Institut National de la Transfusion Sanguine

Alzheimer's disease is a leading cause of morbidity and mortality among the elderly. Several families have been described in which Alzheimer's disease is caused by an autosomal dominant gene defect. The chromosomal location of this defective gene has been discovered by using genetic linkage to DNA markers on chromosome 21. The localization on chromosome 21 provides an explanation for the occurrence of Alzheimer's disease-like pathology in Down syndrome. Isolation and characterization of the gene at this locus may yield new insights into the nature of the defect causing familial Alzheimer's disease and possibly, into the etiology of all forms of Alzheimer's disease.

This study evaluates polymorphonuclear neutrophil (PMN) cell performance in 61 diabetic patients free of infection (40 Type 1, 21 Type 2), using tests that explore all the functional steps of PMN: (1) adherence: expression of adhesion molecules, CD 11a, CD 11b, CD 11c; nylon fiber adherence test; (2) chemotaxis under agarose towards the bacterial oligopeptide FMLP and complement fractions, used as attracting agents; (3) phagocytosis of opsonized latex microbeads; (4) bactericidal activity: chemiluminescence assessment of the oxidative killing potential before and after stimulation by opsonized zymosan and PMA; nitroblue tetrazolium reduction test. Results were analysed according to potentially influential factors: metabolic control (HbA1C, glycaemia), age of patient, type of diabetes, disease duration, and existence of vascular complications. PMN chemotaxis was significantly lower in patients than in healthy controls (p < 0.001) and associated with spontaneous adherence and increased expression of adhesion molecules (CD 11b, CD 11c). The increased response to chemiluminescence reflects spontaneous activation of PMN cells and increased free radical production; after stimulation, response was lower than in controls. The type of diabetes, the age of patients, HbA1C level and disease duration did not affect the responses. Chemotaxis and chemiluminescence were further reduced in patients with vascular complications and hyperglycaemia. We conclude that all steps of PMN functioning are altered in diabetic patients, which may increase the risk of vascular complications and infectious episodes.

Sequences of 234 complete genomes and 631 hepatitis B surface antigen genes were used to assess the worldwide diversity of hepatitis B virus (HBV). Apart from the described two subgenotypes each for A and F, also B, C, and D divided into four subgenotypes each in the analysis of complete genomes supported by significant bootstrap values. The subgenotypes of B and C differed in their geographical distribution, with B1 dominating in Japan, B2 in China and Vietnam, B3 confined to Indonesia, and B4 confined to Vietnam, all strains specifying subtype ayw1. Subgenotype C1 was common in Japan, Korea, and China; C2 in China, South-East Asia, and Bangladesh, and C3 in the Oceania comprising strains specifying adrq-, and C4 specifying ayw3 is encountered in Aborigines from Australia. This pattern of defined geographical distribution was less evident for D1-D4, where the subgenotypes were widely spread in Europe, Africa, and Asia, possibly due to their divergence having occurred a longer time ago than for genotypes B and C, with D4 being the first split and still the dominating subgenotype of D in the Oceania. The genetic diversity of HBV and the geographical distribution of its subgenotypes provide a tool to reconstruct the evolutionary history of HBV and may help to complement genetic data in the understanding of the evolution and past migrations of man.

The BCL-2 proto-oncogene encodes a mitochondrial protein that blocks programmed cell death. High amounts of bcl-2 protein are found not only in lymphoid malignancies, but also in normal tissues characterized by apoptotic cell death, including bone marrow. Using a monoclonal antibody to bcl-2 protein, we analyzed 82 samples of newly diagnosed acute myeloid leukemia. The number of bcl-2+ cells in each sample was heterogeneous (range, 0% to 95%), with a mean of 23%. The percentage of bcl-2+ cells was higher in M4 and M5 types, according to French-American-British classification, and in cases with high white blood cell counts. bcl-2 expression was also correlated with that of the stem cell marker CD34. In vitro survival of leukemic cells maintained in liquid culture in the absence of growth factors was significantly longer in cases with a high percentage of bcl-2+ cells. High expression of bcl-2 was associated with a low complete remission rate after intensive chemotherapy (29% in cases with 20% or more positive cells v 85% in cases with less than 20% positive cells, P < 10(-5)) and with a significantly shorter survival. In multivariate analysis, the percentage of bcl-2+ cells (or the blast survival in culture), age, and the percentage of CD34+ cells were independently associated with poor survival.

