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Introduction Les anticorps anti-nuclear matrix protein 2 (anti-NXP2) sont des auto-anticorps specifiques de myosites initialement identifies dans les dermatomyosites juveniles ou ils sont associes a des atteintes musculaires severes et des calcinoses. Leur expression clinique chez l’adulte est moins connue. Le but de notre etude est de decrire les caracteristiques des patients adultes presentant des anticorps anti-NXP2. Materiel et methodes Etude retrospective du 1er janvier 2017 au 1er juin 2019 incluant les patients de plus de 16 ans avec anticorps anti-NXP2 detectes par un dot myosite au CHU de Caen. Resultats Sur les 112 patients presentant un dot myosite positif, 6 (5 %) avaient des anticorps anti-NXP2. Il s’agissait uniquement de femmes, avec un âge median au diagnostic de 40,5 ans. Le suivi median etait de 21,5 mois. Quatre des 6 patientes presentaient une dermatomyosite avec une atteinte musculaire et cutanee typique de dermatomyosite : signe de la manucure (n = 4), rash peri-orbitaire (n = 3), signe du châle (n = 3), papules de Gottron (n = 2 ; Figure 1 ), syndrome de Raynaud (n = 2), photosensibilite (n = 2) et alopecie (n = 2). Un seul cas de calcinose cutanee etait observe. Une panniculite lobulaire avec adiponecrose et sans vascularite a ete decrite dans un cas ( Figure 2 ). Les atteintes extramusculaires decrites etaient : une atteinte laryngee (1), une myocardite (1), une pneumopathie interstitielle (1), une vascularite digestive (1) et une atteinte articulaire (1). Ces 4 patientes etaient toutes en remission sous traitement immunosuppresseur. Les 2 autres patientes presentaient une myosite de chevauchement definie par la presence d’atteinte musculaire squelettique sans atteinte cutanee et des manifestations extramusculaires au premier plan : troubles de la deglutition (n = 1), dysphonie (n = 1), pneumopathie interstitielle diffuse (n = 2), hypertension arterielle precapillaire (n = 1), vascularite digestive (n = 1) et une atteinte articulaire (n = 1). Ces 2 patientes ont presente une evolution defavorable avec aggravation respiratoire malgre le traitement immunosuppresseur, causant le deces de l’une d’elles. Chez les 6 patientes, aucun cancer n’etait decrit dans les 5 annees precedant ou suivant le debut des symptomes. Discussion Les myosites avec anticorps anti-NXP2 sont rares chez l’adulte et ne presentent pas les memes caracteristiques cliniques que les cas pediatriques. Il s’agit ici soit d’un tableau de dermatomyosite de bon pronostic, sans calcinose ni neoplasie associees. Des atteintes systemiques par vascularite digestive, atteintes ORL ou par pneumopathie interstitielle diffuse, trouvees notamment dans le cadre de myosites de chevauchement, assombrissent le pronostic en l’absence de traitement immunosuppresseur.

Double-positive vasculitis with anti-polynuclear cytoplasm (ANCA) and anti-glomerular basement membrane (GBM) antibodies is a rare entity of systemic vasculitis defined by the presence of ANCA and anti-GBM antibodies. The gradual accumulation of clinical and therapeutic data shows the usefulness of identifying and differentiating this entity from the two vasculitis respectively associated with the isolated presence of each of these two antibodies. Indeed, the double-positive ANCA and anti-GBM vasculitis appears to associate the characteristics of the demography and the extra-renal and pulmonary involvement of the ANCA-associated vasculitis on the one hand, and of the histological type and severe renal prognosis of the anti-MBG vasculitis on the other hand, with the renal involvement which is the only involvement consistently observed in double-positive vasculitis. The aim of this focus is to describe the epidemiological, clinico-biological, histological and prognostic characteristics of this entity, in light of recent literature and ongoing therapeutic changes in the two eponymous vasculitis.

Introduction Une patiente de 62 ans lombalgique chronique multioperee, a presente en 2017 lors d’une chirurgie rachidienne un choc a 60/40 mmHg et une erythrodermie 5 min apres l’administration de cefazoline, atracurium, dexamethasone, ketamine, xylocaine, sufentanyl, propofol. L’evolution a ete favorable sous adrenaline. La tryptasemie etait en faveur d’une reaction d’hypersensibilite immediate (HI). Du ketoprofene a ete administre 1H apres, sans reaction. En 2018, la prise de ketoprofene, esomeprazole, thiocolchicoside a entraine 1 h plus tard : erythrodermie, prurit, chaleur du visage, dysphonie, asthenie traites par prednisolone et dexchlorpheniramine, sans dosage des mediateurs sanguins de degranulation. En 2019, pour ses lombalgies, la patiente a recu a son domicile du ketoprofene IM et est decedee d’un choc anaphylactique. Methodes Realisation de tests cutanes (TC) a 2 mois de la deuxieme reaction pour toutes les molecules recues en 2017 et 2018 et IDR de controle de la cefazoline fait juste avant le test de reintroduction en 2019. Resultats Les premiers TC etant negatifs (ketoprofene douteux a une concentration irritante), une carte d’allergique provisoire contre indiquant toutes les molecules recues et les AINS a ete remise en attendant la suite des explorations. L’IDR de controle de la cefazoline s’etant positivee, les autres β-lactamines ont ete testees, revelant une reactivite croisee a la penicilline G et a l’amoxicilline. Parmi les dosages des IgE, anti-latex, ammoniums IV, penicilline G, V, amoxicilline, ceftriaxone et cefuroxime, seules les IgE anti-penicilline V etaient positives (1,6 kUA/L ; seuil Discussion Contrairement aux allergies alimentaires ou respiratoires, les allergies medicamenteuses sont rarement multiples. L’HI aux AINS est rare et les TC difficiles a interpreter en raison de possibles reactions irritatives. Nous rapportons le cas d’une patiente ayant fait un choc peroperatoire a la cefazoline qui a possiblement masque une allergie vraie au ketoprofene, dont la re-administration lui a ete fatale. Conclusion Ce cas souligne l’importance de l’education du patient, et pose le probleme du partage de l’information des medicaments contre-indiques entre professionnels de sante.

