Development of model for analysing respective collections of intended hematopoietic stem cells and harvests of unintended mature cells in apheresis for autologous hematopoietic stem cell collection.

Hematopoietic stem cells (HSCs) required to perform peripheral hematopoietic autologous stem cell transplantation (APBSCT) can be collected by processing several blood volumes (BVs) in leukapheresis sessions. However, this may cause granulocyte harvest in graft and decrease in patient's platelet blood level. Both consequences may induce disturbances in patient. One apheresis team's current purpose is to improve HSC collection by increasing HSC collection and prevent increase in granulocyte and platelet harvests. Before improving HSC collection it seemed important to know more about the way to harvest these types of cells. The purpose of our study was to develop a simple model for analysing respective collections of intended CD34+ cells among HSC (designated here as HSC) and harvests of unintended platelets or granulocytes among mature cells (designated here as mature cells) considering the number of BVs processed and factors likely to influence cell collection or harvest. For this, we processed 1, 2 and 3 BVs in 59 leukapheresis sessions and analysed corresponding collections and harvests with a referent device (COBE Spectra). First we analysed the amounts of HSC collected and mature cells harvested and second the evolution of the respective shares of HSC and mature cells collected or harvested throughout the BV processes. HSC collections and mature cell harvests increased globally (p<0.0001) and their respective shares remained stable throughout the BV processes (p non-significant). We analysed the role of intrinsic (patient's features) and extrinsic (features before starting leukapheresis sessions) factors in collections and harvests, which showed that only pre-leukapheresis blood levels (CD34+cells and platelets) influenced both cell collections and harvests (CD34+cells and platelets) (p<0.001) and shares of HSC collections and mature unintended cells harvests (p<0.001) throughout the BV processes. Altogether, our results suggested that the main factors likely to influence intended HSC collections or unintended mature cell harvests were pre-leukapheresis blood cell levels. Our model was meant to assist apheresis teams in analysing shares of HSC collected and mature cells harvested with new devices or with new types of HSC mobilization.

[Prevalence of platelet-specific antibodies in the recipients of platelet units with transfusion adverse event]. / Prévalence des anticorps antiplaquettes spécifiques chez les receveurs de concentrés plaquettaires avec effet indésirable transfusionnel.

PURPOSE OF THE STUDY: In practice, platelet transfusions are frequently performed in patients with haematologic and/or oncologic diseases. Subsequent to these transfusions, platelet specific antibodies may develop and could induce adverse events, such as platelet transfusion refractoriness or post-transfusion purpura. In order to evaluate the prevalence of platelet specific antibodies in these recipients, adverse events with a request for platelet specific antibodies testing, were studied. PATIENTS AND METHODS: Recipients of platelet units with adverse event, excluding platelet transfusion refractoriness or post-transfusion purpura, were evaluated. RESULTS: From January 1st 2009 to October 31st 2011, 125 adverse events with platelet specific antibodies screening, corresponding to 116 recipients (64 females, 52 males) were included. The main aetiology of the adverse event was a febrile non-haemolytic transfusion reaction in 62 cases (49.6%) and allergy in 40 (32.0%). Most samples tested were post-transfusion solely (101 adverse events, 80.8%). Seven of these samples had free platelet specific antibodies, including four anti-HPA-5b, one anti-HPA-2a and two anti-GPIaIIa. In the cross-match test, platelet specific antibodies were found in two pre-transfusion samples (anti-GP IaIIa) and in five post-transfusion samples (anti-GPIaIIa, three cases; anti-GP IbIX, one case; anti-GP IIbIIIa and -GPIbIX, one case). CONCLUSION: According to this study on platelet transfusion related adverse event, few platelet specific antibodies were detected on pre- and post-transfusion samples. The implementation of platelet specific antibodies testing before transfusion would give more accurate data and help prevent adverse events as typed platelets would be given when platelet specific antibodies were found.

[Chimerism analysis after allogeneic haematopoietic stem cell transplantation. Interest of cell sorting: general review]. / Étude de chimérisme post-greffe de cellules souches hématopoïétiques. Intérêt du tri cellulaire : revue générale.

