 mois et demi. Tous les patients, sauf un, etaient satisfaits du resultat de l’intervention (le motif de satisfaction portant essentiellement sur la disparition des douleurs). Les mobilites actives moyennes etaient de 75° en flexion, 65° en abduction (la position moyenne de l’arthrodese etant de 20° en flexion, 25° en abduction et 30° en rotation interne). Deux groupes ont ete individualises pour l’analyse du score de Constant absolu. Le score a ete ameliore de 16 points (24 a 40) pour le groupe des omarthroses sans instabilite (le score de la douleur passant de 3 a 13) et diminue de 14 points (66 a 52) pour le groupe avec instabilite (imputable a la baisse des mobilites, le score moyen des mobilites passant de 38 a 14). Parmi les complications, on retrouvait une paralysie radiale, une pseudarthrodese, et un œdeme declive du membre superieur. L’arthrodese d’epaule n’est pas qu’une intervention dont les objectifs sont limites a l’obtention de l’indolence et de la stabilite. Elle doit pouvoir assurer une recuperation fonctionnelle utile (main-bouche, main-perinee et pince brachio-thoracique). Sa place se situe donc, la ou l’arthroplastie prothetique trouve ses limites (arthrites infectieuses, omarthrose evoluee chez le sujet jeune, perte du stock osseux glenoidien et echec de traitement des instabilites multidirectionnelles avec arthrose). Sa predictibilite et sa constance en terme de resultat, nous font pencher pour ce type d’intervention dans ces indications qui restent rares.

Resume Introduction. – Les mains de « blast » resultent de la manipulation d’explosifs instables, dont le souffle induit des lesions majeures de la premiere commissure. Notre but est d’en definir une classification afin de deboucher sur une strategie therapeutique. Methode. – Nous rapportons une serie de neuf mains de blast chez huit patients d’âge moyen 24 ans. L’etiologie etait cinq fois la fabrication artisanale de petards et quatre fois la manipulation de munitions. Cinq mains presentaient une amputation du pouce associee dans trois cas a celle de l’index voire du medius. Seul un pouce a pu etre revascularise avec succes. Quatre pouces ont ete reconstruits, deux par transfert d’orteil, deux grâce a un « index banque ». Discussion. – Quatre grades lesionnels sont individualisables : Grade 1 : lesions isolees musculocutanees. Un brochage intermetacarpien evite la fermeture de la premiere commissure. La couverture est assuree par un lambeau pedicule regional voire une greffe cutanee. Grade 2 : s’ajoutent des lesions osteoarticulaires du pouce et de l’index respectant la trapezometacarpienne. La reconstruction du pouce repose souvent sur le principe de « l’index banque » Grade 3 : se distingue par une atteinte pejorative de la trapezometacarpienne. Grade 4 : Amputation ou devascularisation du pouce. Des lesions vasculaires diffuses imposent des pontages en zones saines. Les amputations le plus souvent proximales necessitent une reconstruction prealable du premier metacarpien et de son enveloppe cutanee avant un transfert d’orteil. Les translocations sont rendues difficiles car l’index est tres souvent lese et les teguments cicatriciels.

We report on Galactic Center L'-band observations made during NAOS/CONICA Science Verification. Colors of the inner 2" stars reveal an infrared excess of S2, the closest star to the black hole, that could sign the first thermal infrared detection of Sgr A*. A multi-wavelength maximum likelihood analysis has allowed us to eliminate all but two of the candidates for gravitational lensing proposed by Alexander (2001). Our observations of the thin and intersecting structures of the Northern Arm could trace several shocks heating the neighbooring dust rather than a stream of matter in orbit around the central mass as previously thought., Comment: 5 pages, 2 figures, submitted to Astronomy and Astrophysics: 16 April 2003, accpeted: 14 November 2003, updated references

A new research and diagnosis technique has emerged during the past 10 years at the hospital and in the research laboratories: confocal laser endomicroscopy (CLE), which combines the advantages of in situ and in vivo imaging with the power of confocal microscopy. This article recalls the principles of this new technique and illustrates its applications.