Introduction Les hypersensibilites (HS) aux proteines de liquide amniotique bovin (LAB) sont des affections professionnelles peu frequentes. Les tests cutanes (TC) aux liquides biologiques d’origine animale ne sont pas toujours contributifs ni possibles. De plus, la physiopathologie et les allergenes en cause ont ete peu etudies. Nous presentons 3 cas de patients atopiques, ayant presente des reactions apres des velages. Methodes Nous avons realise des TC (prick-to prick, patch et open test), dosages d’IgE specifiques (ImmunoCap® Phadia) et tests d’activation des basophiles (TAB) (Buhlmann) en reponse a divers allergenes de mammiferes ( Tableau 1 ). Un test de proliferation lymphocytaire (TPL) en presence de LAB ou serum de veau fœtal (SVF) a aussi ete realise (positif si index de stimulation patient/temoin > 3). Resultats Les TAB et TPL ont permis de confirmer l’HS au LAB et d’objectiver les mecanismes impliques avec une reponse mixte IgE mediee et cellulaire retardee a ses proteines. Conclusion Les tests cellulaires peuvent completer utilement les TC non disponibles ou ininterpretables. Ils peuvent aussi objectiver une sensibilisation au LAB, prealable a la declaration d’allergie professionnelle. Une etude chez des veterinaires va debuter pour estimer la prevalence de ces allergies et etudier les allergenes et allergies croisees.

Double-positive vasculitis with anti-polynuclear cytoplasm (ANCA) and anti-glomerular basement membrane (GBM) antibodies is a rare entity of systemic vasculitis defined by the presence of ANCA and anti-GBM antibodies. The gradual accumulation of clinical and therapeutic data shows the usefulness of identifying and differentiating this entity from the two vasculitis respectively associated with the isolated presence of each of these two antibodies. Indeed, the double-positive ANCA and anti-GBM vasculitis appears to associate the characteristics of the demography and the extra-renal and pulmonary involvement of the ANCA-associated vasculitis on the one hand, and of the histological type and severe renal prognosis of the anti-MBG vasculitis on the other hand, with the renal involvement which is the only involvement consistently observed in double-positive vasculitis. The aim of this focus is to describe the epidemiological, clinico-biological, histological and prognostic characteristics of this entity, in light of recent literature and ongoing therapeutic changes in the two eponymous vasculitis.

Introduction La fluoresceine est frequemment utilisee en ophtalmologie, ses effets indesirables sont rares mais il existe un risque de choc anaphylactique evalue a ≈0,08 % par injection IV. Methodes Cas concernant un homme de 81 ans ayant fait un arret cardio-respiratoire 2 min apres injection IV de fluoresceine. Clinique : pas de prodrome, perte de connaissance sans mouvements tonico-cloniques associes, sans hypertonie ni vomissements. Appel du SAMU, a son arrivee pas de ventilation spontanee, arret respiratoire necessitant un massage cardiaque externe. Lors du transport vers l’hopital, injection de 0,5 mg/h de noradrenaline puis intubation et remplissage par Ringer lactate, NaCl et gelofusine. A l’arrivee aux urgences : patient inconscient, hypotendu a 85 mmHg de systolique, tachycarde a 115bpm et desaturation. Transfert en reanimation, l’hemodynamie revient a la normale. Amelioration en quelques jours. Resultats Tryptasemie prelevee a 1 h de l’apparition des signes cliniques : 174 μg/L ; a 2 h : 131 μg/L ; a 24 h : 12 μg/ml. Degranulation mastocytaire en faveur d’une reaction d’hypersensibilite immediate. Discussion Pas de consultation allergologique ni de tests cutanes mais imputabilite evidente. A l’heure actuelle, 52 cas d’anaphylaxie a la fluoresceine IV dont 3 deces sont publies. L’incidence des chocs a la fluoresceine en IV est rare (0,08 %) et n’est pas determinee pour les chocs aux collyres qui sont rarissimes (3 cas publies). L’alternative proposee aux ophtalmologues est le vert d’indocyanine pour lequel l’effet indesirable grave decrit le plus souvent est l’hypotension (0,05 %), qui pourrait parfois relever de l’anaphylaxie. Les incidences des reactions allergiques aux autres principaux colorants responsables d’anaphylaxie en IV (tous grades) sont : Bleu patente V 0,9 %, Bleu isosulfan 0,8 %. Les structures des bleus different mais des sensibilisations aux deux colorants chez un meme patient ont ete rapportees. Conclusion Malgre leur faible frequence, il faut avoir connaissance de l’existence de chocs anaphylactiques dus a la fluoresceine. Son importante utilisation dans les cabinets d’ophtalmologie expose a un risque d’anaphylaxie dont les utilisateurs doivent etre informes pour etre aptes a agir dans l’urgence.