Mesenchymal stem cells (MSCs) are considered as emergent "universal" cells and various tissue repair programs using MSCs are in development. In vitro expansion of MSCs is conventionally achieved in medium containing fetal calf serum (FCS) and is increased by addition of growth factors. However, for widespread clinical applications, contact of MSCs with FCS must be minimized since it is a putative source of prion or virus transmission. Therefore, because platelets are a natural source of growth factors, we sought to investigate in vitro MSC expansion in response to platelet lysates (PL) obtained from platelet-rich plasma. Human MSCs were expanded in FCS (+/-bFGF)- or PL-supplemented medium through a process of subculture. We demonstrated that PL-containing medium is enriched by growth factors (platelet-derived growth factors (PDGFs), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-beta), insulin-like growth factor-1 (IGF-1) ...) and showed that PL is able to promote MSC expansion, to decrease the time required to reach confluence, and to increase CFU-F size, as compared to the FCS medium. Furthermore, we demonstrated that MSCs cultured in the presence of PL maintain their osteogenic, chondrogenic, and adipogenic differentiation properties and retain their immunosuppressive activity. Therefore, we propose that PL may be a powerful and safe substitute for FCS in development of tissue- and cellular-engineered products in clinical settings using MSCs.

In October 2018 a large number of international experts with complementary expertise came together in Taormina to participate in a workshop on occult hepatitis B virus infection (OBI). The objectives of the workshop were to review the existing knowledge on OBI, to identify issues that require further investigation, to highlight both existing controversies and newly emerging perspectives, and ultimately to update the statements previously agreed in 2008. This paper represents the output from the workshop.

To evaluate the clinical value of the expression of multidrug resistance P-glycoprotein (P-170) on the surface of acute nonlymphoblastic leukemia (ANLL) cells, we analyzed specimens from 150 newly diagnosed patients for staining with MRK16, a monoclonal antibody (MoAb) that binds to an external epitope of P-170. Other surface markers (CD13, CD14, CD15, and CD34) were studied by the same technique. A marker was considered positive when 20% or more cells were stained. Of 150 samples, 71 were P-170-positive. These cases did not differ from P-170-negative cases with regard to age, sex, initial white blood cell (WBC) counts, or French-American-British (FAB) type (except for M3 ANLL, which were more frequently negative). However, leukemias arising from previous myelodysplastic syndrome (MDS) and therapy-induced leukemias were more frequently P-170-positive. CD34 and P-170 expression were significantly associated. All patients were treated by intensive chemotherapy. Complete remission (CR) rates were significantly lower in P-170-positive (23/71, 32%) than in P-170-negative cases (64/79, 81%) (P less than 10(-5)). CD34 positivity was also associated with a low remission rate (P less than 10(-5)). Survival was shorter for P-170- and CD34-positive patients (P less than 10(-5)). The prognostic value of both markers was confirmed in multivariate analysis. CR duration was also shorter for P-170-positive cases, but the difference is less significant (P = .05). It is concluded that P-170 analysis may be an important tool for predicting the outcome of intensive chemotherapy in ANLL patients.

ADP is a key agonist in hemostasis and thrombosis. ADP-induced platelet activation involves the purinergic P2Y(1) receptor, which is responsible for shape change through intracellular calcium mobilization. This process also depends on an unidentified P2 receptor (P2cyc) that leads to adenylyl cyclase inhibition and promotes the completion and amplification of the platelet response. P2Y(1)-null mice were generated to define the role of the P2Y(1) receptor and to determine whether the unidentified P2cyc receptor is distinct from P2Y(1). These mice are viable with no apparent abnormalities affecting their development, survival, reproduction, or the morphology of their platelets, and the platelet count in these animals is identical to that of wild-type mice. However, platelets from P2Y(1)-deficient mice are unable to aggregate in response to usual concentrations of ADP and display impaired aggregation to other agonists, while high concentrations of ADP induce platelet aggregation without shape change. In addition, ADP-induced inhibition of adenylyl cyclase still occurs, demonstrating the existence of an ADP receptor distinct from P2Y(1). P2Y(1)-null mice have no spontaneous bleeding tendency but are resistant to thromboembolism induced by intravenous injection of ADP or collagen and adrenaline. Hence, the P2Y(1) receptor plays an essential role in thrombotic states and represents a potential target for antithrombotic drugs.