Ceramide can induce apoptosis through a caspase independent pathway. Bax has been described as able to kill cells in the absence of caspase activity, therefore we measured Bax in situ during ceramide-induced apoptosis using anti-Bax antibodies and flow cytometry analysis. An early ( 2 h) accompanying nuclear apoptosis, and was inhibited by Z-VAD-fmk. Bax immuno-reactivity was found to be related to intracellular pH (pHi), and C2-Ceramide (C2-Cer) induced a very early (

The response to therapy of leukemic cells is largely determined by their capacity of proliferation and apoptosis in presence of the administered drugs. We describe here the main markers used in flow cytometry (FCM) and involved in the assessment of cell cycle parameters: single labeling by Propidium Iodide (PI) and double labeling anti-Bromodeoxyuridine (BrdUrd)/PI which, both in vitro and in vivo, gives cell percentages in the different cell cycle phases. The markers of cell cycle progression can be divided into proliferation markers such as PCNA (proliferating cell nuclear antigen) or Ki-67 and cell cycle progression markers. The latter, which are the core of the cell cycle machinery, are molecules recently characterized (Cyclins, CDKs (cell dependent kinases), CDIs (cyclin-dependent kinase inhibitors)) and their cell expression can be analyzed using FCM. FCM is also one of the best means to detect and quantitate apoptotic cells. Several techniques are described: Nuclear labeling using Hoechst 33342: mitochondrial labeling using DiOC6(3): detection of DNA fragmentation using 1) labeling of fixed and permeabilized cells with a DNA marker or 2) labeling of the free 3' DNA ends using incorporation of labeled deoxynucleotides; detection in apoptotic cells (Bcl-2, Fas, phospholipids...). At last, we analyzed flow cytometry methods to study the cell resistance to Ara-C and anthracyclins. In combination with cell kinetic studies and detection of apoptotic cells, they should increase the efficiency of the acute leukemia treatment.

The relationship between heparin and fibrinolysis is strongly suggested. We have studied the influence on fibrinolysis of standard heparin (SH), Calciparin and low molecular weight heparin (LMWH), Fraxiparin, given preventively in an elderly population. Patients were randomized into two groups (SH, LMWH). We investigated fibrinolytic parameters (ECLT, t-PA antigen, PAI-1 activity and antigen, t-PA/PAI-1 complexes) before treatment (D0) and at D30 and D60, before and after venous occlusion (VO). Values at D0, D30 and D60 were compared within each group. A significant and marked increase in t-PA and t-PA/PAI-1 complexes at D30 and D60 before and after VO was noted only in the SH group. The mechanism and clinical relevance of this increase remains to be established.

The stiffness of the thoracic aorta can be assessed non-invasively. If aortic stiffness can be shown to be related to coronary heart disease, perhaps it can be used to identify which patients with hypercholesterolaemia are most likely to have atheromatous changes and thus to be selected for intensive cholesterol-lowering treatment. Hence the distensibility of the transverse aortic arch was measured by echocardiography of the aortic arch in four groups of patients--symptom-free patients with normal serum cholesterol; symptom-free patients with raised serum cholesterol; patients with coronary heart disease (all with raised serum cholesterol), and post-heart-transplant patients. In all groups distensibility fell with age. The regression slope was steeper (p less than 0.05) for patients with known coronary disease than for either of the disease-free groups, and among cardiac transplant recipients there was also a segregation of distensibility values between those with and without atheroma in their native hearts. The results indicate that aortic distensibility might be an indicator of coronary heart disease and that it might be useful in identifying which symptom-free subjects with modest hypercholesterolaemia should be treated aggressively.