Introduction La vascularite dite double-positive ANCA et anticorps anti-membrane basale glomerulaire (MBG) est une entite rare de vascularite systemique cumulant la presence d’ANCA et d’anticorps anti-MBG. Les caracteristiques epidemiologiques, clinico-biologiques et therapeutiques de cette entite restent mal connues. Materiels et methodes Nous avons realise une revue systematique de la litterature (de 1989 -date du premier cas rapporte- a janvier 2019) anglophone et francophone pour les vascularites double-positives a partir de la base de donnee PubMed via les mots-cles suivants : (« Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis » [MeSH] OR « Antibodies, Antineutrophil Cytoplasmic » [MeSH]) AND (« Anti-Glomerular Basement Membrane Disease » [MeSH] OR « anti-MBG » [All Fields]). Resultats Au total, 449 cas de vascularites double-positives ont ete identifies. Le sex-ratio des patients etait legerement en faveur des hommes (55,1 %) avec un âge moyen au diagnostic de 60,7 ans. Cette entite representait 34,4 % des vascularites a anti-MBG et 6,84 % des vascularites a ANCA. Les ANCA etaient de facon predominante diriges contre la myeloperoxydase (71,3 %), tandis que de rares cas de triple positivite (3,81 %) etaient rapportes. La duree moyenne d’evolution des symptomes chez ces patients etait de 12,7 semaines. L’atteinte renale etait constante et le plus souvent severe, caracterisee par une hematurie et une proteinurie. La creatininemie moyenne au diagnostic etait de 544 μmol/L (76,7 % des patients presentaient une creatininemie > 500 μmol/L au diagnostic) et une majorite de patients (72,1 %) presentait une glomerulonephrite avec proliferation extra-capillaire a l’histologie. L’immunofluorescence montrait des depots lineaires d’IgG et de C3 dans la majorite des cas (71,9 %) et parfois des depots granuleux d’IgG et de C3 (14 %). L’atteinte respiratoire (59 %) etait dominee par les hemorragies intra-alveolaires (53,1 %). Concernant les autres manifestations, les patients double-positifs presentaient une alteration de l’etat general avec une asthenie (42,9 %), une perte de poids (42,9 %), une hyperthermie (42,9 %) et d’autres manifestations systemiques plus rares incluant une eruption cutanee (18,4 %), des arthralgies (15,4 %), des myalgies (12,2 %), des atteintes neurologiques peripheriques (9 %), des atteintes ORL (27,7 %) ou ophtalmologiques (9 %). Le pronostic a 1 an de ces patients etait sombre avec seulement 42,7 % de patients sevres de la dialyse, un taux de mortalite et un taux de rechute respectivement de 41,1 % (parmi 95 patients) et 9,7 % (parmi 93 patients). Ces patients ont recu un traitement immunosuppresseur dans 88 % des cas et/ou des echanges plasmatiques dans 55,6 % des cas. Conclusion La vascularite dite double-positive associe les caracteristiques des deux vascularites eponymes, partageant avec la vascularite a ANCA un plus vaste spectre clinique ainsi qu’un taux de rechute extra-renal plus eleve, et avec celle a anti-MBG la quasi-constance de l’atteinte renale qui est de plus forte severite et de moins bon pronostic. Son individualisation au sein des vascularites primitives est donc licite et justifie la mise en place d’etudes cliniques therapeutiques a part entiere.

Introduction Les reactions d’hypersensibilite immediate sont liees a la liberation de mediateurs par les mastocytes et/ou basophiles, dont l’histamine et la tryptase, utilises comme marqueurs diagnostiques. Contrairement a l’histamine, la tryptase est un marqueur repute robuste mais non informatif si la reaction est peu grave. Les conditions pre-analytiques peuvent modifier les concentrations mesurees. Nous avons evalue l’impact des conditions d’acheminement des prelevements de sang sur ces dosages. Methodes Les prelevements de sang sur EDTA de 14 patients lors d’une reaction de grade 2 ou 3 et de 10 sujets sans reaction ont ete divises en plusieurs aliquots des reception au laboratoire. L’un a ete immediatement traite selon les conditions usuelles. Les autres ont ete laisses a +4 °C ou temperature ambiante (TA) de 2 h a 72 h avant centrifugation. La tryptase a ete mesuree par technique ImmunoCap (ThermoFisher) et l’histamine par radioimmunologie (Beckman Coulter). Resultats Aucune variation des concentrations de tryptase n’a ete constatee a +4 °C ni a TA. Pour les sujets sans reaction, a +4 °C, les concentrations d’histamine n’ont pas ete modifiees apres 2 h ou 6 h ; une augmentation moyenne de 1,3 nmol/L (SD = 0,61 ; p 10 nmol/L. Pour les patients, les concentrations d’histamine n’ont pas varie significativement apres conservation a +4 °C ou TA pendant 24 h ou 72 h. Conclusion Il n’y a pas d’impact des conditions de conservation du sang pour la tryptase. Pour l’histamine, si les prelevements ne peuvent etre achemines rapidement vers le laboratoire, ils doivent etre conserves a +4 °C. Il y a un risque de faux-positifs a taux faible pour les prelevements non decantes au-dela de 24 h a +4 °C ou 6 h a TA et le biologiste devra interpreter les resultats en fonction des conditions d’acheminement. Mais tous les prelevements doivent etre acceptes par le laboratoire car ils ne sont pas renouvelables.

Introduction Des reactions d’hypersensibilite graves (HSI) mediees par des IgE anti-galactose-α-1,3-galactose (αGal) peuvent survenir a la premiere injection de cetuximab chez certains patients. Le depistage des IgE anti-cetuximab realise au laboratoire d’immunologie du CHU de Caen (technique Elisa [1] ) est a disposition des cliniciens pour adapter la prise en charge dans le but de diminuer ce risque. L’objectif de l’etude est de comparer les performances de l’ImmunoCap® o215/αGal a ce test. Methodes Une premiere cohorte de 90 patients [1] , ayant permis la mise au point de l’Elisa, a ete analysee pour comparer les performances des 2 tests. Tous les echantillons ont ete analyses sur Phadia 250 avec un seuil de positivite > 0,1 kUA/L. Un test d’inhibition par cetuximab atteste de la reactivite du Cap o215 envers le cetuximab. Puis, 95 patients, tires au sort dans une cohorte prospective de 247 patients [2] , ont ete utilises pour etudier la prediction du risque d’HSI. Resultats Dans la premiere cohorte, 7/8 HSI ont ete detectees par les 2 tests. La sensibilite et la valeur predictive negative sont egales (Se = 88 %, VPN = 99 %). Cependant, le Cap o215 possede une meilleure specificite et valeur predictive positive que l’Elisa (Sp = 94 % vs 80 %, VPP = 58 % vs 30 %, respectivement). L’analyse ROC permet d’optimiser un seuil de positivite a 0,525 kUA/L. Dans la seconde cohorte, 5/8 HSI sont detectees par les deux tests (Se = 62,5 %). La prediction d’HSI est meilleure avec le Cap o215 (seuil > 0,1 kUA/L : risque d’HSI = 50 %, p = 0,00021 ; seuil ≥ 0,525 kUA/L : risque d’HSI = 80 %, p = 0,0011) qu’avec le test Elisa (risque d’HSI = 25 %, p = 0,0095). Conclusion Le Cap o215 permet une meilleure prediction du risque d’HSI au cetuximab lors de la premiere injection. Son utilisation est adaptee en routine pour les laboratoires equipes d’un automate Phadia. Le seuil de positivite de ce test meriterait cependant d’etre valide dans une etude prospective elargie.

