Identification of a novel HLA allele, HLA-B*53:39, in a Guadaloupean individual.

The novel HLA-B*53:39 allele differs from HLA-B*53:01 by a single nucleotide substitution at codon 45 (ACG>AAG).

Differentiation of human dendritic cell subsets for immune tolerance induction.

OBJECTIVES: Since no further progress was achieved, in order to improve the long-term organ transplantation outcome, the immune tolerance appears as an interesting therapeutic goal. Dendritic cells (DCs) are specialized cells participating in the homeostasis of the immune response. Moreover, subsets of DCs, identified in humans, appear to have their respective competences in immune response modulation. Our objective is to purify from PBMC or to differentiate DC subsets from monocytes using several strategies and evaluate their IL10 secretion. METHODS: CD14+ cells were purified from peripheral blood mononuclear cell (PBMC) by affinity beads and cultured with cytokines up to 7 days. The pDCs were purified with anti-BDCA-2 beads from PBMC fraction enriched by Percoll® gradient. The moDCs, pDCs and moLCs subsets were analyzed by phenotype labelling and FACS analyses and IL10 secretion measured by ELISA. RESULTS: The moDCs were characterized by the CD209 expression and a lower expression of CD1a markers. Expression of CD207 and CD1a markers characterized moLCs and CD123+/BDCA-2+ pDCs. Variable IL-10 secretions were shown between the three DC subsets, both at basal and activated levels. CONCLUSIONS: As the several DC populations studied have different capacities of IL-10 synthesis, they might play, among others, distinct roles in the induction of immune tolerance.

Identification of a novel HLA allele, HLA-B*41:50, in a French individual.

The novel HLA-B*41:50 allele differs from HLA-B*41:01:01 by a single nucleotide substitution at codon 116.

Identification of a novel HLA allele, HLA-B*15:416, in a bone marrow hematopoietic stem cell donor.

The novel HLA-B*15:416 allele differs from HLA-B*15:01:01:01 by a single nucleotide substitution at codon 114.

Identification of a novel HLA allele, HLA-B*08:163, in a platelet donor.

The novel HLA-B*08:163 allele differs from HLA-B*08:01:01:01 by a single nucleotide substitution at codon 105.

EFS Centre-Atlantique donor's biobank: Ten years of samples usage.

INTRODUCTION: The French Establishment of Blood Centre Atlantique (EFSCA) is one of the French regional blood transfusion centers. Donor's biobank is a mandatory activity leading to the storage of biological samples taken from the blood donor. Samples of each blood donation are preserved for a 5-year period at Châteauroux in the form of two straws of 500microliters stored in liquid nitrogen. The aim of this study was to analyze the samples usage by studying quantitative, qualitative and economic criteria. MATERIAL AND METHOD: We analyzed all the requests of stored blood samples from 2005 to 2014. They were coming either from the blood donor qualification laboratory (BDQL), in order to perform complementary tests, or from hemovigilance inquiry. RESULTS: Among the blood donation samples, 2,144,636 (whole blood, plasma or platelets apheresis) were preserved during these ten years. During this period, 548 (0.025%) requests for samples were received; 78% were in relation with a request of the BDQL and 22% in relation with a request of hemovigilance. For the straws, the mean exit delay with regard to the blood donation date was 11.5 months (2-55). The cost of samples exit includes only working hours of a laboratory technician. On average, the annual working time dedicated to this activity was 23h. Also, the average price for one-year issuing activity was 620.31 euros. CONCLUSION: In our study, the donor's biobank was little used. The part of hemovigilance was weak but essential for the blood safety.

Patient labels as a quality indicator of identitovigilance in French hospitals.

