[How do I assess requirement of a blood bank and its kind for a healthcare establishment?]. / Comment j'évalue la nécessité d'un dépôt de sang et son type pour un établissement de santé ?

Access to blood components is required for healthcare establishments, particularly for emergency situation and hospital blood bank was often a response to this requirement. However, the complexity of regulation and economic pressures lead healthcare establishment to review regularly their need for a blood bank. This assessment requires analysis of need for transfusions in terms of delay, quantity and clinical situations to which they must respond. When a blood bank is required, three kinds could be under consideration: emergency blood bank, intermediate blood bank and issuance blood bank. According to requirements, advantages and disadvantages of each kind, healthcare establishments would select the most suitable one.

[Transfusion transmitted bacterial infectious (TTBI) with blood component accountability score 2: Retrospective analysis of the French e-fit database 2000-2007. Groupe de travail­validation des IBTT (Working group for validation of transfusion-transmitted bacterial infections)]. / Les infections bactériennes transmises par transfusion avec imputabilité 2 du PSL : analyse rétrospective de la base e-FIT de 2000 à 2007.

PURPOSE OF THE STUDY: Transfusion transmitted bacterial infection is an adverse reaction occurring in a patient during blood transfusion and due to the presence of bacteria in the blood component. For each transfusion transmitted bacterial infection suspicion, clinical and biological investigations should allow to either affirm the accountability of the transfused product in the occurrence of the infection (accountability score 4) or exclude it (accountability score 0). However, among 60,175 adverse reaction sheets extracted from the French e-FIT database (AFSSAPS), 143 are classified as transfusion transmitted bacterial infection diagnosis and 97 of them show a score of accountability 2 (possible). This study aims to analyze these 97 adverse reaction sheets and search for the reasons that led the haemovigilance network actors not to refine the degree of accountability in line with an exclusion or a confirmation of transfusion origin. METHOD: During collective reading sessions, each adverse reaction sheet among the 97 extracted was re-analyzed with an accountability criteria grid, built beforehand, and proposed in the technical guide sheet for transfusion transmitted bacterial infection (e-Fit AFSSAPS). RESULTS: Among the 97 analyzed adverse reaction sheets with a score accountability of 2: 12.4 % were considered as "non-analysable"; 54% were reclassified in another diagnosis category: non haemolytic febrile reaction (n=12), unknown diagnosis (n=17); patient infection before transfusion (n=23); blood component's "smear" (n=9); retrograde contamination of blood component (n=5). Finally, only 18.5% adverse reaction sheets (n=18) were maintained with a true diagnosis of transfusion transmitted bacterial infection an accountability score of 2. These cases were in agreement with those described in number 2, 3 or 4 in the annex sheet "Fiche Technique TTBI". 70% of adverse reaction sheets reclassified under another diagnosis as transfusion transmitted bacterial infection had been declared between 2000 and 2004. In order to improve transfusion transmitted bacterial infection suspicions diagnosis approach and to guide the French haemovigilance network in the investigations following a transfusion transmitted bacterial infection suspicion, the group propose recommendations after each adverse reaction sheets category analysis. CONCLUSION: The improvement measures taken as part of the French haemovigilance declaration framework allowed to perfect the data quality of transfusion transmitted bacterial infection. Progresses are still to be made to improve clinical and biological declaration, in order to precise the accountability of a blood component in the occurrence of an adverse transfusion transmitted bacterial infection effect. Tracking transfusion transmitted bacterial infection notifications by a group of experts at the national level is still recommended.

Influence of blood prestorage conditions and white blood cell filtration on the bacterial load of blood deliberately inoculated with Gram-positive and Gram-negative pathogens.