Background The aim of this study was to analyze humoral xenoreactivity of various Old World primate species sera against pig islets and the effects of these sera on pig islet viability and function after culture. Methods. Freshly isolated or cultured adult pig islets were analyzed by immunohistology or by cytofluorimetry for Old World primate xenoreactive natural antibody (XNA) binding and complement deposition. Complement-mediated cytotoxicity was evaluated by 51 Cr release assays. After 4 days of culture in 50% sera from Old World primates, the morphology and in vitro metabolic function of pig islets were also analyzed. Results. Chimpanzee, Macaca mulatta (rhesus), or baboon XNA binding was detectable only on intra-islet endothelial cells (ECs). Incubation of pig islets with sera from all Old World primate species tested showed C3 and C4 deposition on ECs and on some surrounding endocrine cells. However, membrane attack complex (MAC) showed a pattern of positivity similar to XNA binding, i.e., restricted to ECs only. No deposition of factor B was detected. Although complement cascade was activated, no cytotoxicity was observed after incubation of islets with chimpanzee serum, whereas between 10% and 35% 51 Cr specific release was obtained with rhesus, baboon, or Macaca fascicularis sera. Despite this cytotoxic effect, purified pig islets showed a normal morphology and a well-preserved insulin release in response to an acute glucose stimulus, after prolonged culture with 50% serum obtained from all primate species considered. Conclusions. Despite the fact that pig β-cell function was not affected by the serum of any of the primate species tested, some of them yielded significant lysis of islet cells, presumably as a result of a cytotoxic effect on intra-islet ECs. These data show that Old World primate sera from different species do not have equivalent effect on pig islets; these differences should be taken into account in preclinical trials of pig islet xenotransplantation.

Introduction L’histamine et la tryptase beta sont les mediateurs mastocytaires utilisables pour confirmer la nature anaphylactique d’un choc. Le dosage de la tryptase n’est pas specifique de l’isoforme beta et implique de comparer la valeur mesuree au pic a la valeur a l’etat basal. Une augmentation de la tryptase n’est pas toujours detectable dans les reactions de grade 1. Le dosage de l’histamine de demi-vie courte necessite un prelevement precoce (idealement er signes). La mise a jour de la nomenclature des actes de biologie medicale considere cet acte comme obsolete. Nous avons voulu evaluer la pertinence du dosage de l’histamine dans l’exploration d’un choc en complement de la tryptase. Methodes Une etude retrospective menee par 7 laboratoires francais entre 2011 et 2015 a permis l’analyse de 273 reactions (dont 38 provenaient de l’etude francilienne NASA). Pour 269, l’agent responsable etait un medicament. Les reactions ont ete classees en grade 1 (15 %), 2 (25 %), 3 (27 %), 4 (5 %) ou indetermine (28 %). Seuls les patients avec tests cutanes positifs (162, 60 %) ont ete classes allergiques. Resultats Les courbes ROC calculees avec les valeurs au pic pour la tryptase (ThermoFisher) et pour l’histamine (Beckman Coulter RIA/ELISA) donnent des AUC a 0,88 et 0,86 respectivement. L’index de Youden permet de calculer un seuil optimal de positivite de 8 μg/L pour la tryptase et de 18 nmol/L pour l’histamine. L’analyse de la cinetique (pic > taux basal × 1,2 +2 μg/L), y compris quand le pic de tryptase est Conclusion L’analyse des variations de cinetique de la tryptase et l’optimisation du seuil de positivite a 8 μg/L permettent d’obtenir une bonne sensibilite pour ce seul mediateur. Mais, en absence d’augmentation par rapport au taux basal, le dosage de l’histamine peut etre informatif pour l’allergologue.

T lymphocyte expansion is triggered through interaction of interleukin 2 (IL-2) with its high-affinity receptor (IL-2R). This molecule is a heterodimer comprising an antigen-inducible component, the Tac chain (P55). Activation of T lymphocytes also generates a soluble form of this P55 called S-IL-2R. S-IL-2R is elevated in many T-cell-related pathologies (leukemia, autoimmunity, etc.). In graft recipients, rejection is a result of T-cell activation by graft antigens and therefore might induce a release of S-IL-2R in the circulation; this parameter is now said to be a good indicator of rejection. We have performed a study in renal graft recipients in order to assess the usefulness of circulating S-IL-2R particularly to discriminate the origin of renal failure in cases of rejection or of cyclosporin-A (CsA)-induced nephrotoxicity. We demonstrated that there are no differences between isolated values in the clinical groups at the time of diagnosis. Variations in S-IL-2R are increased compared to steady-state periods during rejection and cytomegalovirus infections, although not in CsA toxicity episodes. However, at the individual level there are too many false-positive and false-negative results, making this parameter no more meaningful than serum creatinine levels alone or even in association (as tested in logistic discriminant analysis). In addition, it seems that the variations in S-IL-2R are partly related to renal function itself, as suggested by the correlation between S-IL-2R levels and serum creatinine levels.(ABSTRACT TRUNCATED AT 250 WORDS)

A truncated soluble form of the human interleukin-2 receptor p55 chain (T-S-IL-2R) was expressed to high levels in RODENT (mammalian) cells and affinity-purified. Its biochemical behavior was analyzed by polyacrylamide gel electrophoresis (PAGE), gel filtration, and sucrose gradient centrifugation. It migrated as a single 40-kDa band on sodium dodecyl sulfate-PAGE (reducing or nonreducing conditions), whereas it ran as a 80-kDa component on native PAGE or as a 86-kDa component on gel filtration. The combination of gel filtration and density gradient sedimentation gave a Stokes radius of 4.0 nm and a sedimentation coefficient of 3.72 S. The deduced molecular mass was 67 kDa, and the fractional ratio was 1.516. These data therefore indicated that the T-S-IL-2R was secreted as an homodimer of two noncovalently associated 40-kDa subunits. Cross-linking experiments using bifunctional reagents enabled the materialization of the dimeric structure on sodium dodecyl sulfate-PAGE. Stoichiometric binding studies using two monoclonal antibodies (mAbs 33B3.1 and 11H2) reacting with separate epitopes on the p55 chain also strongly supported the dimeric structure. Indeed, there was one binding site for the 33B3.1 mAb (and Fab fragment) per T-S-IL-2R 40-kDa subunit, whereas the 11H2 mAb (or Fab fragment) could bind only half a site per subunit, a result which could only be explained when assuming more than one subunit for the native T-S-IL-2R. Soluble interleukin-2 receptor species were also purified from culture supernatants of either L cells transfected with the full-length p55 cDNA or a normal alloreactive T cell clone. Similar biochemical behavior and reactivities with the two mAbs were found. Finally, cell-surface p55 chains expressed either by pgL21 or 4AS cells bound the 33B3.1 and 11H2 mAbs in a 2:1 ratio, suggesting that the p55 chains are also associated as homodimers when imbedded in the membrane.