Haematopoietic stem cells transplantation, widely used these last decades, represent the ultimate treatment resource for patients with haematological malignancies. Long range success of this treatment is particularly affected by relapse of the initial disease, graft rejection or graft versus host disease. Chimerism analysis after transplantation had been used since several years to document engraftment, to determine the risk of relapse and to adapt therapy promptly when necessary. Usefulness of this analysis for the outcome of transplanted patients, as well as the impact of using high sensitive techniques coupled with specific cell populations sorted have been demonstrated by retrospective studies. Follow-up of chimerism would allow to operate efficiently before the onset of clinical signs in leukaemic patients with high risk of relapse and to control the expression of minimal residual disease when specific molecular markers could not be monitored.

[Monitoring of chimerism in myeloid cells sorting of transplanted patients with acute myeloid leukaemia: a study from Lyon (France)]. / Suivi de chimérisme des populations cellulaires myéloïdes chez des patients allogreffés atteints de leucémie aiguë myéloïde : étude lyonnaise.

Chimerism analysis after allogeneic haematopoietic stem cell transplantation has been used to document engraftment and to adapt therapy promptly. The aim of this study was to document engraftment and to detect as soon as possible relapse in patients with acute myeloid leukaemia who underwent stem cell transplantation. Real-time quantitative polymerase chain reaction is a highly sensitive and reproducible technology. It is useful in some disease to target selected sub-populations in order to have an earlier detection of relapse on cell fractions. In the acute myeloid leukaemia (n=65), analysis of the chimerism on whole peripheral blood cells and bone marrow cells, CD3+ cells, specific myeloid CD33+ cells (from blood) and CD34+ cells (from bone marrow) is of importance. After transplant, 25 patients relapsed (38%), three massively, with chimerism detection in whole blood and bone marrow and 22 insidiously following two different schemes (GRI and GRII). In GRI, (n=13): chimerism of CD33+ and CD34+ cellular fractions allowed an early detection of relapse in 100% of cases undetected in whole cells whereas in GRII (n=9): myeloid cells could identified relapse in 89% of cases when whole blood cells and CD3+ cells expressed a mixed chimerism. This study highlighted the importance of sub-cellular population chimerism documentation enable to ascertain a stable engraftment and to detect early relapse. The selection of sub-cellular population studied with high sensitive technology allows a rapid and efficient intervention before the onset of clinical signs in patient with acute myeloid leukaemia and could improve the patient's follow-up.

Multiplexed immunoassay for the rapid detection of anti-tumor-associated antigens antibodies.

TAAs (tumor-associated antigens) microarrays were designed to detect auto-antibodies directly in patient sera. Twelve different probes were chosen according to their described occurrence in cancer pathologies (Cyclin B1, Cyclin D1, Complement factor H, c-myc, IMP1, p53, p62, survivin, Her2/neu, Koc, NY-ESO-1 and PSA). Microarrays of these 12 proteins were immobilized within the nitrocellulose/cellulose acetate membrane of a 96-well filtering microtiter plate bottom. The captured auto-antibodies were detected using a staining approach based on alkaline phosphatase labeling. Thus, the presence of specific auto-antibodies in samples was visualized through the positive staining of the corresponding TAA spots. The TAA HiFi microarrays were shown to be able to capture specific purified anti-TAA antibodies. In real samples, 9 proteins from the 12 TAAs panel were shown to generate specific signal and 5 antigens (p53, NY-ESO-1, IMP1, cyclin B1 and c-myc) were shown to have interaction with more than 10% of the positive sera from cancer patients. This protein subpanel was proven to be able to detect 72.2% of the cancer patients tested (within a 34 panel of 18 patients and 16 healthy donors).

[Autoimmune haemolytic anemia: diagnosis strategy and new treatments]. / Anémies hémolytiques auto-immunes: diagnostic biologique et nouvelles approches thérapeutiques.

