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International audience; Antibodies of non-human mammals are glycosylated with carbohydrate antigens, such as galactose-α-1-3-galactose (α-Gal) and N-glycolylneuraminic acid (Neu5Gc). These non-human carbohydrate antigens are highly immunogenic in humans due to loss-of-function mutations of the key genes involved in their synthesis. Such immunogenic carbohydrates are expressed on therapeutic polyclonal rabbit anti-human T-cell IgGs (anti-thymocyte globulin; ATG), the most popular induction treatment in allograft recipients. To decipher the quantitative and qualitative response against these antigens in immunosuppressed patients, particularly against Neu5Gc, which may induce endothelial inflammation in both the graft and the host. We report a prospective study of the antibody response against α-Gal and Neu5Gc-containing glycans following rabbit ATG induction compared to controls. We show a drop in the overall levels of anti-Neu5Gc antibodies at 6 and 12 months post-graft compared to the pre-existing levels due to the major early immunosuppression. However, in contrast, in a cross-sectional study there was a highly significant increase in anti-Neu5Gc IgGs levels at 6 months post-graft in the ATG-treated compared to non-treated patients(P = 0.007), with a clear hierarchy favouring anti-Neu5Gc over anti-Gal response. A sialoglycan microarray analysis revealed that the increased anti-Neu5Gc IgG response was still highly diverse against multiple different Neu5Gc-containing glycans. Furthermore, some of the ATG-treated patients developed a shift in their anti-Neu5Gc IgG repertoire compared with the baseline, recognizing different patterns of Neu5Gc-glycans. In contrast to Gal, Neu5Gc epitopes remain antigenic in severely immunosuppressed patients, who also develop an anti-Neu5Gc repertoire shift. The clinical implications of these observations are discussed.

We give existence and uniqueness results of the weak-renormalized solution for a class of nonlinear Boussinesq's systems. The main tools rely on regularity results for the Navier-Stokes equations with precise estimates on the solution with respect to the data in dimension $2$ and on the techniques involved for renormalized solutions of parabolic problems.

Given a parabolic cylinder Ω × (0, T), where Ω is a bounded domain of $${\mathbb{R}^N}$$ , we consider IBV problems involving equations of the type $$b(u)_{t} - \Delta_{p} u = \mu$$ where b is a increasing C 1-function and μ is a diffuse measure. We prove the existence and uniqueness of a renormalized solution for this class of nonlinear parabolic equations.

This Note deals with the modeling of a structure made of straight elastic rods whose thickness tends to 0. We show that, upon an adequate scaling, the infimum of the total elastic energy tends to the minimum of a functional which depends on fields defined on the centerlines of the rods.

We introduce a simplified model for the minimization of the elastic energy in thin shells. The thickness of the shell remains a parameter in this new model.

BACKGROUND: As an alternative to the cumbersome Kleihauer-Betke test (KBT), flow cytometry represents a powerful method for the identification and quantification of fetal red blood cells (RBCs) in maternal circulation. STUDY DESIGN AND METHODS: The aim of this study was to evaluate the Fetal Cell Count kit (IQ Products), an innovative flow cytometric method, based on the combination of antibodies directed, respectively, against fetal hemoglobin (HbF) and carbonic anhydrase (CA), a marker expressed after birth, to discriminate fetal RBCs from adult F cells containing HbF. The investigation was performed by two French laboratories that compared the data obtained by flow cytometry and KBT in 455 pregnant or just-delivered women as well as in 124 artificial mixtures containing from 0.01 to 5.00 percent cord cells. RESULTS: The FL1/FL2 histogram allowed distinction between fetal RBCs (HbF+, CA–), F cells (HbF+, CA+), and adult RBCs (HbF–, CA+). The limits of detection and quantification were determined at 0.03 and 0.10 percent or 0.02 and 0.05 percent when analyzing 100,000 or 200,000 events, respectively. Linearity was demonstrated between 0.01 and 5.00 percent fetal cells in the mixtures (r = 0.95, p

We consider a set of elastic rods periodically distributed over a 3d elastic plate (both of them with axis $x_3$) and we investigate the limit behavior of this problem as the periodicity $\varepsilon$ and the radius r of the rods tend to zero. We use a decomposition of the displacement field in the rods of the form u=U+v where the principal part U is a field which is piecewise constant with respect to the variable $(x_1,x_2)$ (and then naturally extended on a fixed domain), while the perturbation v remains defined on the domain containing the rods. We derive estimates of U and v in term of the total elastic energy. This allows to obtain a priori estimates on u without solving the delicate question of the dependence, with respect to $\varepsilon$ and r, of the constant in Korn’s inequality in a domain with such a rough boundary. To deal with the field v, we use a version of an unfolding operator which permits both to rescale all the rods and to work on the same fixed domain as for U to carry out the homogenization process. The above decomposition also helps in passing to the limit and to identify the limit junction conditions between the rods and the 3d plate.