The advanced glycation end products (AGEs) are a heterogeneous class of molecules, including the following main subgroups: bis(lysyl)imidazolium cross-links, hydroimidazolones, 3-deoxyglucosone derivatives, and monolysyl adducts. AGEs are increased in diabetes, renal failure, and aging. Microvascular lesions correlate with the accumulation of AGEs, as demonstrated in diabetic retinopathy or renal glomerulosclerosis. On endothelial cells, ligation of receptor for AGE (RAGE) by AGEs induces the expression of cell adhesion molecules, tissue factor, cytokines such as interleukin-6, and monocyte chemoattractant protein-1. A chief means by which AGEs via RAGE exert their effects is by generation of reactive oxygen species, at least in part via stimulation of NADPH oxidase. Diabetes-associated vascular dysfunction in vivo can be prevented by blockade of RAGE. Thus, agents that limit AGE formation, increase the catabolism of these species, or antagonize their binding to RAGE may provide new targets for vascular protection in diabetes.

Immunization with heat shock proteins (HSPs) induces Ag-specific CTL responses. The specificity of the immune response is based on peptides associated with HSPs. To investigate how exogenous HSP/peptide complexes gain access to the MHC class I-restricted Ag presentation pathway, we incubated the monocytic cell line P388D1 and the dendritic cell line D2SC/1 with gold-labeled HSPs gp96 and HSC70. We show that HSPs bind specifically to the surface of these APCs and are internalized spontaneously by receptor-mediated endocytosis, demonstrating the existence of specific receptors for HSPs on these cells. In addition, we observe colocalization of internalized HSPs and surface MHC class I molecules in early and late endosomal structures. These findings provide possible explanations for the immunogenicity of HSP/peptide complexes and for the transfer of HSP-associated peptides onto MHC class I molecules.

Malaria therapy, experimental, and epidemiological studies have shown that erythrocyte Duffy blood group-negative people, largely of African ancestry, are resistant to erythrocyte Plasmodium vivax infection. These findings established a paradigm that the Duffy antigen is required for P. vivax erythrocyte invasion. P. vivax is endemic in Madagascar, where admixture of Duffy-negative and Duffy-positive populations of diverse ethnic backgrounds has occurred over 2 millennia. There, we investigated susceptibility to P. vivax blood-stage infection and disease in association with Duffy blood group polymorphism. Duffy blood group genotyping identified 72% Duffy-negative individuals (FY*B(ES)/*B(ES)) in community surveys conducted at eight sentinel sites. Flow cytometry and adsorption-elution results confirmed the absence of Duffy antigen expression on Duffy-negative erythrocytes. P. vivax PCR positivity was observed in 8.8% (42/476) of asymptomatic Duffy-negative people. Clinical vivax malaria was identified in Duffy-negative subjects with nine P. vivax monoinfections and eight mixed Plasmodium species infections that included P. vivax (4.9 and 4.4% of 183 participants, respectively). Microscopy examination of blood smears confirmed blood-stage development of P. vivax, including gametocytes. Genotyping of polymorphic surface and microsatellite markers suggested that multiple P. vivax strains were infecting Duffy-negative people. In Madagascar, P. vivax has broken through its dependence on the Duffy antigen for establishing human blood-stage infection and disease. Further studies are necessary to identify the parasite and host molecules that enable this Duffy-independent P. vivax invasion of human erythrocytes.