Interleukin-2 is a major growth factor for activated T lymphocytes, and antibodies reacting with the Tac-chain component of the interleukin-2 receptor can prevent allograft rejection in animals. Because Tac chains are expressed only on a small fraction of activated lymphocytes, monoclonal antibodies against the interleukin-2 receptor may offer a more specific means of immunosuppression than polyclonal antilymphocyte globulin in prophylaxis against graft rejection. Therefore, we compared the immunosuppressive effect of 33B3.1, a rat monoclonal antibody against the human Tac chain, with the effect of a rabbit polyclonal antithymocyte globulin in a randomized study of 100 recipients of first renal transplants. Injections of 33B3.1 (10 mg per day) were tolerated well, whereas major side effects in 15 of 47 patients (32 percent) receiving antithymocyte globulin required discontinuation of treatment before day 14. The incidence of rejection episodes was not statistically different in the two groups at days 14, 30, 60, and 90 after transplantation. Patient and graft survival was also equal in the two groups at one year (96 and 85 percent, respectively, in both groups), and graft function was similar. The total number of infectious episodes within the first three months was lower in the 33B3.1 group than in the antithymocyte group (47 vs. 72). The drop in peripheral-blood lymphocyte concentrations was significantly larger in the patients treated with antithymocyte globulin. The level of circulating Tac-chain-bearing lymphocytes remained below 1 percent during 33B3.1 treatment, as compared with 4 to 5 percent during antithymocyte-globulin treatment (P not significant). We conclude that 33B3.1 is as effective as antithymocyte globulin in the prevention of renal-transplant rejection, and its use results in fewer infections and side effects.

Introduction Le neuro-lupus (NL) peut se manifester par des symptomes psychiatriques isoles tels que des hallucinations, une humeur depressive ou une anxiete. Ainsi, une presentation psychiatrique du lupus pourrait potentiellement conduire a tort, a une hospitalisation en milieu psychiatrique. La prise en charge du NL est une urgence, son traitement repose sur l’association d’une corticotherapie et d’un immunosuppresseur. Aucune etude ne s’est, jusqu’ici, interessee au depistage de NL dans un service de psychiatrie. Patientes et methodes Nous avons realise, entre 2012 et 2014, une etude monocentrique, prospective, au sein de 3 services de psychiatrie du CHU de Caen, dans le but de depister d’eventuels NL. Un depistage par le dosage des anticorps antinucleaires (AAN), des anticorps anti-ADN et des anti-antigenes nucleaires solubles, etait propose de maniere systematique a toute patiente, âgee de 18 a 50 ans, hospitalisee en psychiatrie. Resultats Cent patientes d’âge moyen 33,1 ± 8,44 ans [18–50] ont ete inclues dans l’etude. Une patiente a ete exclue car elle presentait un antecedent de lupus. Les pathologies psychiatriques etaient reparties comme suit : depression (46 %), schizophrenie (13 %), trouble de la personnalite (10 %), et trouble anxieux (2 %). Un quart des patientes n’avaient pas de pathologie psychiatrique averee. Le nombre moyen d’annees depuis le diagnostic de la pathologie psychiatrique etait de 9,89 ± 22,08 [0–23,6]. Soixante-dix pour cent des patientes etaient hospitalisees dans une unite de long sejour et 30 % l’etaient dans une unite de court sejour. Trente-deux pour cent avaient des AAN positifs (≥ 1/160) dont deux patientes avaient un titre au 1/1280. Aucune patiente ne presentait d’anticorps anti-ADN et une patiente avait des anti-SSA positif. Trois patientes ont ete vues en consultation de medecine interne, du fait d’anomalie biologique (anti-SSA positif ou AAN positifs a taux eleve), aucun NL n’a ete diagnostique. Conclusion Cette etude n’a pas permis de depister de NL dans une population de femmes jeunes hospitalisees en psychiatrie. Ainsi le dosage systematique des AAN, lors d’hospitalisation en psychiatrie ne parait pas probant.

Transplantation faces several major obstacles that could be overcome by expression of immunomodulatory proteins through application of gene therapy techniques. Gene therapy strategies to prolong graft survival involve gene transfer of immunosuppressive or graft-protecting molecules. Very promising results have been obtained in small animal experimental models with inhibitors of co-stimulatory signals on T cells, immunosuppressive cytokines, donor major histocompatibility antigens and regulators of cell apoptosis or oxidative stress. The application of gene therapy techniques to transplantation offers a great experimental and therapeutic potential. Local production of immunosuppressive molecules may increase their therapeutic efficiency and reduce their systemic effects. When compared with other clinical situations, gene therapy in transplantation offers several potential advantages. Gene transfer into the graft can be performed ex vivo, during the transit between the donor and the recipient, thus avoiding many of the hurdles encountered with in vivo gene transfer. Furthermore, the difficulties associated with immune responses to the gene transfer vectors and transient gene expression may be easier to overcome when gene therapy protocols are applied to transplantation than when applied to other clinical situations. The next century should witness a rapid increase in the application of gene therapy techniques to large animal pre-clinical models of transplantation and later to clinical trials. Gene Therapy (2000) 7, 14-19.