In [4], the first two authors studied a nonlinear monotone problem in a multidomain composed of a part [Formula: see text], with a highly oscillating boundary, placed upon an asymptotically flat part of thickness hε. More precisely, [Formula: see text] is a "forest" of cylinders with fixed height and small cross section of size ε, distributed with ε-periodicity upon the flat domain. The analysis was achieved under the assumption εp/hε → 0 (p - 1 is the growth order of the operator at infinity), as ε tends to 0, and for rescaled external forces hε fε converging to 0 in the (rescaled) flat domain. In the present paper, we present the analysis under the assumption εp/hε → l, with l ∈ [0, +∞], and for general limit forces in the flat domain. When l ∈ ]0, +∞[, we show that a discontinuity in the Dirichlet transmission condition may occur between the limit domain filled by the oscillating boundary and the plate. This discontinuity is derived through solving a nonlinear problem (in general for a different monotone operator) in the unit cell of the oscillating boundary. When l = +∞, we show that a deterministic limit model may hardly be expected.

We consider a class of quasi-linear diffusion problems involving a matrix A(t,x,u) which blows up for a finite value m of the unknown u. Stationary and evolution equations are studied for L 1 data. We focus on the case where the solution u can reach the value m. For such problems we introduce a notion of renormalized solutions and we prove the existence of such solutions.

Methamphetamine and ecstasy are addictive drugs that cause major health problems in young people. Here we report on the development of high-affinity monoclonal antibodies to methamphetamine and its analogues, which may constitute powerful tools for antibody-based therapy. Six haptens, methamphetamine and ecstasy analogues, were synthesized, linked to a carrier protein and injected into mice. Several specific monoclonal antibodies were subsequently obtained following fusion of splenocytes from the immunized animals, with Sp2/O cells. Antibody specificity was fully investigated by competition ELISA, using a series of analogues, to identify specific amphetamine and/or ecstasy-specific antibodies. Antibody affinity was estimated to be in the range of 10(8) M(-1) with an enantiomeric hapten. Finally, two characteristic hybridoma clones (DAS-M243-6H5 and DAS-M278-4B12), secreting specific and potent mAbs were isolated. The development of drug-specific antibodies as in this study may provide promising therapeutic insight into how to neutralize methamphetamine in vivo during acute intoxication.

We prove existence and uniqueness of a solution for a class of quasi-linear problems with L 1 data. The diffusion matrix A ( x , u ) is allowed to degenerate with respect to the unknown u. We obtain uniqueness of the solution under a weak assumption on A ( x , u ) that permits to consider highly oscillating or/and increasing coefficients (with respect to u).

Cocaine abuse is a major health problem, with the number of overdose-related incidents on a constant increase. Monoclonal antibodies against cocaine and its major toxic metabolite cocaethylene, have been developed for immunotherapeutical neutralization in vivo. A series of monoclonal antibodies with high affinity for cocaethylene and cocaine were obtained. Clones DASm244-4D8A4A4 (4D8) and DASm244-5B3C3C6 (5B3) were selected and fully characterized. The antibodies secreted exhibited 1.40 x 10(8) and 3.69 x 10(7) M(-1) affinity constants for [3H]-cocaine and cocaethylene, respectively. In addition to cocaine, they bound to cocaethylene and did not recognize non-toxic cocaine metabolites. They did not bind to blood cells, indicating that they may be potential tools for cocaine neutralization in vivo in cases of overdose.

We consider a quasilinear equation (see (1.1)) with L1 data and with a diffusion matrix A(x,u), which is not uniformly coercive with respect to u (see Assumptions (H3)–(H4)). Under such assumptions it is not realistic, in general, to search for a solution which is finite almost everywhere. We introduce two equivalent notions of solutions which take into account the possible values +∞ and -∞ (see Definitions 2.1 and 2.3). Then we prove that there exists at least one such solution. At last we establish an uniqueness result in the class of simultaneous infinite valued solutions.

Catalytic antibodies with a metalloporphyrin cofactor or «hemoabzymes», used as models for hemoproteins like peroxidases and cytochrome P 450, represent a promising route to catalysts tailored for selective oxidation reactions. A brief overview of the literature shows that until now, the first strategy for obtaining such artificial hemoproteins has been to produce antiporphyrin antibodies, raised against various free-base, N-substituted Sn-, Pd- or Fe-porphyrins. Five of them exhibited, in the presence of the corresponding Fe-porphyrin cofactor, a significant peroxidase activity, with k cat / K m values of 3.7×10 3 –2.9×10 5 M −1 min −1 . This value remained, however, low when compared to that of peroxidases. This strategy has also led to a few models of cytochrome P 450. The best of them, raised against a water-soluble tin(IV) porphyrin containing an axial α-naphtoxy ligand, was reported to catalyze the stereoselective oxidation of aromatic sulfides by iodosyl benzene using a Ru(II)-porphyrin cofactor. The relatively low efficiency of the porphyrin–antibody complexes is probably due, at least in part, to the fact that no proximal ligand of Fe has been induced in those antibodies. We then proposed to use, as a hapten, microperoxidase 8 (MP8), a heme octapeptide in which the imidazole side chain of histidine 18 acts as a proximal ligand of the iron atom. This led to the production of seven antibodies recognizing MP8, the best of them, 3A3, binding it with an apparent binding constant of 10 −7 M. The corresponding 3A3–MP8 complex was found to have a good peroxidase activity characterized by a k cat / K m value of 2×10 6 M −1 min −1 , which constitutes the best one ever reported for an antibody–porphyrin complex. Active site topology studies suggest that the binding of MP8 occurs through interactions of its carboxylate substituents with amino acids of the antibody and that the protein brings a partial steric hindrance of the distal face of the heme of MP8. Consequently, the use of the 3A3–MP8 complexes for the selective oxidation of substrates, such as sulfides, alkanes and alkenes will be undertaken in the future.