The pattern of autoimmune hemolytic anemia has changed significantly these last 15 years. With regard to the diagnosis strategy, the use of gel filtration technique to perform the direct antiglobulin test (DAT) has decreased the number of autoimmune haemolytic anemias with negative tests results. In recent years, autoimmune haemolytic anemia increased in patients receiving purine nucleoside analogues, blood transfusions, solid organ transplantation or hematopoietic stem cells transplantation. These difficult autoimmune haemolytic anemia cases need to use new kinds of treatments. With regard to the treatment, very little progress was made this latter 50 years. The discovery of the efficacy of anti-CD20 antibody in this disease represents a breakthrough. Nowdays, the second-line treatment includes rituximab or splenectomy. Sometimes, the anti-CD20 treatment could be proposed in first-line but some clinical trials are needed.

The first results demonstrating efficiency and safety of a double-column whole blood method of LDL-apheresis.

LDL-apheresis is a treatment for familial hypercholesterolemia in addition to diet and drug therapy. In the past, LDL-apheresis techniques consisted in separating plasma from blood and adsorbing plasma LDL-C whereas recent methods remove LDL-C directly from whole blood. The whole blood system developed by Kaneka consists of a single-column (Liposorber DL-75) treatment (SCWB) but a double-column whole blood (DCWB) method has recently been developed (Liposorber DL-50 x 2). When 1.6 blood volumes (plus 1l) were processed, acute reductions of total cholesterol and LDL-C were 67.9+/-6% and 80.2+/-4.5%, respectively. The performances of the DCWB method were compared to other LDL-apheresis methods. Assessed in 10 patients, the DCWB method is more efficient than the SCWB method with higher reduction rates of LDL-C (79.7+/-4.9 vs. 68.2+/-5.0% p<0.0001) and apolipoprotein-B (79.5+/-5.4 vs. 67.4+/-5.4% p<0.0001). In a sub group of five patients having the highest LDL-C baseline levels, the LDL-C reduction rates obtained by the DCWB method are equivalent to those obtained by the conventional LDL-apheresis method consisting of preliminary plasma separation followed by plasma LDL-C adsorption and used as first line apheresis therapy (80.5+/-4.5 vs. 79.0+/-5.9%). The safety of DCWB was demonstrated in 12 patients with only a low frequency of mild and transient adverse effects (4%). In conclusion, the DCWB LDL-apheresis method provides efficient removal of LDL-C, a low level of adverse effects, and a shortened duration of the procedure.

[Towards an industrial control of the cloning of lymphocytes B human for the manufacturing of monoclonal antibodies stemming from the human repertoire]. / Vers une maîtrise industrielle du clonage des lymphocytes B humains pour la fabrication des anticorps monoclonaux issus du répertoire humain.

Monoclonal antibodies (mAbs) are efficient drugs for treating infectious, inflammatory and cancer diseases. Antibodies secreted by human lymphocytes that have been isolated from either peripheral blood or tissues present the definite interest of being part of the physiological or disease-related response to antigens present in the human body. However, attempts to generate hybridomas with human B cells have been largely unsuccessful, and cloning of human B cells has been achieved only via their inefficient immortalization with Epstein Barr Virus (EBV). However, recent progress in our understanding of the molecular mechanisms of polyclonal B cell activation has dramatically increased the capacity to clone human B cells. In particular, activation of human naïve and memory B cells through CD40 or memory B cells only through TLR9 was shown to greatly facilitate their immortalization by EBV. Industrial development based on these observations will soon provide large collections of high affinity human mAbs of every isotype directly selected by the human immune system directed to recognize epitopes relevant for individual patients. Moreover, after CD40 activation, these mAbs will cover the full human repertoire, including the natural auto-immune repertoire. Full characterization of the biological activity of these mAbs will in turn bring useful information for selecting vaccine epitopes. This breakthrough in human B cell cloning opens the way into new areas for therapeutic use of mAbs.

Severe HPA-15b related neonatal alloimmune thrombocytopenia.