Several lines of evidence have previously indicated that the RhD, c, and E blood group antigens are most likely carried by three distinct but homologous red blood cell membrane proteins. To determine whether these polypeptides are encoded by one or several related genes, we have performed Southern blot analysis of genomic DNA prepared from donors of different Rh phenotypes. Using an entire Rh cDNA probe and several exon-specific probes covering the cloned gene from its 5' to 3' ends, we have shown that the Rh locus carried by the genome of RhD-positive individuals is composed of two different but strongly related genes of identical general organization whether they expressed the C or c and E or e antigens, and, surprisingly, even when they do not express these epitopes, as in the D-- phenotype. The only antigenic variation found to be associated with a consistent genomic polymorphism corresponded to the RhD-positive/RhD-negative phenotypes. Indeed, one of the two Rh genes was completely lacking when the genomes of several unrelated RhD-negative donors were analyzed. From the present study we conclude that one of the two genes of the Rh locus encodes the RhC/c and RhE/e polypeptides while the other encodes the RhD protein. The absence of any D gene and of its postulated allelic form d in the RhD-negative genome explains finally why no Rhd antigen has ever been shown.

The surface (S) genes of 12 hepatitis B viruses (HBVs) encoding nine different serotypes of hepatitis B surface antigen (HBsAg) were amplified by the polymerase chain reaction and sequenced. These represented the eight strains of HBV, P1 to P8, defined at an international workshop on HBsAg subtypes in Paris in 1975, and the adrq- subtype. The S genes from additional HBV strains, one ayw4, one adw4 and one ayw1, of sub-Saharan African origin, were also sequenced. The relationship of these 12 new S gene sequences to those of the 20 published previously was investigated by constructing a phylogenetic tree, which confirmed a previous classification into four groups, designated A to D, based on 18 complete HBV genomes. When relating our sequenced S genes to these genomic groups, ayw1 of African origin and P6 (adw2) were both allocated to group A, the reference P1 (ayw1 of Vietnamese origin) was allocated to group B, P5 (ayr), P8 (adr) and adrq- were all related to group C, and P2 (ayw2) and P3 (ayw3) could both be allocated to group D. Interestingly, the S genes of w4 serotype viruses, i.e. P4 (ayw4) and P7 (adw4q-), differed by 4% or more from both previous groups and from each other, suggesting their classification into two new groups, for which the designations E and F are proposed. Genomes specifying ayw were also found in groups A and B; previously sequenced genomes specifying the ayw subtype have all been confined to group D. There were indications that the epitope for subdeterminants of w resided at amino acid positions 125 to 127. Thus, at positions 125 and 127, ayw1, ayw2 and adw2 had T and P residues, respectively, whereas M and T residues were at the corresponding positions of ayw3. Both ayw4 and adw4 had L at residue 127, and all strains expressing r, apart from P5, had an I instead of a T residue at position 126.

KD mice showed increased and reduced neuronal damage, respectively. Altogether, our findings indicate that microglia-astrocyte interaction, involving a purinergic signal, is essential for the formation of neuroprotective astrocytes.

Human lymphocyte antigen (HLA) class I proteins of the major histocompatibility complex are largely dependent for expression on small peptides supplied to them by transporter associated with antigen processing (TAP) protein. An inherited human deficiency in the TAP transporter was identified in two siblings suffering from recurrent respiratory bacterial infections. The expression on the cell surface of class I proteins was very low, whereas that of CD1a was normal, and the cytotoxicity of natural killer cells was affected. In addition, CD8+ alpha beta T cells were present in low but significant numbers and were cytotoxic in the most severely affected sibling, who also showed an increase in CD4+CD8+ T cells and gamma delta T cells.