We study a class of quasilinear elliptic problems with diffusion matrices that have at least one diagonal coefficient that blows up for a finite value of the unknown; the other coefficients being continuous with respect to the unknown (without any growth assumption). We introduce two equivalent notions of solutions for such problems and we prove an existence result in these frameworks. Under additional local assumptions on the coefficients, we also establish the uniqueness of the solution. In that case, and when the non-diagonal coefficients are bounded, this unique (generalized) solution is also the unique weak solution strictly less than the value where the diagonal coefficient blows up.

Background microRNAs (miRNAs) play crucial roles in major biological processes and their deregulations are often associated with human malignancies. As such, they represent appealing candidates as targets of innovative therapies. Another interesting aspect of their biology is that they are present in various biological fluids where, advantageously, they appear to be very stable. A plethora of studies have now reported their potential as biomarkers that can be used in diagnosis, prognosis and/or theranostic issues. However, the application of circulating miRNAs in clinical practices still requires the identification of highly efficient, robust and reproducible methods for their isolation from biological samples. In that context, we performed an independent cross-comparison of three commercially available RNA extraction kits for miRNAs isolation from human blood samples (Qiagen and Norgen kits as well as the new NucleoSpin miRNAs Plasma kit from Macherey-Nagel). miRNAs were further profiled using the Taqman Low Density Array technology. Results We found that, although these 3 kits had equal performances in extracting miRNAs from peripheral blood mononuclear cells, the Macherey-Nagel kit presented several advantages when isolating miRNAs from sera. Besides, our results have indicated that, depending on the quantity of the biological samples used, the extraction procedure directly impacted on the G/C composition of the miRNAs detected. Conclusion Overall, our study contributes to the definition of a reliable framework for profiling circulating miRNAs. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-395) contains supplementary material, which is available to authorized users.

Cell proliferation is controlled by cdk2 which in association with cyclin E and A regulates G1/S transition and S phase progression. cdk2 activation is dependent on its localization in the nucleus where regulatory mediators are found. We report that activation of cdk2 is associated with the formation of cdk2/MAP Kinase complexes. cdk2 associates with both inactive and activated MAP Kinase. Prevention of MAP Kinase activation by the MEK inhibitor PD98059 inhibits both activation and nuclear localization of cdk2 and S phase entry. These findings indicate that the nuclear translocation of cdk2 is associated with the formation of molecular complexes containing active MAP Kinase and is dependent on MAP Kinase activation. Oncogene (2000) 19, 4184 - 4189

Resume Nous donnons un resultat d'existence et d'unicite d'une solution renormalisee pour le probleme d'evolution ∂ u /∂ t − div( a(t,x,u ,Du) + Φ(u)) = ƒ - div g , ou le second membre appartient a L 1 ((0, T ) × Ω) + L p ′(0, T ; W −1 , p ′(Ω)) et ou l'operateur −div( a ( t,x,u , D u )) est un operateur de Leray-Lions qui croit comme ¦D u ¦ p − 1 en D u mais dont la croissance n'est pas controlee en u .

Manganese ions block apoptosis of phagocytes induced by various agents. The prevention of apoptosis was attributed to the activation of manganous superoxide dismutase (Mn-SOD) and to the antioxidant function of free Mn2+ cations. However, the effect of Mn2+ on B cell apoptosis is not documented. In this study, we investigated the effects of Mn2+ on the apoptotic process in human B cells. We observed that Mn2+ but not Mg2+ or Ca2+, inhibited cell growth and induced apoptosis of activated tonsilar B cells, Epstein Barr virus (EBV)-negative Burkitt's lymphoma cell lines (BL-CL) and EBV-transformed B cell lines (EBV-BCL). In the same conditions, no apoptosis was observed in U937, a monoblastic cell line. Induction of B cell apoptosis by Mn2+ was time- and dose-dependent. The cell permeable tripeptide inhibitor of ICE family cysteine proteases, zVAD-fmk, suppressed Mn2+-induced apoptosis. Furthermore, Mn2+ triggered the activation of interleukin-1beta converting enzyme (ICE/caspase 1), followed by the activation of CPP32/Yama/Apopain/caspase-3. In addition, poly-(ADP-ribose) polymerase (PARP), a cellular substrate for CPP32 protease was degraded to generate apoptotic fragments in Mn2+-treated B cell lines. The inhibitor, zVAD-fmk suppressed Mn2+-triggered CPP32 activation and PARP cleavage and apoptosis. These results indicate that the activation of caspase family proteases is required for the apoptotic process induced by Mn2+ treatment of B cells. While the caspase-1 inhibitor YVAD was unable to block apoptosis, the caspase-3 specific inhibitor DEVD-cmk, partially inhibited Mn2+-induced CPP32 activation, PARP cleavage and apoptosis of cells. Moreover, Bcl-2 overexpression in BL-CL effectively protected cells from apoptosis and cell death induced by manganese. This is the first report showing the involvement of Mn2+ in the regulation of B lymphocyte death presumably via a caspase-dependent process with a death-protective effect of Bcl-2.