The aim of this study was to analyze humoral xenoreactivity of various Old World primate species sera against pig islets and the effects of these sera on pig islet viability and function after culture.Freshly isolated or cultured adult pig islets were analyzed by immunohistology or by cytofluorimetry for Old World primate xenoreactive natural antibody (XNA) binding and complement deposition. Complement-mediated cytotoxicity was evaluated by 51Cr release assays. After 4 days of culture in 50% sera from Old World primates, the morphology and in vitro metabolic function of pig islets were also analyzed.Chimpanzee, Macaca mulatta (rhesus), or baboon XNA binding was detectable only on intra-islet endothelial cells (ECs). Incubation of pig islets with sera from all Old World primate species tested showed C3 and C4 deposition on ECs and on some surrounding endocrine cells. However, membrane attack complex (MAC) showed a pattern of positivity similar to XNA binding, i.e., restricted to ECs only. No deposition of factor B was detected. Although complement cascade was activated, no cytotoxicity was observed after incubation of islets with chimpanzee serum, whereas between 10% and 35% 51Cr specific release was obtained with rhesus, baboon, or Macaca fascicularis sera. Despite this cytotoxic effect, purified pig islets showed a normal morphology and a well-preserved insulin release in response to an acute glucose stimulus, after prolonged culture with 50% serum obtained from all primate species considered.Despite the fact that pig beta-cell function was not affected by the serum of any of the primate species tested, some of them yielded significant lysis of islet cells, presumably as a result of a cytotoxic effect on intra-islet ECs. These data show that Old World primate sera from different species do not have equivalent effect on pig islets; these differences should be taken into account in preclinical trials of pig islet xenotransplantation.

Several immune responses are either limited to or concentrated in a given organ. Cytokines produced during ongoing immune responses have organ-localized effects that can be only partially mimicked upon their systemic delivery. Recombinant adenoviruses are efficient vectors to induce transient organ-localized cytokine expression. This allows in vivo analysis of the effects of cytokines produced spatially and temporally in a manner comparable to that observed during immune responses. The authors generated recombinant adenovirus for rat IL-4 (AdIL-4) and IL-10 (AdIL-10) to analyse the in vivo effects of these two important immunoregulatory molecules after gene transfer in the liver. It was first established that AdIL-4 and AdIL-10 were able to direct the production of biologically active cytokines by different rat cell types in vitro. Intraportal injection of doses of up to 10 10 pfu of AdIL-10 or control non-coding recombinant adenovirus were well tolerated, and hepatic histology showed only mild alterations. Conversely, animals receiving more than 2.5×10 9 pfu of AdIL-4 showed dose-dependent mortality, with clinical signs of hepatic dysfunction. Liver histology in animals receiving 2.5×10 9 pfu of AdIL-4 showed severe acute hepatitis with maximal lesions between day 7 and 14 and almost complete normalization by day 28 after gene transfer. The leukocyte infiltrate was composed primarily of mononuclear cells, but eosinophils and mast cells were significantly increased as compared to control animals. Hepatic function was also altered in animals that received AdIL-4, with kinetics similar to that of histological lesions. Our study describes a model for investigating cytokine function in vivo through liver-localized transgene expression mediated by adenoviral vectors and demonstrates that liver production of IL-4 but not IL-10 results in acute severe hepatitis.

The expression of xenogeneic epitopes and the activation of human complement by adult pig islets after prolonged culture have hitherto not been described.Freshly isolated and cultured islets were analyzed by fluorescence-activated cell sorter analysis, fluorescence microscopy, and immunohistology for expression of Gal(alpha1,3)Gal epitopes, binding of human xenoreactive natural antibodies (XNA), and complement deposition.Freshly isolated and cultured islets showed detectable Gal(alpha1,3)Gal expression and human XNA binding limited to intraislet capillary endothelial cells. No significant modification in Gal(alpha1,3)Gal expression and human XNA binding levels was detected in adult pig islets cultured for up to 4 days compared with freshly isolated islets. Incubation of pig islets with human serum demonstrated the deposition of C3, C4, and membrane attack complex, but not factor B with a similar pattern to XNA. However C3 and C4 showed a more widespread deposition. Despite complement activation, no cytotoxic effect on islets was detected after 4 hr of incubation with human serum capable of killing porcine endothelial cells. Even after 4 days of culture in 50% intact human serum, pig islets retained both their normal morphology and a normal insulin response to glucose stimulation.Neither islet cell lysis nor, more importantly, any alteration in beta cell function occurred, which suggests that adult pig islets may not be directly damaged by serum after xenotransplantation in humans. Nevertheless, complement activation in vivo could trigger rapid cellular rejection mechanisms through islet cell opsonization and release of bioactive fragments.

B-F5, a mouse IgG1 anti-CD4 MoAb, was used in recipients of a first cadaveric kidney allograft. Eighteen patients received 30 mg/day MoAb with a quadruple sequential therapy. All but one kidney were functioning at 6 months, with a mean serum creatinine of 153 micromol/L. However, 50% of the patients had an acute rejection episode within the first three months, and most of the early episodes (i.e.,1 month) occurred in patients with low levels of circulating MoAb. The biological analysis showed a strong depleting effect on the CD4+ cell counts, a saturation by the MoAb of the remaining circulating CD4+ cells, and no detectable immunization against B-F5. Although the biological parameters indicate an action of B-F5 in vivo, the clinical data associated with poor MoAb bioavailability suggest the need for an improved pharmacokinetic behavior of the MoAb to determine its use for prophylaxis of early rejection.

The focus of progress in transplantation immunosuppression is to achieve more specific immunosuppression with monoclonal antibodies. We have already shown that the efficacy of 33B3.1, a rat monoclonal Ig2A directed against the human IL-2 receptor, was similar to that of rabbit antithymocyte globulin in the prevention of acute rejection in first kidney transplants. A similar comparative analysis has been made in 40-sec renal transplants. ATG (1 mg/kg/day) or 33B3.1 (10 mg/day) was administered during the first 10 days postgrafting in association with corticosteroids and azathioprine. Cyclosporine was introduced on day 9 and azathioprine/CsA constituted the patient's maintenance treatment after day 45. Rejection treatment consisted of equine antilymphocyte globulin in both cases and of steroid boluses when patients were under Cyclosporine. One patient in each group died. Graft survival was 90%, 85%, and 79% in the ATG group (n = 20) and 100%, 89%, and 89% in the 33B3.1 group (n = 20) at 3, 12, and 24 months, respectively. Of the ATG group patients, 45% and 40% in the 33B3.1 group had at least one rejection episode, half the episodes in the MoAb cohort occurring under 33B3.1, vs. none in the ATG group. Transplant function was similar in both groups. Viral infections appeared to be more frequent with ATG (60%) than with 33B3.1 (12%), with CMV accounting for half of these in the ATG group, and none in the MoAb group. Tolerance of both agents was good. Of the 33B3.1 recipients, 70% developed anti-33B3.1 antibodies. From these data, we conclude that this anti-IL-2 receptor MoAb seems less effective than rabbit ATG as induction treatment in second kidney transplant patients.