INTRODUCTION: Identity risk is frequent and serious. Between 2007 and 2010, 25.6% of 1572 serious adverse events declared in France are related to identitovigilance. No regulation clearly defines an ideal patient label even when a delivery refusal is applied in case of absent or incomplete identity (absence of surname and/or first name and/or birth date). The aim of the study was to draw up the current situation of patient labels in hospitals connected with our blood transfusion center and being used for blood products delivery and immuno-hematology analyses. MATERIALS AND METHODS: We defined an ideal label with 5 items which must be present and clearly identified: surname, usual or marital name, first name, birth date and sex. It contains also an identifier, if possible with a bar code. We compare it with labels used in our hospitals. RESULTS: Only 22% (17/76) had a patient label in compliance with our ideal label. Most of the items, even if they were not clearly identified on the label, were present. The surname was present and clearly indicated in 75% of cases (57/76). In approximately 50% of cases, there was a barcoded permanent and/or stay identifier. CONCLUSION: Our results, with only 22% of labels considered as 'ideal', show all the work which remains to be done. A temporary solution can be the elaboration by hospitals of an identification guide of their present labels.

Blood donors--Serious adverse reactions (SAR) 2010-2014 EFS Châteauroux, France.

BACKGROUND: In 2013, the national French incidence of serious adverse reactions (SAR) was 155.7 per 100,000 donations and 82% of SAR were grade 2 (French classification of SAR related to blood donors) AIMS: The purpose of our study was to describe the profile of blood donator candidate which had a SAR in our center. METHODS: The study contains all the SAR superior to grade 1 occurred on the site EFS Châteauroux (site and mobile blood collection) from January 2010 to October 31, 2014. We analyzed 37 parameters from the e-fit files (e-site French blood vigilance) and In-log software. RESULTS: We identified 82 SAR for 72,553 blood donations (incidence: 113.02 SAR per 100,000 donations). Forty-one men and 41 women, middle age 39 years (18-66). Average height: 1.68 m (1.49-1.85); average weight: 68 kg (50-98); body mass index (kg/m(2)): 24,13(18.6-31.9). All donors were Caucasian and 30% unemployed. We found 74 vasovagal syncope (VVS), 5 hematomas, 2 arterial injuries and an adverse reaction to citrate. In 90%, the SAR was immediate and of grade 2 in 85% of cases. Thirty-seven percent of SAR were first donation in connection with whole blood in 87% of cases. Regarding the seniority of donors, the number of average donations (whole blood, plasma, platelets) was 16.5. An SAR determined the stop of blood donation in 65% of cases with nearly 80% stoppage if it was a first donation. Seventy-three percent of SAR as a VVS took place during blood collection or within 5 minutes following the end of the donation. Sixty-one percent were men. Forty-four percent of cases were a first donation and 83% occurred in mobile blood collection. Average age was 36 years. The result was a permanent stop of all type of donations in 76% of cases. Twenty-seven percent of SAR as a VVS took place beyond 5 minutes after the end of the donation. Seventy-five percent were women. Thirty percent of cases were a first donation and 95% of SAR occurred in mobile blood collection. Average age was 42 years. The result was a permanent stop of all type of donations in 40% of cases. CONCLUSIONS: When the SAR as a VVS occurs during or within 5 minutes following the end of the donation, it leads to a permanent stop of any type of donation in 76% of cases.

Illiteracy and brain damage--1. Aphasia testing in culturally contrasted populations (control subjects).

One hundred neurologically healthy adults were tested for their pointing (choosing one of four or six line drawings as the match to an auditorily presented linguistic stimulus), naming (from line drawings), and repetition abilities. All subjects were unilingual adult right-handers. Fifty-seven subjects were totally unschooled illiterates and 43 were fluent readers. Statistically significant differences were found to exist between the scores of the illiterate and literate subpopulations across all tasks. With the focus being placed on these cultural differences, the discussion bears on: (a) the interaction between linguistic and iconographic factors in certain types of naming and pointing tasks currently used in clinical and research aphasiology, (b) some of the linguistic parameters which are apparently at stake in repetition behavior, and (c) the circumstances in which aphasiological research dealing with groups of patients cannot yield reliable data without reference to neurologically healthy controls. It is argued that, when testing brain-damaged patients of different cultural backgrounds, one runs the risk of over- or underestimating the frequency of aphasia if one does not refer to norms which explicitly take educational level into account.