A new method to quantify red blood cells (RBCs) containing fetal hemoglobin (HbF), or F cells, by flow cytometry was developed. The use of formaldehyde as fixative agent and sodium dodecyl sulfate (SDS) to permeabilize fixed RBCs resulted in a low staining background, a negligible Hb leakage, and minimal cell clumping. HbF was detected by immunofluorescence with a murine monoclonal antibody directed to the γ chain of human Hb. The accuracy of the F-cell count was evaluated on mixed-field populations of adult and cord RBCs. The results of flow cytometry were highly correlated with HbF dosage in the hemolysate by high-pressure liquid chromatography (HPLC) (R > 0.99). The sensitivity of the method allowed the study of HbF distribution at the single-cell level in normal controls and patients with sickle-cell disease (SCD). All patients exhibited a heterocellular distribution of HbF, with highly variable percentages of F cells and non-F cells. As an additional and valuable biological parameter in SCD, the mean HbF content per F cell could be deduced from the measurement of HbF level by HPLC and F-cell count by flow cytometry. Results were unchanged after storing the cells for several weeks in an optimized resuspending solution. Since it does not need uncommon reagents or devices and allows the simultaneous detection of membrane antigens by two-color flow cytometry, this method is convenient for routine laboratories as well as for research purposes. Cytometry 32:186–190, 1998. © 1998 Wiley-Liss, Inc.

An existence and uniqueness result of a renormalized solution for a class of doubly nonlinear evolution equations is established. The data belong to L 1 and no growth assumption is made on the nonlinearities. A comparison result is also proved for such solutions.

Resume Nous presentons deux cadres equivalents de solutions pour un probleme quasi-lineaire du type diffusion avec coefficients qui explosent pour une valeur finie de l'inconnue. Nous demontrons, sous l'hypothese de continuite des coefficients (sur leurs domaines de definition) par rapport a l'inconnue, un resultat d'existence d'une solution dans ces cadres equivalents. Sous des hypotheses plus fortes de regularite locale sur les coefficients, nous donnons un resultat d'unicite de cette solution, qui est aussi, dans ce cas, l'unique solution faible strictement plus petite que la valeur pour laquelle les coefficients explosent.

We prove two existence results of a solution for a nonlinear system in thermoviscoelasticity in which the mechanical dissipation is not linearized. The techniques of renormalized solutions for parabolic equations with L1 data are used to handle the energy conservation law.

Rather efficient catalysis of the decomposition of 5-nitro-benzisoxazole to the cyanophenol was observed with antibodies elicited against a cationic hapten structurally unrelated to the benzisoxazole substrate. The rate enhancement by the most active antibody is better than 104 and the reaction is catalyzed by a carboxylate group associated with a hydrophobic binding site.

Control of complement deposition on autologous cells is mediated by a group of complement regulatory membrane proteins acting at different levels of the complement cascade. Decay accelerating factor (CD55) prevents the assembly of C3 convertases and CD59 membrane inhibitor of reactive lysis (MIRL) restricts homologous complement lysis by the membrane attack complex of complement (MAC) by inhibition of C5b-8 catalyzed insertion of C9. The aim of this work was to study the eventual expression of CD55 and CD59 on human skeletal muscle fibers. Highly sensitive immunoblotting using murine monoclonal antibodies showed that CD59, but not CD55, was present in skeletal muscle fibers. Immunocytochemistry with a monoclonal antibody against CD59 demonstrated a dense granular immunostaining mainly localized at the level of the sarcolemma. Thus, CD59, but not CD55, is expressed on normal skeletal muscle fibers. CD59 may play a prominent role in preventing MAC deposition and subsequent complement-mediated damage in myopathies where the complement system activation is involved.

With the aim to obtain specific anti-cocaine antibodies directed against cocaine and active metabolites for use in immunotherapy, a series of six haptens were prepared, based on the structure of cocaine. The haptens differed by 3 positions of linkers: nitrogen, carboxyl group, and aromatic nucleus. The haptens were grafted onto 3 carrier proteins: bovine serum albumin, tetanus toxoid or keyhole limpet hemocyanin according to different methods of coupling: carbodiimide or mixed anhydride techniques. The immuno-conjugates were administered to rabbits and the antisera elicited were analyzed in term of titer, affinity and specificity. Variation in antisera properties were observed and attributed to the site of coupling the hapten, to the carrier proteins, and to the method of coupling. Antisera titers were in the range of 1/1 (no significant response) to 1/12,832, with antisera affinity up to 5.9 × 10(11) M-1. This strategy allowed the selection of a new hapten, which after coupling on carrier proteins, led to the production of antisera with a high specificity toward cocaine and cocaethylene, but exclude the inactive metabolites of cocaine.

The epitope Fy6 recognized by two monoclonal antibodies (i3A and BG6), which inhibit binding of chemokines to the Duffy antigen, was characterized by means of peptides synthesized on pins (Epitope Scanning Kit) and deletion mutagenesis. Both antibodies showed very similar specificities. They recognized a linear epitope, the essential portion of which was the heptapeptide Gln-Leu-Asp-Phe-Glu-Asp-Val comprising amino acid residues 21-27, located between two glycosylation sites of the Duffy protein. All the amino acid residues of the epitope, except Glu, were essential for antibody binding, since they could not be replaced by any other amino acid residues or by only one or two. The Glu residue could be replaced by most other amino acid residues, and its replacement by 10 amino acid residues gave a distinct increase in the antibody binding. The results were in full agreement with the finding that the mutant of the Duffy antigen, lacking amino acid residues 23-25 (-Asp-Phe-Glu-), did not bind the i3A antibody, but bound the anti-Fy3 monoclonal antibody similarly to the wild type of the Duffy antigen. The apparent affinity constants of both anti-Fy6 antibodies were determined by surface plasmon resonance, using immunopurified Duffy protein as a ligand.