Erythropoiesis occurs mostly in bone marrow and ends in blood stream. Mature red blood cells are generated from multipotent hematopoietic stem cells, through a complex maturation process involving several morphological changes to produce a highly functional specialized cells. In mammals, terminal steps involved expulsion of the nucleus from erythroblasts that leads to the formation of reticulocytes. In order to produce mature biconcave red blood cells, organelles and ribosomes are selectively eliminated from reticulocytes as well as the plasma membrane undergoes remodeling. The mechanisms involved in these last maturation steps are still under investigation. Enucleation involves dramatic chromatin condensation and establishment of the nuclear polarity, which is driven by a rearrangement of actin cytoskeleton and the clathrin-dependent generation of vacuoles at the nuclear-cytoplasmic junction. This process is favored by interaction between the erythroblasts and macrophages at the erythroblastic island. Mitochondria are eliminated by mitophagy. This is a macroautophagy pathway consisting in the engulfment of mitochondria into a double-membrane structure called autophagosome before degradation. Several mice knock-out models were developed to identify mitophagy-involved proteins during erythropoiesis, but whole mechanisms are not completely determined. Less is known concerning the clearance of other organelles, such as smooth and rough ER, Golgi apparatus and ribosomes. Understanding the modulators of organelles clearance in erythropoiesis may elucidate the pathogenesis of different dyserythropoietic diseases such as myelodysplastic syndrome, leukemia and anemia.

A 681 nucleotide fragment of the hepatitis B virus (HBV) genome was sequenced that corresponded to the complete gene for hepatitis B surface antigen (HBsAg) in 80 HBsAg- and hepatitis B e antigen (HBeAg)-positive sera of diverse geographical origins. These and 42 previously published HBV sequences within the S gene were used for the construction of a dendrogram. In this comparison, each of the 122 HBsAg genes was found to be related to one or other of the six previously identified genomic groups of HBV, A to F. The HBV strains within each genomic group showed a characteristic geographical distribution. Group A genomes were represented by 23 strains mainly originating in northern Europe and sub-Saharan Africa. The group B and C genomes, represented by 17 and 28 strains respectively, were confined to populations with origins in eastern Asia and the Far East. The group D genomes, represented by 38 strains, were found worldwide, but were the predominant strains in the Mediterranean area, the Near and Middle East, and in south Asia. Group E genomes, represented by nine strains, were indigenous to western sub-Saharan Africa as far south as Angola. There were indications that the F group, made up of six strains, represented the genomic group of HBV among populations with origins in the New World. Thus, HBV has diverged into genomic groups according to the distribution of mankind in the different continents. As well as giving information on the genetic relationship of HBV strains of different geographical origin, this study also provides information on the primary structure of HBsAg in different regions of the world. Such data might prove valuable in explaining the reported failures to obtain protection with current HBV vaccines.

B19 virus is a human virus belonging to the genus Erythrovirus: The genetic diversity among B19 virus isolates has been reported to be very low, with less than 2% nucleotide divergence in the whole genome sequence. We have previously reported the isolation of a human erythrovirus isolate, termed V9, whose sequence was markedly distinct (>11% nucleotide divergence) from that of B19 virus. To date, the V9 isolate remains the unique representative of a new variant in the genus Erythrovirus, and its taxonomic position is unclear. We report here the isolation of 11 V9-related viruses. A prospective study conducted in France between 1999 and 2001 indicates that V9-related viruses actually circulate at a significant frequency (11.4%) along with B19 viruses. Analysis of the nearly full-length genome sequence of one V9-related isolate (D91.1) indicates that the D91.1 sequence clusters together with but is notably distant from the V9 sequence (5.3% divergence) and is distantly related to B19 virus sequences (13.8 to 14.2% divergence). Additional phylogenetic analysis of partial sequences from the V9-related isolates combined with erythrovirus sequences available in GenBank indicates that the erythrovirus group is more diverse than thought previously and can be divided into three well-individualized genotypes, with B19 viruses corresponding to genotype 1 and V9-related viruses being distributed into genotypes 2 and 3.