Illiteracy and brain damage. 3: A contribution to the study of speech and language disorders in illiterates with unilateral brain damage (initial testing).

This report bears on the behavior of 188 unilateral stroke subjects when administered an aphasia screening test comprising a short interview as well as naming, repetition, word-picture matching and sentence-picture matching tasks. All subjects were unilingual lusophone adult (40 yr of age or older) right-handers. Furthermore, they were either totally unschooled illiterates or they had received school education and thereafter retained writing skills and reading habits. Subjects were tested less than 2 months after a first unilateral stroke. In all tasks, global error scores were greater among left and right brain-damaged illiterate and literate subjects than among their controls. In repetition and matching, these differences were statistically significant for the left but not for the right-stroke groups, irrespective of the literacy factor. In naming, on the other hand, significant differences were found not only for the two left-stroke groups but also for the right-stroke illiterate group although not for the right-stroke literate one. Likewise, some degree of word-finding difficulty and of reduction in speech output as well as sizeable production of phonemic paraphasias were observed in the interviews of several right-stroke illiterates, clearly less in those of right-stroke literates. These findings lead us to suggest that cerebral representation of language is more ambilateral in illiterates than it is in school educated subjects although left cerebral "dominance" remains the rule in both.

Polyreactive autoimmune response induced by PC 12 cell grafts into rat striatum.

In order to better characterize the autoantibodies induced by PC12 cells grafted into rat brain, we have tested sera from these animals by immunoblotting with several preparations, including phosphorylated and dephosphorylated neurofilaments, keratins, PC12 cells and proteins from various rat tissues, and by immunofluorescence of rat spinal cord neurons in culture. Sera from grafted rats reacted with several antigens present in all tissues tested and stained in cultured neurons not only NF but also cell bodies and membranous granular structures. These observations suggest either the polyreactivity of autoantibodies or the induction of a polyclonal B cell activation consecutive to the release of central nervous system antigens into the blood stream. These results are discussed with regard to the role of NF autoantibodies in neurodegenerative diseases.

Differential behaviour of PC12 cells grafted into rat hippocampus and striatum.

To investigate the mechanisms involved in graft survival, a rat cell line (PC12) that differentiates into sympathetic-like neurons by exposure to trophic factors has been grafted into rat striatum and hippocampus, two structures which differ in their amounts of trophic factors. Our results show that grafted PC12 cells behave differently depending on the area of implantation; they display a differentiated morphology in the hippocampus and proliferate as a tumor in the striatum. A qualitatively similar immunological reaction occurs in both structures, characterized by the invasion of T and B lymphocytes, macrophage-like cells and by the expression of major histocompatibility complex class I and II antigens around the graft.

n-6 polyunsaturated fatty acids increase the neurite length of PC12 cells and embryonic chick motoneurons.

We have tested the action of three n-6 polyunsaturated fatty acids, either free or in the form of ethyl esters, on the neurite outgrowth in two neuronal models: a rat pheochromocytoma cell line (PC12) and embryonic chick motoneurons, after 7 days in culture. An inverted microscope coupled with the 'VIDS 4' software was used for measuring the neurite length. Free fatty acids were found to be cytotoxic at 10(-3) M and the maximal increase of the neurite length was obtained at 10(-5) M. In contrast, fatty acids in the form of ethylesters were not cytotoxic and at 10(-3) M induced the maximal increase in the neurite length. This increase (1.2 to 2 fold) significantly differed from the control and was dose-dependent. These results were discussed in relation to the action of fatty acids on enzyme activation and membrane fluidity.

Autoantibodies against H- and M-subunits of neurofilaments are induced by PC12 cell grafts or lesions into different sites of rat brain.