Recently, fibrinoid of the human placenta has been described as being composed of two main types differing in origin and chemical composition. Fibrin-type fibrinoid is mostly a blood clot product. Matrix-type fibrinoid was defined as the extracellular matrix secreted by extravillous trophoblast cells. The structure and composition of matrix-type fibrinoid was addressed in this study, focusing on fibronectins as one major constituent. A panel of antibodies directed against different fibronectin isoforms generated by different mRNA splicing, as well as antibodies recognizing oncofetal carbohydrate epitopes, were used on cryostat, paraffin and Lowicryl sections of placental tissue from different stages of pregnancy. The oncofetal carbohydrate epitopes studied comprised the blood group precursor antigens i and I. We identified the blood group-related antigen i as an additional marker for matrix-type fibrinoid. The antigen was detected on a glycoprotein that was also recognized by the fibronectin antibodies in western blots. Immunohistochemically this i-glycosylated oncofetal fibronectin-like molecule of about 55 kDa is expressed only by the invasive phenotype of extravillous trophoblast. Long chain carbohydrate moieties with a structure fulfilling the criteria for i reactivity on human placental fibronectin are known to have anti-adhesive properties and to enhance resistance of the protein chain to proteolysis. These properties underline the functional relevance of glycosylation of fibronectins in matrix-type fibrinoid and suggest matrix-type fibrinoid is a typical matrix of invasive cells. In contrast, the more mature blood group precursor I could be detected after sialidase pretreatment of sections. This antigen was expressed by villous, non-invasive trophoblast.

A 37-kDa protein was immunopurified from human erythrocytes as a complex with a monoclonal antibody directed against the Kell blood group protein of 93 kDa. A rabbit antibody raised against the purified complex reacted on a Western blot with the 93-kDa and 37-kDa proteins and was able to immunoprecipitate the 37-kDa component from K,, erythrocytes which express large amount of the Kx antigen, but not from erythrocytes of patients suffering from McLeod syndrome, a X-linked disorder in which the Kx antigen is lacking. Additional studies have shown that the 37-kDa protein is not glycosylated, and permitted the sequence of the 22 first N-terminal amino acids to be established. This sequence was identical to the predicted protein product of the XK gene cloned recently, which is deleted or mutated in McLeod patients [Ho, M., Chelly, J., Carter, N., Danek, A., Crocker, P. & Monaco, A. P. (1994) Cell 77, 869-8801. Our findings provide strong evidence that the 37-kDa red cell membrane protein is identical to the Kx protein produced by the XK structural gene and demonstrate that Kx and Kell proteins are two subunits expressed as a complex hold by disulfide bond(s) at the red cell surface.

The binding of human natural antibodies to porcine endothelial cells is the first step leading to activation of complement and lysis of the cells. The kinetic of incorporation of propidium iodide into porcine endothelial cell line SVPAEC/6A or peripheral blood lymphocytes was investigated to monitor the permeabilization of target cells by membrane attack complex. In less than 5 min, more than 90% of porcine cells were highly fluorescent upon incubation with human sera, showing they incorporate propidium iodide. The lysis occurred neither with adult sera pretreated with dithiothreitol nor with umbilical cord sera, suggesting that lytic antibodies belong to the IgM class. By using antibodies purified from human sera, the bulk of lytic activity was found to be associated with IgM specific for the Galal-3Gal epitope, whereas IgG were not able to lyse the porcine cells. These antibodies could be readily absorbed on rabbit red blood cells known to express the main epitope target of xeno human antibodies. Finally, our results show that permeabilization of porcine endothelial cells is a very early phenomenon, probably directly associated with the biological process responsible for the hyperacute rejection.

Resume L'alloimmunisation anti-HPA-1a chez les femmes HPA-1b homozygotes est fortement associee au phenotype HLA-DR3, DRw52a, ce qui suggere que les molecules HLA de classe II jouent un role crucial dans la reponse immune contre l'antigene HPA-1a. Le systeme diallelique HPA-1 est porte par la glycoproteine IIIa et resulte d'un polymorphisme unique de l'acide amine 33. Un mecanisme de presentation de l'antigene HPA-1a par ces molecules de classe II peut etre envisage. Nous avons donc teste la liaison « in vitro , par une technique sur plaque et en solution, de peptides de synthese couvrant la region 25–42 de la GPIIIa a des molecules HLA-DR3 et DRw52a purifiees. Aucune liaison n'a ete observee, indiquant que la region cruciale pour la liaison aux molecules HLA de classe II n'est pas la region 25–42 de la GPIIIa ou que d'autres phenomenes sont requis « in vivopour la liaison. Ces resultats peuvent aussi suggerer un role indirect de l'acide amine 33 conduisant a la stimulation des cellules T.