BACKGROUND AND OBJECTIVES: Currently, the bacterial contamination of blood constitutes one of the major infectious risks of transfusion. The aim of this study was to evaluate the bactericidal effect of blood on various bacterial species and to determine the influence of prestorage conditions and white blood cell (WBC) filtration on the reduction of the bacterial load in isolated red blood cells (RBCs). MATERIALS AND METHODS: The growth kinetics of eight different species of bacteria were studied at 20 degrees C in deliberately contaminated RBC units. Further experiments evaluated the effect of prestorage conditions and WBC filtration on the viability of two model bacteria (Klebsiella oxytoca and Staphylococcus epidermidis) in comparison to previous results obtained with Yersinia enterocolitica. RESULTS: For bacteria susceptible to the bactericidal effect of blood (mainly Gram-negative rods), a reduction of the bacterial load was obtained within 2 h of prestorage at 20 degrees C. When the prestorage period was prolonged beyond 3 h at 20 degrees C, rapid growth was observed with some Enterobacteriaceae. Whereas WBC filtration reduced dramatically the viability of Y. enterocolitica, it had only a minimal effect on the viability of S. epidermidis and K. oxytoca. However, the two latter species of bacteria did not survive prolonged storage at 4 degrees C. CONCLUSIONS: Experiments conducted under realistic conditions are needed to determine whether it would be worthwhile recommending the rapid storage of RBCs at 4 degrees C after WBC reduction of the blood product.

Bovine thrombospondin-2: complete complementary deoxyribonucleic acid sequence and immunolocalization in the external zones of the adrenal cortex.

Given the variety of biological functions in the adrenal cortex that are controlled by ACTH, we hypothesized that some extracellular proteins act as biological relays for this systemic hormone. One candidate protein [corticotropin-induced secreted protein (CISP)] was purified from the conditioned medium of bovine adrenocortical cells on the basis of a 5- to 14-fold increase in its synthesis after the addition of ACTH. We report here the cloning of overlapping complementary DNAs that span the sequence encoding the full-length protein (1170 amino acids). The deduced CISP protein sequence is 89% identical to that of human thrombospondin-2 (TSP2), but only 61% identical to that of bovine TSP1, confirming that CISP is the bovine ortholog of TSP2. The bovine TSP2 sequence aligned perfectly with human, mouse, and chicken TSP2 sequences, except for a gap of 2 amino acids located in a linker region. All 58 cysteine residues that are conserved in other species were present in the bovine sequence as well as most of the functional domains. Most endocrine tissues (adrenal cortex, testis, ovary, and placenta) appeared to express TSP2, as determined by Western blot analysis. The highest levels of TSP2 protein were found in the adrenal cortex, followed by the heart, spleen, brain, and kidney. A differential extent of N-glycosylation or tissular proteolytic maturation may be responsible for the mol wt differences observed between bovine TSP2 detected in the medium from primary cultures and that in fresh tissue extracts. The immunohistochemical analysis of the distribution of TSP2 in the bovine adrenal gland revealed that the protein is much more abundant in the external zones (zona glomerulosa and zona fasciculata) than in the internal reticularis zone, a pattern similar to that reported for ACTH receptors. This distribution clearly suggests that TSP2 is a candidate relay protein for a subset of ACTH actions in the adrenal cortex.

Opposite regulation of thrombospondin-1 and corticotropin-induced secreted protein/thrombospondin-2 expression by adrenocorticotropic hormone in adrenocortical cells.

Corticotropin-induced secreted protein (CISP) is a trimeric glycoprotein secreted by primary cultures of bovine adrenortical cells in response to adrenocorticotropic hormone (ACTH). This protein was recently purified in our laboratory, and its N-terminal amino-acid sequence revealed a significant similarity with thrombospondin-2 (TSP2). We report here the nucleotide sequence of a 386 bp RT-PCR fragment specific for CISP. The deduced protein sequence shares 84% identity with the N-terminal portion of mature human TSP2, suggesting that CISP is its bovine counterpart. Northern analysis of adrenocortical cell RNA using the above cDNA fragment as a probe revealed a 6.0 kb CISP/TSP2 mRNA whose abundance was increased nearly fivefold following a 24 h cell treatment with 10(-7) M ACTH. Under the same conditions, the expression of TSP1 mRNA was reduced by tenfold. The protein levels of TSP1 and CISP/TSP2 varied accordingly with their respective mRNA levels, as shown by immunoprecipitation and immunofluorescence experiments. Taken together, these data show that ACTH induces a dramatic shift in the pattern of adrenocortical cell thrombospondin expression from TSP1 to CISP/TSP2. This observation suggests that these two members of the thrombospondin family exert distinct biological functions in the adrenal cortex. This hypothesis is further supported by the observation that anti-CISP antibodies inhibit the maintenance of the morphological changes of bovine adrenocortical cells induced by ACTH, whereas anti-TSP1 antibodies do not.