UNLABELLED: The HPA-15 platelet (PLT) group was recently described. Severe neonatal thrombocytopenia due to alloimmunization by HPA-15b has very rarely been observed. A 22-year-old mother, gravida 1/para 1, gave birth to a male infant who presented with a severe thrombocytopenia, the PLT count recorded to be 3 x10(9)/L. A few hours after birth, he developed purpura with extensive haematomas but without visceral or intracranial haemorrhage (ICH). Two PLT transfusions were given including one using maternal PLTs. The infant's PLT count was 267 x 10(9)/L on day 6. The maternal platelet group was HPA-15a/a and her infant was HPA-15a/b. Anti-HPA-15b antibodies was found in maternal serum. CONCLUSION: HPA-15b maternal alloimmunization may induce severe neonatal thrombocytopenia. In order to establish the frequency of neonatal alloimmune thrombocytopenia (NAIT) due to anti-HPA-15b antibodies, an improved detection method is necessary.

Adaptability of protein A-immunoadsorption allows temporary reduction of anti-VIII antibodies and realisation of high-risk haemorrhagic surgery.

We report the successful treatment by protein A-immunoadsorption (IA) of an hemophilic man with anti-F VIII antibodies (Abs) who needed high-risk bleeding surgery. This patient had developed high levels of anti-F VIII Abs preventing substitution by clotting factor and preventing high-risk bleeding surgery. Because of rebound in Abs levels or complications, IA procedures were modified several times leading to appropriate decrease of anti-F VIII inhibitor Abs allowing bilateral knees surgery. IA procedure is enough adaptable to be modified to prevent complications. Collaboration between clinical, biological, apheresis and surgical teams implied has permitted surgery and prevented life-threatening bleeding complications.

Alloreaction increases or restores CD40, CD54, and/or HLA molecule expression in acute myelogenous leukemia blasts, through secretion of inflammatory cytokines: Dominant role for TNFbeta, in concert with IFNgamma.

We have previously reported that alloreaction can lead to activation of dendritic cells through secretion of inflammatory cytokines. Here, we addressed whether alloreaction-derived cytokines may also lead to acute myelogenous leukemia (AML) blast differentiation. With this aim, supernatant (sn) harvested from major or minor histocompatibility antigen-mismatched mixed lymphocyte reaction (MLR) were used to culture French American Bristish (FAB) type M4 or M5 AML blasts. Our results showed that the secreted factors induced upregulation of CD40, CD54, and/or HLA molecules in AML blasts. Protein fractionation, blockade experiments and exogenous cytokine reconstitution demonstrated the involvement of TNF in the upregulation of CD54, CD40 and HLA-class II molecules, and of IFNgamma in the increase of HLA-class I and class II molecule expression. But, in line of its much higher levels of secretion, TNFbeta, rather than TNFalpha, was likely to play a preponderant role in AML blast differentiation. Moreover TNFbeta and IFNgamma were also likely to be involved in the AML blast differentiation-mediated by HLA-identical donor T-cell alloresponse against recipient AML blasts. In conclusion, we show herein that upon allogeneic reaction, TNFbeta secretion contributes, in concert with IFNgamma, to increase or restore surface molecules involved in AML blast interaction with T cells.

With a little help from a friend: increasing HIV transduction of monocyte-derived dendritic cells with virion-like particles of SIV(MAC).

Modification of dendritic cells (DCs) is a promising avenue for gene therapy purposes, given the versatility and the multiplicity of functions of these cells. In this study, we show that preincubation of monocyte-derived DCs with low amounts of non-infectious virion-like particles derived from the simian immunodeficiency virus (SIV(MAC) VLPs) increases up to 10-fold the efficiency of transduction by HIV-1 lentiviral vectors at low multiplicity of infections yielding up to 90% of transduced cells, in the absence of alterations of DCs behavior. This effect is restricted to DCs and specified by the viral accessory protein Vpx. Thus, preincubation with empty VLPs of SIV(MAC) can be used in transduction protocols to increase the efficacy of HIV-1-mediated modification of DCs.

[Solitary fibrous tumor of the orbit: an unusual cause of unilateral proptosis. Case report with a review of the literature]. / Tumeur fibreuse solitaire de l'orbite: une cause inhabituelle d'exophtalmie unilatérale. A propos d'un cas avec revue de la littérature.