Resume Des anticorps monoclonaux diriges contre les glycoproteines membranaires DAF (antigene CD55) et MIRL (HRF20 ou antigene CD59) ont ete utilises pour identifier les populations erythrocytaires CD55− et CD59− des patients atteints d'hemoglobinurie nocturne paroxystique (HNP). Les anticorps NaM16-4D3 (CD55, IgG2a) et NaM77-1E5 (CD59, IgG3) sont faiblement agglutinants et representent des outils de choix pour la quantification par cytometrie en flux indirecte des populations erythrocytaires normales (HNPI) et anormales (HNPII et HNPIII) des patients HNP. Les anticorps NaM125-7H10 (CD55) et NaM123-6G12 (CD59), de classe IgM ont ete selectionnes pour leur pouvoir agglutinant et permettent de separer les cellules HNPI des cellules HNPII et HNPIII par la technique d'agglutination en gel. Une relation directe a ete etablie entre les erythrocytes fluorescents (cytometrie en flux) et les erythrocytes agglutines (agglutination en gel) pour des populations DAF+ et DAF− artificielles et pour une serie de patients HNP connus. Ces methodes utilisant des anticorps monoclonaux specifiques representent une alternative aux methodes classiques d'hemolyse. Sur la base de l'analyse de plus de 100 echantillons dont 12 cas de HNP, nous proposons : 1) la caracterisation rapide des erythrocytes CD55− et CD59− par agglutination en gel a l'aide des anticorps de classe IgM ; 2) la quantification des populations erythrocytaires normales et anormales par cytometrie en flux.

Resume Dans les thrombopenies neonatales allo-immunes, le systeme PI A est le plus frequemment implique. Cependant, dans environ 30% des cas, aucun anticorps circulant anti-PI A1 ne peut etre decele dans le serum maternel malgre les diverses techniques utilisees. Nous proposons d'ameliorer la detection sur membrane de nitrocellulose par l'utilisation de peptides provenant d'extraits triton X-114 de plaquettes traitees a l'α-chymotrypsine. La lyse par ce detergent non-ionique permet un enrichissement en peptides hydrophobes issus de la glycoproteine IIIa (GPIIIa) portant la specificite antigenique PI A1 . Sur les repliques obtenues apres transfert electrophoretique, les anti-PI A1 reconnaissent deux peptides caracteristiques de masse moleculaire 66 et 54 kDA qui portent l'epitope PI A1 . Cette technique, utilisee pour le depistage et l'identification des anticorps anti-PI A1 se revele etre sensible, fiable et realisable dans un temps relativement court (minimum de 6 h).

Summary Murine monoclonal antibodies (MoAbs) were produced against the blood group KEL1 glycoprotein (93 kD component) immunopurified from human erythrocytes. One monoclonal antibody, 5A11 (IgGl, K), detects by immunoblotting a 93 and 184 kD component from KEL: 1, -2 or KEL: - 1,2 red cell membrane preparations, separated by SDS polyacrylamide gel electrophoresis (PAGE) under non-reducing conditions. The 184 kD component was not detected under reducing conditions, suggesting that it represented a dimer of the 93 kD KEL glycoprotein. Neither the 93 nor the 184 kD could be detected from KO or McLeod erythrocyte membrane preparations, indicating that the monoclonal antibody reacts with the KEL glycoprotein previously identified as a 93 kD species. Since this antibody does not agglutinate native or protease-treated erythrocytes, it is likely that it reacts with the cytoplasmic domain of the KEL glycoprotein. This was also substantiated by showing that 5A11 could immunoprecipitate the 93 kD component from intact membranes and inside-out vesicles but not from right-side-out vesicles. Immunostaining of membrane proteins prepared from human blood cells (platelets, lymphocytes, monocytes and granulocytes) and non-human erythrocytes revealed that the 93 kD molecule was only present on human red cells. Several other murine monoclonal antibodies obtained from the same fusion experiment gave identical results, but competition analyses on immunoblots indicated that these antibodies reacted with distinct epitopes on the KEL glycoprotein.

This paper is concerned with the mathematical study of a nonlinear system modelling an irreversible phase change problem. Uniqueness of the solution is proved using the accretivity of the system in (L1)2. Expressing one of the two unknowns as an explicit functional of the other reduces the system to a single nonlinear evolution equation and ultimately leads to an existence theorem.In this paper the existence and uniqueness of the solution of a nonlinear system modelling some irreversible phase changes is established.

The alloimmunization against the platelet PL-A1 antigen is strongly associated with a HLA class II structure in mothers of thrombocytopenic neonates. Most of the immunized women have first been shown to possess the DR3 specificity and subsequently the DRw52 allele. The 18 immunized mothers studied here by restriction fragment length polymorphism analysis had the DRw52a specificity at the DRB3 locus whatever their HLA-DRB1 gene product. This finding strongly suggests that the DRB3 chain is directly involved in the presentation of the PL-A1 antigen to the specific T cell. In addition, the similarities between DR3 and DRw52 structures due to a hypothetical gene conversion event should be considered in order to understand the high frequency of DR3 among the DRw52a-responding women. Alternatively, the high frequency of DR3 among the DRw52-responding mothers might be due to the high responder status associated with the former specificity.