Scapulohumeral arthrodesis is mainly used for the treatment of sequelar injury after brachial plexus palsy. Indications are however controversial and limited to patients with non-neurological shoulders. We report a series of eight shoulder arthrodeses performed on non-neurological shoulders in order to determine and detail the current role of this procedure.The series included eight patients, five men and three women, mean age 47 years (23-87). The dominant side was operated on in two patients and the non-dominant side in six. Seven patients had had at least one shoulder procedure prior to arthrodesis. Arthrodesis was performed for foreign body reaction on a tendon prosthesis in one patients, posttraumatic head necrosis in two, off-centered degenerative joint disease with full thickness rotator cuff tear in three, and multi-directional instability on degenerative joint disease in two. Arthrodesis was performed via a posterior approach in all patients but one using screw fixation associated with external fixation left in place for 2.5 months on average.All patients except one were satisfied with the outcome (basically because of pain relief). Mean active motion was 75 degrees flexion, 65 degrees abduction (arthrodesis at 20 degrees flexion, 25 degrees abduction and 30 degrees internal rotation). Two groups were identified to analyse the absolute Constant score. The score improved 16 points (from 24 to 40) in the group of patients without instability (pain score improved from 3 to 13) and decreased 14 points (from 66 to 52) in the group with instability (due to decreased motion, the mean motion score declining from 38 to 14). Complications included one radial palsy, one nonunion, and one gravity edema of the upper limb.Shoulder arthrodesis is more than a salvage method to reduce pain and gain stability. The objective should be to recover useful function (hand-mouth, hand-perineum, brachio-thoracic function). It should be used when prosthetic arthroplasty is not possible (infectious arthritis, advanced degenerative disease in young subjects, loss of glenoid bone stock, failure after treatment of multidirectional instability with degenerative disease). Shoulder arthrodesis still has rare indications because of the predictability of sustained outcome.

Reprint author: Bacou F. bacou@supagro.inra.fr; International audience; Skeletal muscle demonstrates a force deficit after repair of injured peripheral nerves. Data from the literature indicate that myoblast transfer enhances recovery of muscle function. Thus, we tested the hypothesis that transfer of adult myoblasts improves the properties of reinnervated rabbit tibialis anterior (TA) muscles in both the short term (4 months) and long term (14 months). Two months after transection and immediate suture of the common peroneal nerve, TA muscles were made to degenerate by cardiotoxin injection and then transplanted with adult myoblasts cultured for 13 days. Under these conditions, muscles studied at 4 months were heavier, contained larger fibers, and developed a significantly higher maximal force than muscles that had only been denervated-reinnervated. In the long term, although muscles made to degenerate were heavier and developed a significantly higher maximal force than denervated-reinnervated muscles, myoblast transfer failed to improve these parameters. However, the overall characteristics of long-term operated muscles tended clearly to approach those of the controls. Taken together, these results may have significant implications in certain orthopedic contexts, particularly after immediate or delayed muscle reinnervation.

Blast injuries of the hand result from the manipulation of handmade explosives, the blast causes most damage in the first web. Our purpose is to propose a classification of these injuries so as to lead to a therapeutic strategy.We report a series of nine blast injuries of the hand in eight patients of average age 24 years. The aetiologie was handmade explosive manufacture (five cases) and the manipulation of munitions (four cases). Five hands presented an amputation of the thumb together with the index and in three of them, the long finger was involved as well. Only one thumb was revascularised successfully. Four thumbs were reconstructed, two by toe transfer, and two by "index bank".We observed four different stages: Stage 1: Isolated musculo-cutaneous injury. An intermetacarpal pin avoids first web contracture. Skin coverage is achieved by a pedicled local flap or a skin graft. Stage 2: Osteo-articular injuries of the thumb and second ray but sparing the trapezo-metacarpal joint. The thumb reconstruction is often based upon rinciple. Stage 3: Characterised by a destructive injury of the trapezo-metacarpal joint. Stage 4: Amputation or devascularization of the thumb. Extensive vascular injuries neccessitate a bypass from a healthy zone. The amputations, which are mostly proximal, require a preliminary osteocutaneous reconstruction of the first metacarpal before any toe transfer. Finger translocations are made difficult because of the high rate of serious injuries of the index and palm.