Transforming growth factors beta stimulate both thrombospondin-1 and CISP/thrombospondin-2 synthesis by bovine adrenocortical cells.

We recently observed that adrenocortical cells secrete, under ACTH treatment, a large trimeric glycoprotein (CISP) presenting amino acid sequence similarity with thrombospondin-2. We also observed that the same cells synthesize and secrete thrombospondin-1 whereas under smaller amounts. The aim of this study was to investigate the regulation of these two secreted proteins by members of the TGF beta family of regulatory peptides. We developed an appropriate immunoprecipitation technique that allowed us to quantitate synthesis of thrombospondin-1 and CISP/thrombospondin-2 in a single assay. Using this assay, we observed that thrombospondin-1 and CISP/thrombospondin-2 syntheses were respectively stimulated threefold and twofold by a 24-h treatment with 2 ng/ml TGF beta 1. These inductions were dose-dependent (half-maximal effect: 0.2 ng/ml) and time-dependent (detectable after 5 h and plateauing between 15 and 25 h of treatment). They were not observed when transcription was blocked by RNA polymerase inhibitors such as 5,6-dichlorobenzimidazole riboside or actinomycin D. Among members of the TGF beta family, TGF beta 1 and TGF beta 2 and to a lesser extent activin could stimulate thrombospondin-1 and CISP/thrombospondin-2 synthesis, whereas inhibin and Müllerian inhibiting substance were inactive. Taken together, these data represent the first study on the regulation of both thrombospondin-1 and CISP/thrombospondin-2 by TGF betas. They further support the concept that TGF beta is a local regulator of adrenocortical functions.

Distinct effects of thrombospondin-1 and CISP/thrombospondin-2 on adrenocortical cell spreading.

Corticotropin-induced secreted protein (CISP) is a trimeric protein secreted by bovine adrenocortical cells in response to ACTH, that is likely to represent the bovine form of thrombospondin-2 (TSP2). This study was aimed at delineating the respective effects of CISP/TSP2 and TSP1 (thrombospondin-1) on adrenocortical cell attachment and spreading. TSP1 and CISP/TSP2 were found to slightly reduce the attachment of adrenocortical cells to plastic in the presence of serum but exhibited a pronounced differential effect on cell spreading. CISP/TSP2 inhibited adrenocortical cell spreading in a dose-dependent manner (maximal effect with 40 micrograms/ml) whereas TSP1 (up to 100 micrograms/ml) did not influence this process. The inhibition of spreading was observed whether plates were coated with CISP/TSP2 alone or with a mixture of CISP/TSP2 and fibronectin. We suggest that the inhibition of in vitro adrenocortical cell spreading by CISP/TSP2 is indicative of an implication of this protein in the migration of adrenocortical cells in vivo.

The molecular structure of corticotropin-induced secreted protein, a novel member of the thrombospondin family.

CISP (corticotropin-induced secreted protein) is a secreted protein recently purified in our laboratory from the conditioned medium of ACTH-treated bovine adrenocortical cells. Partial amino acid sequencing of CISP revealed homology with thrombospondins (TSPs), a family of adhesive proteins and in particular with TSP2. We report here the characterization of the molecular structure of CISP. Analysis of CISP by polyacrylamide gel electrophoresis in the absence or presence of SDS indicated an apparent molecular mass approximately equal to 600 kDa for the unreduced protein and an apparent molecular mass of 195 kDa after reduction by 2-mercaptoethanol. The sedimentation coefficient of CISP determined by ultracentrifugation on sucrose gradients was shifted from 9.7 S in the absence to 5.7 S in the presence of 2-mercaptoethanol. These data are consistent with a trimeric organization of the CISP molecule in which 195-kDa monomers would be linked together by disulfide bonds. The trimeric structure of CISP could be observed by rotary shadowing/electron microscopy, where CISP appeared to be composed of three equally electron-dense nodules and of a fourth nodule formed by the close association of three smaller fragments. The overall size of the molecule was 60 nm. We also observed that CISP is sulfated and glycosylated. Using glycosylation inhibitors, we could determine that CISP is synthesized as a 175-kDa core protein, is then matured into a 190-kDa high-mannose form and secreted as a 195-kDa mature protein. Inhibition of sulfation by chlorate did not prevent CISP secretion, whereas inhibition of glycosylation by tunicamycin blocked it. Taken together, these data indicate that the TSP2-related CISP molecule presents both structural and functional properties very similar to those of TSP1. CISP differs greatly, however, from TSP1 by the inducibility of its synthesis by cAMP.