We have conducted a multicenter randomized controlled trial comparing two doses of recombinant human alpha-interferon for efficacy in 60 patients with chronic non-A, non-B hepatitis. The source of infection appeared to be transfusion in 30 patients, intravenous drug abuse in 16 patients and was unknown in 14 patients. Patients were randomly assigned to no treatment or to treatment with either 1 or 3 MU of alpha-interferon given three times a week for 24 wk. Forty-five patients (75%) were positive for antibody to hepatitis C virus. During the 24-wk treatment period, mean serum ALT levels decreased in both treatment groups, but the decrease was statistically significant only in the 3 MU group. However, at 24 wk, the proportion of patients with normal ALT levels was similar in the 3 MU group (39%) and the 1 MU group (45%), and both were significantly higher than in controls (0%). Repeat liver biopsy specimens showed a significant decrease in the severity of histological changes in the 3 MU group but not in the 1 MU group or in controls. Responses to alpha-interferon did not correlate with patient's age, gender, source of infection, pretreatment serum ALT, presence of anti-hepatitis C virus or cirrhosis. After treatment, the mean ALT levels rose in both treated groups. The proportion of patients with normal ALT levels at wk 48 was 28% in the 3 MU group and 20% in the 1 MU group. In conclusion, a dose of 3 MU was superior to 1 MU of alpha-interferon given three times weekly for 24 wk in inducing improvements in serum ALT levels and liver histological examinations.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract High resolution electron microscopy has made possible the visualization of transport and storage iron in the form of ferritin, both in dispersed form and in aggregates and in the form of "iron micelles" in mitochondria. Hemosiderin was found to consist either of pure ferritin in crystalline clusters or, more frequently, of ferritin associated with other substances, including a lipid component in the form of myelinic figures and PAS positive material. In the following paragraphs we have summarized the new morphologic findings and what appears to us the most likely interpretation in the light of known biochemical and isotopic studies. Alternative interpretations have been discussed in the body of the paper. Electron microscopy has established the erythroblastic island as a morphologic and functional unit of the bone marrow. A central reticular "nurse cell" appears to impart nutrients to surrounding rows of erythroblasts by the process of rhopheocytosis. Transfer of ferritin by this process is probably a passive phenomenon, since the amount transferred parallels the amount of iron present in the central reticular cell. Ferritin is increased both in the reticular cell and in erythroblasts in hemochromatosis. It is absent in iron deficiency, although rhopheocytosis remains prominent. Normally all erythroblasts (proerythroblasts and normoblasts) and reticulocytes contain ferritin. Only the larger aggregates can be visualized by the Prussian blue reaction in sideroblasts and siderocytes. Ferritin generally disappears when reticulocytes mature, even in hemochromatosis and infections, two conditions in which there is an excess of ferritin in erythroblasts. Interestingly, the increase in infections is entirely in form of dispersed ferritin and cannot be visualized by the Prussian blue reaction; i.e., sideroblasts are absent, in contrast to hemochromatosis where they are normal or increased. It appears most likely that ferritin disappears from normal maturing reticulocytes because it is utilized for hemoglobin formation. It persists in mature red cells in Cooley’s anemia, hypersideremia, hypochromic anemia and lead poisoning where hemoglobin formation is disturbed. The origin of the ferritin in the nurse cells and the extent to which ferritin rather than siderophilin contributes to hemoglobin synthesis are unsolved problems. Isotopic studies indicate that almost all of the iron used for hemoglobin synthesis is derived from siderophilin and hemoglobin synthesis can proceed without any visible ferritin, as in iron deficiency anemia. These facts must be reconciled with the electron microscopic observations which suggest that normally some iron reutilization within the marrow proceeds by way of erythrophagocytosis, fragmentation, intracellular hemolysis of red cells, formation of ferritin and ropheocytosis. Iron derived from erythrophagocytosis elsewhere in the body probably reaches the marrow bound to siderophilin. Such iron can be incorporated into ferritin of reticular cells as may be seen in hyperferremia and following injection of iron compounds. The process of rhopheocytosis would then lead to utilization of at least part of this ferritin iron for hemoglobin synthesis. In certain pathologic states, accumulation of ferritin and related visible dispersed or conglomerated iron micelles may point to the sites where hemoglobin synthesis or iron transport is blocked. In Cooley’s anemia and the hypersideremic hypochromic (non-thalassemic) anemias, iron accumulates in the mitochondria, which are known to be involved in hemoglobin synthesis. In lead poisoning, the mitochondria are markedly abnormal, and probably correspond to the areas of punctate basophilia. However, the iron accumulates in other areas of the cell, suggesting a different type of block.