We present K, L and M diffraction-limited images of NGC 1068 obtained with NAOS+CONICA at VLT/YEPUN over a 3.5" field around the central engine. Hot dust (Tcol = 550-650 K) is found in three different regions : (a) in the true nucleus, seen as a slightly NS elongated, core of extremely hot dust, "resolved" in K and L with respective diameters of ~5 pc and 8.5 pc ; (b) along the NS direction, as a "spiral arm" and a southern tongue ; (c) as a set of parallel elongated nodules ("wave-like") bracketting the jet. Several structures observed on radio maps, mid-IR or HST UV-visible maps are seen, so that a precise registration can be done from UV to 6 cm. These results do support the current interpretion that source (a) corresponds to emission from dust near sublimation temperature delimiting the walls of the cavity in the central obscuring torus. Structure (b) is thought to be a mixture of hot dust and active star forming regions along a micro spiral structure that could trace the tidal mechanism bringing matter to the central engine. Structure c)which was not known, exhibits too high a temperature for "classical'' grains ; it is most probably the signature of transiently heated very small dust grains (VSG) : "nano-diamonds", which are resistant and can form in strong UV field or in shocks, are very attractive candidates. The "waves'' can be condensations triggered by jet induced shocks, as predicted by recent models. First estimates, based on a simple VSG model and on a detailed radiative transfer model, do agree with those interpretations, both qualitatively and quantitatively., Submitted : 15 March 2003 ; accepted : 15 May 2003

International audience; Near-infrared observations of line emission from excited H 2 and in the continuum are reported in the direction of the Orion molecular cloud OMC1 , using the European Southern Observatory Very Large Telescope UT4 , equipped with the NAOS adaptive optics system on the CONICA infrared array camera. Spatial resolution has been achieved at close to the diffraction limit of the telescope (0. 08 −0. 12) and images show a wealth of morphological detail. Structure is not fractal but shows two preferred scale sizes of 2. (1100 AU) and 1. 2 (540 AU) , where the larger scale may be associated with star formation. Key words. ISM : individual objects : OMC1 – ISM : circumstellar matter – ISM : kinematics and dynamics – ISM : molecules – infrared : ISM

We report the detection of a possible subsolar mass companion to the triple young system TY CrA using the NACO instrument at the VLT UT4 during its commissioning. Assuming for TY CrA a distance similar to that of the close binary system HD 176386, the photometric spectral type of this fourth stellar component candidate is consistent with an ~M4 star. We discuss the dynamical stability of this possible quadruple system as well as the possible location of dusty particles inside or outside the system., 4 pages, 2 figures postscript

Comprehension of cell cycle regulation mechanisms has progressed very quickly these past few years and regulators of the cell cycle have gained widespread importance in cancer. This review first summarizes major advances in the understanding of the control of cell cycle mechanisms. Examples of how this control is altered in tumoral cells are then described.The typical mammalian cell cycle consists of four distinct phases occurring in a well-defined order, each of which should be completed successfully before the next begins. Progression of eukaryotic cells through major cell cycle transitions is mediated by sequential assembly and activation of a family of serine-threonine protein kinases, the cyclin dependent kinases (CDK). The timing of their activation is determined by their post-translational modifications (phosphorylations/dephosphorylations), and by the association of a protein called cyclin, which is the regulatory subunit of the kinase complex. The cyclin family is divided into two main classes. The 'G1 cyclins' include cyclins C, D1-3, and E, and their accumulation is rate-limiting for progression from the G1 to S phase. The 'mitotic or G2 cyclins', which include cyclin A and cyclin B, are involved in the control of G2/M transition and mitosis. The cyclins bind to and activate the CDK, which leads to phosphorylation (and then inhibition) of the tumor suppressor protein, pRb. pRb controls commitment to progress from the G1 to S phase, at least in part by repressing the activity of the E2F transcription factors known to promote cell proliferation. Both the D-type cyclins and their partner kinases CDK4/6 have proto-oncogenic properties, and their activity is carefully regulated at multiple levels including negative control by two families of CDK inhibitors. While members of the INK4 family (p16INK4A, p15INK4B, p18INK4C, p19INK4D) interact specifically with CDK4 and CDK6, the CIP/KIP inhibitors p21CIP1/WAF1, p27KIP1 and p57KIP2 inhibit a broader spectrum of CDK. The interplay between p16INK4A, cyclin D/CDK, and pRb/E2F together constitute a functional unit collectively known as the 'pRb pathway'. Each of the major components of this mechanism may become deregulated in cancer, and accumulating evidence points to the 'pRb pathway' as a candidate obligatory target in multistep oncogenesis of possibly all human tumor types.Major advances in the understanding of cell cycle regulation mechanisms provided a better knowledge of the molecular interactions involved in human cancer. This progress has led to the promotion of new therapeutic agents presently in clinical trials or under development. Moreover, the components of the cell cycle are probably involved in other non-cancerous diseases and their role must be defined.