BACKGROUND: The solitary fibrous tumor (SFT) is a spindle-cell tumor that very rarely involves the orbit. We report a new case that we compare to reports in the literature. CASE: A 72-year-old woman presented a conjunctival inflammation of the right eye developing over 5 months with progressive proptosis. Magnetic resonance imaging revealed an extraconal and homogeneous mass, which showed hypointensity on T1-weighted images and hyperintensity on T2-weighted images, without specificity. Histological examination of the lesion removed by anterior orbitotomy confirmed the diagnosis of the SFT of the orbit. The patient was doing well without recurrence after 9 months. DISCUSSION: The diagnosis of SFT is histological. It is a mesenchymal tumor. Immunohistochemically, the tumor cells are strongly positive for CD34 and vimentin. CONCLUSION: The SFT of the orbit is a very rare and generally benign tumor. It must be immunohistochemically differentiated from other spindle-cell tumors of the orbit. The treatment is a complete surgical excision, and long-term follow-up is necessary because recurrence may appear long after treatment.

[Implantation of preserved human amniotic membrane for the treatment of shield ulcers and persistent corneal epithelial defects in chronic allergic keratoconjunctivitis]. / Greffe de membrane amniotique et traitement des ulcères de cornée lors des kératoconjonctivites chroniques allergiques.

PURPOSE: To determine whether amniotic membrane implantation is a safe and effective alternative treatment for shield ulcers and persistent corneal epithelial defects associated with ulcers in chronic allergic keratoconjunctivitis (vernal or atopic keratoconjunctivitis). METHODS: Amniotic membrane implantation was performed in four consecutive patients with persistent corneal epithelial defects or vernal plaques unresponsive to conventional medical treatment lasting an average of 18 weeks. Surgery was done under general anesthesia using amniotic membrane as a therapeutic contact lens. RESULTS: A significant decrease in symptoms and complete reepithelialization of the corneal ulcers were observed in all cases within the first 7 days. These remained stable during a mean follow-up of 12 weeks, with no intraoperative or postoperative complications. Early detachment occurred in all cases with no negative consequences on ulcer healing. CONCLUSION: Patients with severe chronic allergic keratoconjunctivitis derive benefits from amniotic membrane implantation used as a therapeutic contact lens in the treatment of persistent corneal epithelial defects and vernal plaques unresponsive to conventional medical treatment.

[Treatment of conjunctival epithelial tumors: brachytherapy with ruthenium-106]. / Traitement des tumeurs épithéliales de la conjonctive: intérêt de la curiethérapie au ruthénium-106.

INTRODUCTION: Treatment of conjunctival epithelial tumors is not standardized because it is difficult to compare large series in this rare disease. Surgical excision is usual, but the recurrence rate has led several authors to propose alternative therapies. PATIENTS AND METHODS: During the past 20 years, brachytherapy using ophthalmic applicators has been developed and the results of different studies have confirmed the usefulness of this therapy. We report a retrospective study of 13 patients presenting with a conjunctival epithelial tumor treated with ruthenium106 applicators and followed up in our department since 1987. RESULTS AND CONCLUSION: There was no recurrence during a mean follow-up of 48 months. Complications depended on the size of the area treated and the dose of radiation.

Cyclosporin A inhibits dendritic cell maturation promoted by TNF-alpha or LPS but not by double-stranded RNA or CD40L.

Here, we investigated the influence of cyclosporin A (CsA) on dendritic cell (DC) generation. With this aim, human DC were propagated from monocytes in serum-free medium with granulocyte macrophage-colony stimulating factor and interleukin-4. DC were then exposed to tumor necrosis factor alpha (TNF-alpha) for maturation. Our results show that CsA does not impair commitment of monocytes into DC, as assessed by loss of CD14 and increase of CD40 and CD1a. However, TNF-alpha-induced DC maturation was affected, as CsA-treated DC expressed lower levels of human leukocyte antigen and costimulatory molecules but sustained levels of CD1a, and less DC expressed DC-lysosomal-associated-membrane-protein (LAMP) and CD83. Accordingly, CsA inhibited the allostimulatory and accessory cell functions of DC. Surprisingly, when other maturation stimuli were used, we observed that CsA significantly inhibited maturation induced by lipopolysaccharides but not by polyribocytidylic acid or CD40 ligand, as assessed by DC phenotype and functions. Therefore, our results indicate that CsA may differentially affect DC maturation.