Since autoantibodies against neurofilaments (NF) were frequently found in neurodegenerative disorders, this work is an attempt to investigate whether the same phenomenon occurs after intracerebral grafting or lesioning. We have thus either grafted PC12 cells or injected culture medium alone into three sites of rat central nervous system (CNS): olfactory bulb (OB), olfactory anterior nucleus (OAN) and hippocampus (HC), all three sites being impaired in Alzheimer's disease. At day 15, rat sera were collected and tested against NF by western blotting. Sera from grafted rats recognized the H- and M-subunits of NF; we have then quantified the autoantibody response by using an ELISA technique. We show that, in all cases of grafts, the autoantibody response against NF significantly increased when compared to controls (normal rats without grafts or lesions) for total immunoglobulin (Ig) amount. In contrast, concerning the Ig isotypes, some differences appeared depending on the implantation site: for grafts into OB, the immune response was of both the IgG and IgM isotypes, into OAN it was mainly of the IgM isotype and into HC, the isotype of antibodies against NF was mostly IgG. In the case of lesions alone into OAN and HC, no significant enhancement of autoantibody response was observed; in contrast, lesions into OB induced an increase in autoantibody response against NF which significantly differed from controls for all Ig isotypes tested. These data point out the diversity of the autoantibody responses following lesions or grafts according to the rat brain areas.

[Optimization of a haematopoietic stem cell freezing process using a qualification protocol applicable to a programmable freezer]. / Optimisation d'un processus de congélation des cellules souches hématopoïétique par l'application d'un protocole de qualification d'un congélateur programmable.

BACKGROUND: The freezing phase is a critical step of the freezing process of the hematopoietic stem cells. To standardize the decrease of the temperature, the use of a programmable freezer is recommended. There is no available protocol, neither to describe exactly the validation of a programmable freezer, nor to prove the performance of the freezing/thawing step of the grafts. METHOD: We describe a validation protocol with three phases: first a qualification of installation, then an operational qualification and finally, a qualification of performance. The validation is performed in tandem between the freezer which is routinely used (Nicool Plus) and a new one (Freezal). RESULTS: With this protocol, we demonstrate the efficacy of the freezing program and its ability to assure the quality of the grafts reinjected to the patients, particularly in terms of cellular efficiency on CD34+ hematopoietic stem cells. On these cells, we measured a significant increase of cellular efficiency (+10%) after freezing with the Freezal. CONCLUSION: Here, we propose a validation protocol which is able to qualify a programmable freezer. This protocol can optimize the capability of the freezer and is able to prove its performance.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

Heterogeneity in the cognitive profile of normal elderly.

An increasing number of studies point to the fact that distinct cognitive subgroups may be identified among subjects with dementia of the Alzheimer type (DAT). Although such heterogeneity could be the expression of neuropsychological, genetic, or epidemiological factors, the identification of neuropsychological subtypes in DAT could also reflect the existence of cognitive subgroups in a normal aged population. In the present study, the existence of neuropsychological subgroups was sought from among 81 volunteers aged between 55 and 84 years. Subjects were given a neuropsychological battery addressing various aspects of cognitive functioning. Results show that six subgroups could be identified among this population. Subgroups differ primarily in their overall degree of performance. Qualitative differences in cognitive performance are also present, mostly when subgroups which exhibit poor overall performance are considered. Consequently, the presence of such heterogeneity in normal elderly should be taken into account in any attempt to identify neuropsychologically based subgroups in early dementia of the Alzheimer type.

Conception and organisation of a cell therapy unit of Nantes, France.

Manufacturing of cell therapy products has to follow several requirements to obtain sanitary security and quality of the product. Thus, at its conception, the cell therapy unit (CTU) of Nantes choose to integrate a quality assurance system: - The good manufacturing practices (GMP's) are a technical reference for the Unit. They are a quality criteria necessary to guarantee the security of products in term of staff, premises, material, matter and method; - The ISO 9001 standards are a model for quality assurance in design, development, production, installation and servicing. They established a quality system; - The creation, the running and the maintenance of premises are an essential aspect of the quality system and they are described in this paper. Thus, from October 1994 to June 1998, 450 cell processing (with or without cryopreservation and storage of cells) have been realised at the CTU of Nantes, leading to 160 injections without major undesirable effect and without microbiological contamination.