The clinical and morphologic transformation of 3 to 5% of chronic lymphocytic leukemia (CLL) to diffuse large-cell lymphoma (DLCL) is commonly referred to as Richter's syndrome. Richter's syndrome occurs mostly in lymph nodes and may represent a second neoplasm or a transformation from the same clonal population. Clinical features in six patients with digestive Richter's syndrome were recorded. Paired samples of CLL and DLCL were investigated by immunohistological analysis (n = 6) and by polymerase chain reaction (PCR) for immunoglobulin heavy-chain gene rearrangement (n = 4). Histological examination revealed the involvement of the gastrointestinal tract by DLCL of B-cell phenotype (n = 6). The same monoclonal rearrangement between CLL and DLCL was demonstrated by PCR and sequencing analyses in two patients. The monoclonal rearrangement was different between CLL and DLCL in only one case. Median survival was 22 months for five patients receiving chemotherapy, suggesting that digestive Richter's syndrome has a better prognosis than nodal Richter's syndrome. Indeed, appropriate surgical resection combined with chemotherapy led to partial or complete remission in four patients.

Ceramide can induce apoptosis through a caspase independent pathway. Bax has been described as able to kill cells in the absence of caspase activity, therefore we measured Bax in situ during ceramide-induced apoptosis using anti-Bax antibodies and flow cytometry analysis. An early (30 min) increase in Bax labeling was observed after the addition of several ceramide species to several hemopoietic-related cell types. On U937, this increase was not due to antigens synthesis or processing, but rather an increased accessibility or reactivity of Bax antigens for antibodies. This increased immuno-reactivity of Bax was not inhibited by Z-VAD-fmk nor leupeptin, and preceded nuclear fragmentation by several hours. Such an increase in immuno-reactivity was also observed after Fas ligation, but it occurred later (2 h) accompanying nuclear apoptosis, and was inhibited by Z-VAD-fmk. Bax immuno-reactivity was found to be related to intracellular pH (pHi), and C2-Ceramide (C2-Cer) induced a very early (10 min) transitory increase in pHi. Both Bax immunoreactivity and pHi increases were dependent on the mitochondrial permeability transition pore (PTP) status. It was concluded from these results that C2-Cer induced a transitory increase in pHi in relation to the PTP. This rise in pHi led to conformational changes in Bax which could be responsible for further apoptosis in the C2-Cer pathway while it was a consequence of caspase activation in the Fas pathway.

Publisher Summary Flow cytometry (FCM) is widely used to phenotype cases of acute leukemia and is important in their classification. When bone marrow (BM) is largely infiltrated by blasts, there is generally no problem to determine the phenotype of leukemic cells. However, in some cases of acute leukemia (especially most of acute myeloid leukemia, AML), a high degree of heterogeneity is the rule because both leukemic and normal cells are simultaneously present and blast cells themselves can be heterogeneous. The systematic use of leukocyte common antigen (LCA, CD45) marker in a multiple (≥3) immunofluorescence (IF) combination is demonstrated in this chapter to achieve a reliable and highly reproducible discrimination between the blast cell populations and the normal cells, showing that CD45/ side scatter (SSC) gating is a simple method to detect blast cells in large series of acute leukemia. CD45/SSC gating may provide a common platform for uniform data processing during the immunophenotyping of AML at diagnosis because it is considered as a useful bridge between cytology and immunophenotyping of leukemic diseases.