A prospective, randomized trial was conducted to evaluate the short-term and long-term effects of induction immunosuppression with the rat IgG 2a monoclonal antibody 33B3.1, directed against the human alpha chain of the interleukin 2-receptor, following primary, cadaveric, combined pancreas and kidney transplantation. Forty patients were randomly assigned to receive 10 mg/day of 33B3.1 (n = 20) or 1.5 mg/kg/day of rabbit antithymocyte globulin (n = 20) for the first 10 postoperative days. Azathioprine, low-dose corticosteroids, and cyclosporine were given in association with either 33B3.1 or ATG. All 40 patients received the entire 10-day bioreagent course and no episode of rejection was observed during this period. Although the incidence of rejection did not significantly differ within the first, second, and third postoperative months (ten 33B3.1 and 6 ATG patients experienced, respectively, 10 and 6 rejection episodes within the first 3 months), the total number of 33B3.1 patients experiencing rejection throughout the follow-up was significantly higher than that of ATG (13 versus 6; P0.02). Immunological graft failure accounted for 2 pancreas and 2 kidney losses in the 33B3.1 group versus 1 in the ATG one (P = ns). The total number of infectious episodes was similar in both groups (21 versus 23). Two malignancies were observed in the ATG group (1 responsible for patient's death). One 33B3.1 patient died because of infectious pneumonia and 3 ATG patients died because of 2 cardiovascular diseases and 1 cancer. All patients had functioning grafts at the time of death. The 3-month and 36-month patient, pancreas, and kidney actuarial survival rates were, respectively, 100, 65, and 100%, and 95, 50, and 82% in the 33B3.1 group and 95, 80, and 90%, and 80, 70, and 80% in the ATG one (P = ns). These data suggest that, although a significantly higher rejection episode incidence was observed in patients treated with 33B3.1 monoclonal antibody as compared with ATG, similar long-term results can be obtained following primary cadaveric combined pancreas/kidney transplantation.

A truncated soluble form of the human interleukin-2 receptor p55 chain (T-S-IL-2R) was expressed to high levels in RODENT (mammalian) cells and affinity-purified. Its biochemical behavior was analyzed by polyacrylamide gel electrophoresis (PAGE), gel filtration, and sucrose gradient centrifugation. It migrated as a single 40-kDa band on sodium dodecyl sulfate-PAGE (reducing or nonreducing conditions), whereas it ran as a 80-kDa component on native PAGE or as a 86-kDa component on gel filtration. The combination of gel filtration and density gradient sedimentation gave a Stokes radius of 4.0 nm and a sedimentation coefficient of 3.72 S. The deduced molecular mass was 67 kDa, and the fractional ratio was 1.516. These data therefore indicated that the T-S-IL-2R was secreted as an homodimer of two noncovalently associated 40-kDa subunits. Cross-linking experiments using bifunctional reagents enabled the materialization of the dimeric structure on sodium dodecyl sulfate-PAGE. Stoichiometric binding studies using two monoclonal antibodies (mAbs 33B3.1 and 11H2) reacting with separate epitopes on the p55 chain also strongly supported the dimeric structure. Indeed, there was one binding site for the 33B3.1 mAb (and Fab fragment) per T-S-IL-2R 40-kDa subunit, whereas the 11H2 mAb (or Fab fragment) could bind only half a site per subunit, a result which could only be explained when assuming more than one subunit for the native T-S-IL-2R. Soluble interleukin-2 receptor species were also purified from culture supernatants of either L cells transfected with the full-length p55 cDNA or a normal alloreactive T cell clone. Similar biochemical behavior and reactivities with the two mAbs were found. Finally, cell-surface p55 chains expressed either by pgL21 or 4AS cells bound the 33B3.1 and 11H2 mAbs in a 2:1 ratio, suggesting that the p55 chains are also associated as homodimers when imbedded in the membrane.

Gene transfer into grafts of molecules with potentially immunomodulating actions may prolong allograft survival and give a method of analyzing the mechanisms of allograft rejection and tolerance. 1 Interleukin-4 (IL-4) and interleukin-10 (IL-10) have the capacity to drive the immune response from a Thl to a Th2 profile and inhibit macrophage activation, 2 making them candidates to be overexpressed in the local graft microenvironment. 3 The purpose of this study was to analyse the feasibility and conditions for efficient recombinant adenovirus (rAd)-mediated gene transfer into rat liver, kidney, and pancreatic islets, and to validate rAds as efficient vectors for localized cytokine overexpression.

La transplantation d'organes animaux a l'homme a ete tentee a de multiples reprises depuis le debut du siecle mais s'est toujours soldee par des echecs. La xenogreffe avec des organes de porc suscite cependant un tres grand interet. En raison de l'eloignement phylogenique des deux especes, un greffon porcin vascularise ne peut resister a l'intensite du rejet plus de quelques dizaines de minutes. On a recemment identifie les acteurs de la reaction immediate de rejet suraigu : antigenes de l'endothelium du donneur, anticorps naturels et complement de l'hote. Les reactions du rejet secondaire commencent aussi a etre analysees. Parallelement, l'isolement et l'utilisation d'ilots de Langerhans porcins comme alternative a l'insulinotherapie chez les patients diabetiques sont l'objet de recherches intensives. La xenotransplantation necessite non seulement la mise au point de traitements specifiques du rejet, mais aussi la production d'animaux, modifies par transgenese, dont les organes seraient, a terme, toleres par l'homme.