The NeuNAc level of erythrocyte membranes from two related donors exhibiting the Mz variety of St(a+) phenotype within the MNSs blood group system was found to be decreased by about 16%. The quantity of glycophorin A was decreased by about 38%, whereas that of glycophorin B was not significantly different from normal. Mz erythrocyte membranes were also found to contain an abnormal component (molar ratio to glycophorin A about 0.89:1.0) with an apparent molecular mass of about 24,000 Da. Immunoblotting experiments and amino-acid sequence analysis revealed that the novel component (and glycophorin A in one of the donors) carries blood group M activity. Blood group N activity was demonstrable for glycophorin A and glycophorin B from both donors. Amino-acid sequence analysis of chymotryptic, tryptic and cyanogen bromide peptides demonstrated that the novel molecule exhibits the typical structure of a Sta-active molecule. However, since it exhibits blood group M activity, it appears to represent a variant of glycophorin A lacking the residues 27-58 (encoded by exon three of the glycophorin A gene) rather than a glycophorin B-glycophorin A-hybrid molecule of the anti-Lepore type. Since one of the Mz heterozygotes was found to exhibit both M- and N activity on glycophorin A, the Mz gene complex appears to encode a blood group N-active glycophorin A apart from the novel component and a blood group s-active glycophorin B, although the level of glycophorin A in the erythrocyte membranes is decreased by about half.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone-marrow-derived cells facilitate tumour angiogenesis, but the molecular mechanisms of this facilitation are incompletely understood. We have previously shown that the related EG-VEGF and Bv8 proteins, also known as prokineticin 1 (Prok1) and prokineticin 2 (Prok2), promote both tissue-specific angiogenesis and haematopoietic cell mobilization. Unlike EG-VEGF, Bv8 is expressed in the bone marrow. Here we show that implantation of tumour cells in mice resulted in upregulation of Bv8 in CD11b+Gr1+ myeloid cells. We identified granulocyte colony-stimulating factor as a major positive regulator of Bv8 expression. Anti-Bv8 antibodies reduced CD11b+Gr1+ cell mobilization elicited by granulocyte colony-stimulating factor. Adenoviral delivery of Bv8 into tumours was shown to promote angiogenesis. Anti-Bv8 antibodies inhibited growth of several tumours in mice and suppressed angiogenesis. Anti-Bv8 treatment also reduced CD11b+Gr1+ cells, both in peripheral blood and in tumours. The effects of anti-Bv8 antibodies were additive to those of anti-Vegf antibodies or cytotoxic chemotherapy. Thus, Bv8 modulates mobilization of CD11b+Gr1+ cells from the bone marrow during tumour development and also promotes angiogenesis locally.

Catalytic antibodies with a metalloporphyrin cofactor represent a new generation of biocatalysts tailored for selective oxidations. Thus monoclonal antibodies, 3A3, were raised against microperoxidase 8 (MP8), and the corresponding 3A3-MP8 complexes were shown previously to have a high peroxidase activity. This paper shows that those complexes also catalyzed efficiently the nitration of phenol into 2- and 4-nitrophenol by NO2 − in the presence of H2O2. pH dependence studies suggested that no amino acid from the antibody protein participated in the heterolytic cleavage of the O-O bond of H2O2. The inhibition of the reaction by cyanide and radical scavengers suggested a MP8-mediated peroxidase-like mechanism, involving the reduction of high-valent iron-oxo species by NO2 − and phenol producing, respectively, NO2 · and phenoxy radicals, which then reacted to give nitrophenols. Finally, the antibody protein appears to have two major roles: (i) it protects MP8 toward oxidative degradations and (ii) it induces a regioselectivity of the reaction toward the formation of 2-nitrophenol.

In this study, we investigated the involvement of caspases in TGFbeta-induced apoptosis in human B cells. Our results show that TGFbeta-mediated nuclear fragmentation, observed in the Epstein-Barr virus-negative Burkitt's Lymphoma cell line BL41, was abolished in the presence of the tripeptide caspase inhibitor zVAD-fmk or the specific caspase-3 inhibitor DEVD-fmk. Other apoptotic manifestations such as cell shrinkage, surface phosphatidylserine expression and chromatin condensation were strongly inhibited by zVAD-fmk but only partially by DEVD-fmk. This suggests that other caspases in addition to caspase-3 control these apoptotis-associated features. Specific activation of caspase-3 during TGFbeta-induced apoptosis was demonstrated by the DEVD-fmk-sensitive expression of the active p17 subunit of caspase-3 and by in vivo cleavage of PARP. In addition, TGFbeta treatment of BL41 promoted the expression of both dephosphorylated and truncated forms of the retinoblastoma protein. Inhibition of caspase-3 activity abolished both nuclear fragmentation and expression of the truncated retinoblastoma protein, without modifying the G1 cell cycle arrest induced by TGFbeta. Our data thus demonstrate that TGFbeta-induced apoptosis of lymphoma B lymphocytes is dependent on caspase activation and involves caspase-dependent cleavage of the retinoblastoma protein.