Procaspase 3 is a constitutive proenzyme that is activated by cleavage during apoptosis. The resulting enzyme is able to cleave several target proteins after the second aspartate of a DEVD sequence common to all the substrates of caspases 3 and 7 (DEVDase). Because active caspase 3 is a common effector in several apoptotic pathways, it may be a good marker to detect (pre-)apoptotic cells by flow cytometry (FCM). Materials and Methods Apoptosis was induced in U937 or bone marrow mononuclear cells by daunorubicin (DNR), idarubicin (IDA), or camptothecin (CAM). Viable and apoptotic cells were sorted by FCM on the basis of either fluorescein isothiocyante (FITC)-annexin V binding or DiOC6(3) accumulation. DEVDase activity was measured in sorted populations by spectrofluorometry. Cleaved caspase 3 was labeled in situ with phycoerythrin (PE)-conjugated anti-activated caspase 3 antibodies and analyzed by FCM.DEVDase activity was detected in sorted viable CAM- and DNR-treated U937 cells, demonstrating that a partial caspase activation occurred earlier than phosphatidyl-serine exposure and mitochondrial membrane potential dissipation. The presence of a low amount of active caspase 3 in the treated viable cells was confirmed in situ with PE-conjugated anti-active caspase 3 antibodies. The same antibody was used in combination with FITC-annexin V and CD45-PC5 to study caspase 3 activation in acute leukemia blast cells after in vitro DNR and IDA treatment. Both anthracyclines induced a caspase 3-dependent apoptosis that was more efficient in blast cells than in contaminating lymphocytes.These results demonstrate that anti-active caspase 3 labeling can be an alternative to fluorogenic substrates to efficiently detect early apoptosis by FCM in heterogeneous samples. They also confirm that anthracyclines induce blast cell apoptosis by a caspase 3-dependent pathway.

We investigated transfection rates of CD34+ haematopoietic progenitor cells (HPC) or haematopoietic cell lines (TF-1, KG1a and K562) using the LacZ gene as a reporter and cationic liposomes. The transfection efficiency of CD34+ haematopoietic progenitor cells (HPC) or TF-1, KG1a and K562 grown in suspension is very low (average percentage of 0.013 for HPC and 0.03 for cell lines). Adhesion of HPC or cell lines to plates by immunological or physical methods significantly enhances transfection efficiency; however, the percentage of transfected cells still remained low. We found that adhesion of TF-1, KG1a and K562 HC to MS-5 stroma cells or NIH-3T3 fibroblast cells increased transfection efficiency. Under these conditions transfection is achieved in 11.2-25% (mean 18.30%) for the cell lines and 13.6% (range 8.2-24.2%) for CD34+ HPC. These results indicate that liposome-mediated transfection of HC is significantly increased when cells are grown in adherence to stroma or fibroblast monolayers.

Some forms of chemoresistance in leukemia may start from failure of tumour cells to successfully undergo apoptosis and Bcl-2 may play a role in this defect. Therefore, we evaluated the Bcl-2 content and synthesis in relation with the apoptotic potential in leukemic cell lines after anthracycline treatment.U937, HL60, and K562 cells and their drug resistant (DR) variants were treated with varying concentrations of Idarubicin (IDA). Apoptosis was evaluated by fluorescence microscopy after acridine orange staining. Bcl-2 and Bax content were evaluated either by flow cytometry after indirect immunolabelling or by Western blot.High Bcl-2 contents were not related to a poor ability to undergo apoptosis in U937, HL60, K562 and their DR variants. IDA induced a concentration-dependent increase in Bcl-2 content in all cell lines as long as they do not perform apoptosis. Enhanced Bcl-2 expression was inhibited by cycloheximide, actinomycin D, or antisense oligonucleotide directed against bcl-2 mRNA. Bcl-2 expression was also increased in the resistant U937 variant after serum deprivation or C2-ceramide treatment. The synthesis of Bcl-2 led to an increased Bcl-2/Bax ratio solely in the cells with an apoptosis-resistance phenotype.These data suggest that exposure to IDA induces Bcl-2 expression in leukemic cell lines, and that this mechanism could contribute to apoptosis resistance and participate in the acquisition of chemoresistance. They also confirm that the evolution of the Bcl-2/Bax ratio reflects apoptotic ability better than the steady state level of Bcl-2 expression.