Several alloreactive human T cell clones derived from a rejected kidney graft were found to produce in their culture supernatants soluble interleukin 2 receptors (IL-2R) upon specific antigenic challenge (irradiated B cell line from the graft's donor). Among them, the 2B11, a high producer clone, was used to purify a soluble IL-2R preparation which was analyzed, in comparison with the high and low affinity cell-surface IL-2R expressed by 2B11 cells, for its parameters of interaction with a set of anti-IL-2R monoclonal antibodies (mAb) and IL-2. This soluble receptor purified by affinity chromatography (anti-IL-2R mAb column) and sodium dodecyl sulfate gel electrophoresis is composed of a single chain of 35,000 to 45,000 Da. Immunoradiometric assays (IRMA) at equilibrium were set up, using pairs of mAb directed against two separate epitopes on the Tac antigen of the human IL-2R, to measure the respective dissociation constant of these mAb for the soluble IL-2R. They were found to be identical to those found on the cell-surface IL-2R. A 1:1 stoichiometry between the two epitopes were found both on the membrane and soluble species. Competition experiments between membrane and soluble IL-2R for binding the mAb allowed the quantitative analysis of the concentration of soluble IL-2R without the need of amino acid analysis on purified material and set up a quantitative IRMA for the human soluble IL-2R (detection limit 5 pM). The affinity of the soluble IL-2R for IL-2 was determined by various techniques including an IRMA using an anti-IL-2R mAb and radiolabeled IL-2. The results obtained led us to conclude that the soluble IL-2R binds IL-2 with a dissociation constant (KD = 30 nM) identical to that found for the binding of IL-2 to low affinity cell-surface IL-2R (Tac antigen). Whereas 2.5% of cell-surface IL-2R expressed 2 days after antigenic stimulation of 2B11 cells were of high affinity for IL-2 (KD = 25 pM), no (less than 0.07%) high affinity binding sites could be detected on the purified soluble IL-2R. This soluble IL-2R therefore likely corresponds to a truncated, extracellular part of the membrane Tac antigen. The amounts of soluble Tac antigen produced by the 2B11 alloreactive human T cell clone did not exceed 1 nM and, as expected from the binding studies, did not affect IL-2-induced T cell proliferation. The physiologic and pathologic implications of our results are discussed.

A rat IgG2a monoclonal antibody (33B3.1) directed at the human Tac antigen and interacting with the high affinity binding site of IL-2 on its receptor was tested for treatment of kidney allograft recipients. This antibody is efficient in preventing graft rejection in the weeks following transplantation. Its activity compares to polyclonal rabbit anti-thymocyte globulin but it has fewer side effects. The dose of 33B3.1 given is critical for its preventive effect. A majority of the recipients developed IgM and/or IgG against rat Ig. Rejection episodes occurring during 33B3.1 therapy were associated with an early rise in host anti-33B3.1 antibodies and a drop in their monoclonal antibody (MoAb) circulating levels. When 33B3.1 was given to treat ongoing rejection, it had only an inconsistent and/or delayed effect. Various factors are discussed, such as circulating free 33B3.1 levels, host anti-33B3.1 immune response and the presence of inhibiting concentrations of soluble Tac chain, which can interplay with the protective effect of the anti-interleukin-2-receptor monoclonal antibody. The use of bioreagents which interfere only with determinants present on host activated lymphocytes may represent a new step toward selective immunosuppression in allograft recipients.

A human T cell clone (termed 40.2.6) established from a rejected human kidney allograft has been studied for its ability to express membrane IL 2 receptors in response to antigen (irradiated cells from the graft's donor) and recombinant IL 2 (rec-IL 2). On antigenic stimulation, the 40.2.6 clone produced low levels (0.15 U/ml) of IL 2 (peak at 24 hr) and incorporated (3H)thymidine (peak at 48 hr). This incorporation was strongly enhanced on addition of rec-IL 2 and was inhibited by the 33B31 antibody, an anti-human IL 2 receptor monoclonal antibody (Mab). The 125I-labeled 33B31 Mab has been used to quantify the density of IL 2 receptors on 40.2.6 cells. Cells not re-exposed to antigen or rec-IL 2 had a level of 33B31-binding sites which declined rapidly (10% of starting value after 2 days). This level remained much more stable when rec-IL 2 (1 U/ml) was present in the medium (80% at day 2). Antigen induced a three- to eightfold increase in the level of 33B31-binding sites which peaked at 24 hr and then declined. When a similar antigenic stimulation was performed in the presence of rec-IL 2 (1 U/ml), the level of 33B31-binding sites peaked at a higher value (eight- to 20-fold increase at day 2), and its subsequent decline was slower. These potentiating effects of rec-IL 2 were dose-dependent and occurred at low concentrations corresponding to the saturation by rec-IL 2 of high affinity IL 2 receptor sites. Finally, high affinity IL 2 receptors, as measured by the binding of 35S-labeled rec-IL 2, were found to be similarly up-regulated by antigen and rec-IL 2. Together, our results obtained on a monoclonal human T cell population with highly purified rec-IL 2 demonstrate that rec-IL 2 and antigen act in synergy to induce the expression of both high and low affinity membrane IL 2 receptors.

The effect of CsA on antigen-induced IL-2 receptor expression was studied on a human T lymphocyte clone (4AS) obtained from cells infiltrating a rejected human kidney. Stimulation of 4AS clone cells with specific antigen (D.BLCL) was strongly inhibited by CsA (50% inhibition of tritiated thymidine uptake at about 12.5 ng/ml). Addition of recombinant IL-2 only partially restored 4AS growth inhibition, suggesting that another antigen-induced activation signal such as IL-2-receptor expression could be impaired by CsA. Using 125I-labeled human recombinant IL-2 and 125I-labeled 33B3.1 (a MoAb directed against TAC antigen), we found that expression of both high and low affinity sites was decreased when clone cells were stimulated with D.BLCL in the presence of CsA and exogenous IL-2 (about 50% inhibition in the presence of 500 ng/ml of CsA). Northern blot analysis of IL-2-receptor m.RNA (TAC antigen m.RNA) showed that inhibition occurred at least in part at the pretranscriptional level.