Corticotropin-induced secreted protein, an ACTH-induced protein secreted by adrenocortical cells, is structurally related to thrombospondins.

The treatment of primary cultures of bovine adrenocortical cells with nanomolar concentrations of ACTH induces a 10-fold increase in the synthesis of a secreted protein of apparent molecular mass 195 kDa on reducing SDS-polyacrylamide gels. This corticotropin-induced secreted protein (CISP) appears to be an oligomeric calcium-binding protein. Its secretion under serum-free culture conditions is sustained over 4 days in the continuous presence of ACTH. Induction of CISP secretion by ACTH is mimicked by cAMP analogs and adenylate cyclase activators. We report here the purification of CISP to apparent homogeneity with an overall yield of 43% using a combination of heparin-agarose and Mono-Q chromatographies. The NH2-terminal amino acid sequence and the sequence of several tryptic peptides revealed that CISP is structurally related to the members of the thrombospondin (TSP) family. Among these members, bovine CISP appeared to be more homologous to mouse TSP2 (85% identity in the 29 amino acid long NH2-terminal sequence) than to TSP1 (18% identity in the same region). We also observed that CISP binds Ca2+ and is an adhesive protein for bovine adrenocortical cells. Thus, CISP possesses both structural and functional properties of thrombospondins. Whether CISP represents the bovine form of TSP2 or a novel member of the expanding thrombospondin family will need to be elucidated by cloning and sequencing of a larger portion of the molecule.

[Contribution of molecular biology to the diagnosis of familial hypercholesterolemias in children]. / Apport de la biologie moléculaire au diagnostic des hypercholestérolémies familiales chez l'enfant.

The detection of children at high risk in families with a genetic form of hypercholesterolemia is important for acceptance of prevention under medical control. However, usual lipidic parameters are difficult to interpret during infancy. So we studied by a molecular biology approach 11 families presenting with syndrome of pure hypercholesterolemia where a defect of the LDL receptor (LDL-R) gene was suspected (Familial Hypercholesterolemia: FH IIa). Markers of the LDL-R gene (Restriction Fragment Length Polymorphism RFLP) were studied with intragenic probes. The segregation of the abnormal gene was studied in each family. Our results illustrate the limits of such an approach (its heaviness and the fact that, in 2 families, the analysis was not informative), but also its advantages: indeed, in 8 families, the diagnosis was established (particularly in one case at birth). Moreover in 2 families the LDL-R abnormality was excluded. In such case, as an alternative, the hypothesis of an apo B abnormality was envisaged. This differential diagnosis is interesting since it permits the choice of an adequate treatment.

[Autocrine regulation of adrenal steroidogenesis by growth factor transforming TGF-beta 1]. / Régulation autocrine de la stéroïdogénèse corticosurrénalienne par le facteur de croissance transformant TGF bêta 1.

Transforming growth factor beta (TGF beta) is a multifunctional polypeptide presenting various biological activities depending on the cellular context where it is studied. In the adrenal cortex, TGF beta is a potent inhibitor of corticosteroïdogenesis. The sum of data that we have collected in the past years has allowed us to establish that TGF beta (in particular TGF beta 1) is implicated in a physiological autocrine/paracrine regulatory loop. We determined that primary cultures of bovine fasciculatareticularis cells synthesize and secrete about 5 ng/24 h x 10(6) cells of TGF beta but that most of this activity is under a latent form. In the adrenal gland, TFG beta 1 could be localized in the fasciculata-reticularis zone by immunohistochemistry. We also reported that adrenocortical cells possess type I and type III (betaglycan) receptors, whose number is positively regulated by ACTH. Moreover, we have identified 3 major targets of TGF beta in our cell model: cytochrome P-450(17) alpha (17 alpha-hydroxylase) whose gene expression is decreased, angiotensin II receptors and LDL receptors whose number is reduced after TGF beta treatment. As a conclusion, TGF beta appears to participate together with other regulatory peptides (IGF-1, TNF alpha) in the fine tuning of corticosteroid secretion levels which are under the major control of the hypothalamo-pituitary axis.