We studied the feasibility and the sensitivity of fluorescent in situ hybridization (FISH) using leukemic or host/donor-specific probes on flow-sorted cells to assess minimal residual disease (MRD) or chimerism in transplanted patients in complete remission. We first performed experimental models of MRD and chimerism by mixing HL60 cells and normal lymphocytes in different proportions. Over 80% HL60 cells were obtained from mixtures of 5% HL60 cells in peripheral blood mononuclear cells (PBMC). We then evaluated MRD and mixed chimerism in a chronic myelogenous leukemia patient in relapse after allogeneic sex-mismatched bone marrow transplantation (BMT), who had received a donor lymphocyte infusion (DLI). Three months after DLI, mixed chimerism was observed in each bone marrow (BM)-sorted lineage (CD13+, CD14+, CD20+, and CD3+), with the highest level of recipient cells in the granulocytic lineage (CD13+). Five months after DLI, host cells were at a low level but remained detectable in the granulocytic lineage. In the same sample, the bcr-abl gene was detected in the granulocytic lineage and not in the lymphocytes. We also studied chimerism in an aplastic anemia sex-mismatched transplanted female patient. We determined the proportion of recipient total lymphocytes, CD4+ and CD8+ lymphocytes, and CD14+ monocytes under cyclosporin A therapy on five peripheral blood samples and one BM sample over 5 months. Results showed a regular decrease in recipient total lymphocytes (26.6% to 10.6%) and monocytes (20.7% to 8%). CD8(+)-recipient cells decreased rapidly, while CD4+ remained stable (17%). This work demonstrates the feasibility of FISH after cell sorting, combining the sensitivities of both flow cytometry and FISH and the specificities of both immunophenotyping and genotype analysis.

The aim of this study was to compare the effectiveness of two methods of antibiotic susceptibility testing performed on Chlamydia trachomatis-infected cells: a flow cytometric detection method and the standard method, which consists of a microscopic reading of minimal inhibitory concentration (MIC). The L2 reference strain and 13 clinical strains isolated from six patients presenting recurrent infections were tested. McCoy cells infected with an inoculum of 10(5) inclusion forming units (IFU)/ml were incubated with serial dilutions of doxycycline, ofloxacin, and erythromycin. Mean fluorescence intensity (MFI) of cells was determined by flow cytometry after staining of chlamydial inclusions with an anti-Chlamydia fluorescent monoclonal antibody. The end-point values determined by flow cytometry and microscopic reading were equivalent but presented the same imprecision. Calculation of the inhibitory concentration 50 (IC50) by flow cytometry, defined as the antibiotic concentration required to reduce the drug-free control MFI by 50%, allowed a more objective and precise evaluation of antibiotic activity than MIC. Moreover, IC50 values were reproducible, independent of the antibiotic dilution series tested, and could be used to compare the in vitro efficiency of various drugs on C. trachomatis. No resistant strain was found among the 13 clinical isolates of C. trachomatis tested.

The treatment of acute myeloid leukaemia in adults is based on chemotherapy which is divided in two phases: induction (to achieve a complete remission) and post-induction treatment (to maintain the complete remission). Induction chemotherapy usually combines cytosine arabinoside and one anthracycline. The complete remission rate which varies from 50 to 90% is mainly influenced by the age of patients, the presence of certain cytogenetic abnormalities or the presence of a preleukaemic phase. The efficacy of post-induction treatments remains controversial. Maintenance treatment (with low-dose chemotherapy) has probably a marginal efficacy. More intensive (as intensive as induction chemotherapy) chemotherapies seems capable to prolong survival but can only be applied to the youngest patients. The rate of 5-years survivors is about 30-45% and influenced by a very low numbers of factors. The treatment of relapses is very difficult and seems to prolong survival only when the duration of the first remission has been longer than 18 months. In other cases, it can be justified to restrict the treatment to transfusions and oral chemotherapy.

We evaluated the expansion capacity of untreated and 5-fluorouracil (5-FU)-resistant peripheral blood stem cells (PBSC) after 7-day incubation with interleukin-1 (IL-1) plus IL-3 plus stem cell factor (SCF) or with medium alone. We found a significant increase in the proportion of CD34+ cells in the PBSC fraction resistant to 25 micrograms/mL 5-FU after 7-day incubation with IL-1 plus IL-3 plus SCF as compared with the untreated fraction (p = 0.011). We also showed that 5-FU-resistant PBSC have a greater capacity for expansion of IL-1/IL-3/SCF-responsive immature progenitors (p = 0.05), amplification of IL-3 plus GM-CSF responsive progenitors (p = 0.01), and production of committed single growth factor-responsive (granulocyte-macrophage colony-stimulating factor [GM-CSF]) precursors (p = 0.01) than the untreated PBSC. The expansion of all types of progenitors and CD34+ cells was only observed after 7-day incubation with IL-1 plus IL-3 plus SCF. These results suggest that PBSC contain a primitive stem cell population with an enhanced expansion capacity that is identified by 5-FU resistance. As these cells can be expanded in vitro, they may then be suitable for a number of clinical applications.