Genomic organization and restricted expression of the human Mona/Gads gene suggests regulation by two specific promoters.

Monocytic adaptor (Mona) also known as Gads is a Grb2-related adaptor whose expression is restricted to hematopoietic cells. It plays an important role in intracellular signaling in T cells, monocytic cells, and platelets. Here we investigated the regulatory aspects of Mona expression in human hematopoietic cells. This was carried out by combining nucleotide sequence analyzes and experimental approaches. We confirmed that Mona expression is restricted to T-cell, myeloid and platelet lineages. In the various cells examined, we detected two major Mona transcripts (1.9 and 4 kb), likely resulting from the alternative use of two polyadenylation sites. Consequently, Mona transcripts of the same size have identical 3' untranslated region (UTR), irrespective of the cell type. In contrast, Mona transcripts contain either 5' UTR-1A or -1B exons, that were detected in a cell-lineage specific manner. Thus, T cells and several myeloid cell lines express 5' UTR-1A-containing transcripts, whereas platelets and cell lines exhibiting megakaryocytic potential express 5' UTR-1B-containing transcripts. Interestingly, 5' UTR-1A is generated from an exon located approximately 45 kb upstream of exon 1B. This suggested that lineage-restricted transcription of the Mona gene is controlled by specific promoters. Indeed, 2-kb genomic fragments upstream of each 5'-UTR showed lineage-restricted ability to drive expression of luc reporter gene.

Nrh, a human homologue of Nr-13 associates with Bcl-Xs and is an inhibitor of apoptosis.

In search of human homologues of the anti-apoptotic protein Nr-13, we have characterized a human EST clone that potentially encodes a protein, which is the closest homologue of Nr-13 among the Bcl-2 family members, to date known, in humans. Phylogenetic analyses suggest Human nrh, Mouse diva/boo and Quail nr-13 to be orthologous genes. The nrh gene has the same overall organization as nr-13 and diva/boo with one single intron interrupting the ORF at the level of the Bcl-2-homology domain BH2. RT-PCR-based analysis of nrh expression indicated that this gene is preferentially expressed in the lungs, the liver and the kidneys. Interestingly, two in frame ATG codons can lead potentially to the synthesis of two products, one of them lacking 10 aminoacids at the N-terminal end. Sequence alignment with Nr-13 and Diva/Boo in addition to secondary structure prediction of the nrh transcript suggested that the shortest protein will be preferentially synthetized. Immunohistochemical analyses have revealed that Nrh is associated with mitochondria and the nuclear envelope. Moreover, Nrh preferentially associates with the apoptosis accelerator Bcl-Xs and behaves as an inhibitor of apoptosis both in yeast and vertebrate cells.

Generation of helper and cytotoxic CD4+T cell clones specific for the minor histocompatibility antigen H-Y, after in vitro priming of human T cells by HLA-identical monocyte-derived dendritic cells.

BACKGROUND: There is now convincing evidence that minor histocompatibility antigens (mHag) may play a significant role in the pathogenesis of graft-versus-host disease after HLA-identical bone marrow transplantation. Indeed, in this clinical situation, T cells specific for mHag have been isolated. Here, we addressed whether one can generate mHag-specific T cells in vitro, without any in vivo immunization, among healthy blood donors. METHODS: We used monocyte-derived dendritic cells (Mo-DCs) as antigen presenting cells to induce primary responses between healthy HLA-identical siblings, in mixed lymphocyte dendritic cell reactions (MLDCRs). RESULTS: We show that CD4+ T-cell clones, specific for the mHag H-Y, can be generated in vitro. These clones were derived from a gender-mismatched positive MLDCR pair of HLA-identical siblings and were restricted by the HLA DQB1*0502 molecule. In addition, these CD4+ T clones were also able to lyse allogeneic targets with the same pattern of restriction and specificity than helper function. Finally, acute myeloid leukemia (AML) blast cells were susceptible to lysis by these clones. CONCLUSIONS: Altogether, these results predict that Mo-DCs could help to generate class II-associated, mHag-specific, T-cell lines or clones in vitro, between healthy blood donors, without any need of transplantation-mediated immunization.