Human natural “anti-Gal” antibodies are specifically directed to Gal alpha 1–3Gal beta 1–4GlcNAc residues expressed on non-primate mammal and new world monkey cells. We investigated the relative involvement of purified IgG and IgM anti-Gal as xenoreactive natural antibodies (XNA). IgG and IgM were isolated from human plasma, and anti-Gal antibodies were purified by affinity chromatography on a Synsorb-14 column (Chembiomed, Edmonton, Alberta, Canada). Anti-Gal of both IgM and IgG classes represent the bulk of human XNA that bind to porcine platelets in enzyme-linked immunosorbent assay (ELISA). On immunoblots, normal human sera, as well as purified IgM and IgG fractions, reacted with 115- , 125-, 135-, 150-, 180-, 210-, and 240-kd) pig platelet proteins, whereas purified anti-Gal antibodies of both IgM and IgG classes mainly bound to 135-, 150-, 180-, and 210-kD glycoproteins. A low reactivity was observed in ELISA with anti-Gal free IgM and IgG, indicating that xenoantibodies are not solely directed to galactosyl epitopes. These antibodies revealed bands of 115, 125, and 240 kD, alpha-Galactosidase treatment of porcine platelet glycoproteins (gps) enriched by affinity chromatography abrogated the reactivity of 135- and 210-kD proteins. N- and O-glycosidase treatments demonstrated that alpha-galactosyl residues are located on the O-glycans of the 135-kD component. Finally, glycoproteins of 90 and 135 kD were identified by amino acid sequencing as the pig analogs of the human glycoproteins IIIa and IIb, respectively, whereas the 240-kD) component was identified as the porcine fibrinogen, using a new murine monoclonal antibody (naM147–7B6; IgG1) specific for its beta-chain.

Five mAb directed at the IL-2R beta chain were analyzed for their binding and functional properties. They define three epitopes on a recombinant soluble beta chain or on the beta chain expressed at the surface of YT-2C2 cells. Epitope 1 (A41 and 6E8 mAb) is part of the IL-2 binding domain, whereas epitope 2 (CF1 and 6E10 mAb) is not involved in IL-2 binding. Epitope 3 (6B5 mAb) also partly overlaps the IL-2 binding domain but does not overlap epitopes 1 and 2. None of the mAb can by themselves inhibit IL-2 induced proliferation of a human activated T cell clone. Only epitope 1 mAb can synergize with an anti-alpha chain mAb to inhibit this proliferation. Using epitope 1 and 2 mAb as well as a purified, recombinant form of the IL-2R beta chain extracellular domain, an ELISA-based immunoassay was set up which allows the quantitative determination of soluble and detergent solubilized IL-2R beta chains. Epitopes 1 and 2 are in non-competitive interaction: the binding of a mAb to one epitope decreases the affinity of a mAb for the second epitope. Epitope 2 mAb have binding stoichiometries (approximately 16,000 sites/cell) which are approximately 80% higher than that of epitope 1 mAb and IL-2 itself (approximately 9000 sites/cell). Upon binding of epitope 2 mAb, the stoichiometries of epitope 1 mAb and IL-2 are increased to reach the stoichiometry of epitope 2 mAb.(ABSTRACT TRUNCATED AT 250 WORDS)

Flow cytometry represents an alternative method to agglutination assays for the accurate quantification of mixed field populations of erythrocytes observed after bone marrow transplantation. Murine monoclonal antibodies directed against the blood group ABH antigens were selected and processed in order to prepare ready-to-use fluorescent reagents. Anti-A (NaM87-1F6; IgG3), anti-B (NaM9-2E11; IgG3) and anti-H (NaM19-7E11; IgM) were purified, labelled with fluorescein isothiocyanate, and used in a direct flow cytometry assay. Anti-A1 (NaM1-1C9; IgG3) was no longer active after FITC-labelling and then was used in an indirect assay. The agglutination was prevented by formaldehyde pretreatment of erythrocytes. Using artificially-made double populations of erythrocytes, measured values with mixtures of 1-100% of cells were very closely related to expected values, showing both the sensitivity and the accuracy of the method. From careful investigation of a series of bone-marrow transplanted patients, we conclude that engraftments could be demonstrated earlier by flow cytometry than by agglutination, because minor populations (1-10%) of cells could be determined accurately only with labelled reagents. In addition, the disappearance of the donor cells on a long-term follow-up of patients enabled an earlier detection of graft failure in one case. The proposed method provides appreciable help to follow engraftment in patients and may have more general applications for the study of other haemopoietic chimaeras.

The specificity of human natural antibodies directed against blood cells from pigs was investigated by ELISA and immunoblotting. Both IgG and IgM were identified as xenoantibodies reacting with pig platelets adsorbed to microplates. The antibodies could be absorbed on platelets as well as on RBC, suggesting that the corresponding antigens are expressed on the surface of a variety of cells. Galactose (20 mM) and melibiose (10 mM) partially inhibited (approximately 50%) the binding of antibodies to platelets, whereas lactose and cellobiose (300 mM) did not. On immunoblots, platelet glycoproteins of 115, 125, 135, 180, and 210 kDa were specifically revealed with human sera diluted 1/20. In contrast with the results obtained by ELISA, xenoantibodies reactive with blotted glycoproteins were of the IgM class and the binding was not significantly inhibited by galactose or melibiose. "Anti-Gal" antibodies, purified from human sera by affinity chromatography on a melibiose-Sepharose immunoabsorbent, represented only a minor portion of the antibodies reactive with porcine platelets. Purified anti-Gal antibodies bound to the 115- and 135-kDa components, whereas the antibodies in the nonretained fraction revealed the 125-kDa molecule. As deduced from these data, human serum contains natural antibodies of both IgG and IgM classes directed to several porcine antigens. Gal-reactive structures were identified on the 115- and 135-kDa platelet glycoproteins, which might be homologous to their counterpart on endothelial cells. Also, the present work suggests that a majority of the natural antibodies reacted with other unidentified structures.