Ara-C is currently used in the treatment of adult acute myeloid leukemia (AML). The cytotoxicity of Ara-C derives from an inhibition of DNA synthesis which can be determined using flow cytometry from the amount of bromodeoxyuridine (BrdUrd) incorporated into cells after a short exposure to BrdUrd. We developed a computer program to quantify inhibition of the rate of DNA synthesis by analysis of the distribution of BrdUrd/DNA. A resistance index (RI) was expressed as the ratio of the amount of BrdUrd incorporated into S phase cells incubated with Ara-C to that incorporated in the absence of Ara-C. In Ara-C sensitive and resistant HL60 cell lines, a linear relationship between RI and log Ara-C concentration was observed. This technique was applied to 96 bone marrow samples from patients with de novo AML treated by a regimen containing Ara-C. A first group of nine patients with high RI values included only drug resistant (DR) patients; a second group of 63 patients with low RI values included 62 patients who achieved a complete remission (CR); a third group of 24 patients with intermediate RI values included 19 CR and five DR patients. In view of these results, we think that it is possible to detect a majority of DR patients treated by Ara-C.

The techniques of flow cytometry are becoming more and more important for the clinical hematology laboratory. No longer a novelty confined to a few specialized institutions as it was 10 years ago, flow cytometry has blossomed into a mature discipline. The methodology is well-known, the mechanical apparatus is readily available, and the role it plays in clinical hematology is increasingly appreciated. The burgeoning number of scientific articles devoted to this topic attests to the interest it has aroused as a tool for both medical research and patient care. In fact, more than a thousand such papers are now published each year and it would be impossible to deal with all the methodologies and applications of FCM currently utilized or under development. Throughout this paper four relevant hematologic fields are briefly discussed, in which FCM appears to be of great help at present: the immunophenotyping of leukemias and lymphomas, the measurement of proliferative activity and DNA ploidy in hematological malignancies, the detection of drug resistant leukemic cells and the use of FCM in the study of platelets.

The DNR accumulation (DNR Acc) and the verapamil (VRP) index (percent increase of VRP on DNR accumulation) was studied by using flow cytometry. Fresh leukemic mononuclear bone marrow blasts from 80 unselected ANLL patients' samples were incubated with DNR in the presence or absence of VRP. The DNR accumulation was determined by flow cytometry. The median DNR Acc was 28 (range: 4-101) and the median VRP index was 4% (range 0-53). VRP significantly enhanced DNR Acc in 42 of the ANLL samples (52.5%). DNR Acc or VRP index were not influenced by age, sex, or WBC counts. Only the FAB subclassification and the blast immunophenotyping were found to influence the parameters studied here. The lowest DNR Acc was found in M0 and M6 blast cells (15 range 0-46 and 10.5 range 8-13 respectively). M4 and M5 ANLL samples accumulated significantly more DNR than M0 and M6 blast cells. The VRP index was significantly higher in M0 compared with M1 and M2 samples, as well as in M4 compared with M1 samples. A slightly positive correlation was found between the percentage of CD34-positive cells in the CD34-positive samples and DNR Acc. In this study, DNR Acc and the VRP index were not significantly correlated with the response to chemotherapy or survival. In conclusion, this study shows that ANLL leukemic cells differ in anthracyclin accumulation and response to VRP in vitro.

Autologous blood stem cells collected during the recovery phase after intensive chemotherapy are rich in progenitor cells, and, when infused into patients who have received myeloablative therapy, lead to rapid and durable hematopoietic reconstitution [6]. The rate of engraftment has been demonstrated to be correlated with the number of late progenitor cells infused [4].