Detection of prions in the plasma of presymptomatic and symptomatic patients with variant Creutzfeldt-Jakob disease.

Variant Creutzfeldt-Jakob disease (vCJD) is a human prion disease resulting from the consumption of meat products contaminated by the agent causing bovine spongiform encephalopathy. Evidence supporting the presence of a population of silent carriers that can potentially transmit the disease through blood transfusion is increasing. The development of a blood-screening assay for both symptomatic vCJD patients and asymptomatic carriers is urgently required. We show that a diagnostic assay combining plasminogen-bead capture and protein misfolding cyclic amplification (PMCA) technologies consistently detected minute amounts of abnormal prion protein from French and British vCJD cases in the required femtomolar range. This assay allowed the blinded identification of 18 patients with clinical vCJD among 256 plasma samples from the two most affected countries, with 100% sensitivity [95% confidence interval (CI), 81.5 to 100%], 99.2% analytical specificity (95% CI, 95.9 to 100%), and 100% diagnostic specificity (95% CI, 96.5 to 100%). This assay also allowed the detection of silent carriage of prions 1.3 and 2.6 years before the clinical onset in two blood donors who later developed vCJD. These data provide a key step toward the validation of this PMCA technology as a blood-based diagnostic test for vCJD and support its potential for detecting presymptomatic patients, a prerequisite for limiting the risk of vCJD transmission through blood transfusion.

Rapid and Highly Sensitive Detection of Variant Creutzfeldt-Jakob Disease Abnormal Prion Protein on Steel Surfaces by Protein Misfolding Cyclic Amplification: Application to Prion Decontamination Studies.

The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10-8 brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.

Magnetic microparticle-based multimer detection system for the detection of prion oligomers in sheep.

Transmissible spongiform encephalopathies (TSEs) are zoonotic fatal neurodegenerative diseases in animals and humans. TSEs are commonly known as bovine spongiform encephalopathy in cattle, scrapie in sheep and goats, chronic wasting disease in cervids, and Creutzfeldt-Jakob disease in humans. The putative transmissible agents are infectious prion proteins (PrP(Sc)), which are formed by the conversion of the normal prion protein on the glycoprotein cell surface in the presence of other PrP(Sc). Reports of the transmission of TSEs through blood raised considerable concern about the safety of blood and blood products. To address this issue, many laboratories attempted to develop a sensitive and accurate blood diagnostic test to detect PrP(Sc). Previously, we reported that, compared to normal controls, the multimer detection system (MDS) was more efficient in detecting PrP(Sc) in infected hamster brain homogenate, mouse plasma spiked with purified PrP(Sc) from scrapie mouse brain, and scrapie-infected hamster plasmas. MDS differentiates prion multimers from the cellular monomer through the multimeric expression of epitopes on prion multimers, in contrast to the monomeric form. In this study, MDS detected PrP(Sc) in plasma samples from scrapie-infected sheep expressing clinical symptoms, demonstrating 100% sensitivity and specificity in these samples. Plasma samples from asymptomatic lambs at the preclinical stage (8-month-old naturally infected offspring of scrapie-infected parents expressing a highly susceptible genotype) tested positive with 50% sensitivity and 100% specificity. In the first of two coded analyses using clinical scrapie-infected sheep and normal healthy samples, MDS successfully identified all but one of the clinical samples with 92% sensitivity and 100% specificity. Similar results were obtained in the second coded analysis using preclinical samples. MDS again successfully identified all but one of the samples with 87% sensitivity and 100% specificity. The false-negative sample was subjected to a protease pretreatment. In conclusion, MDS could accurately detect scrapie in plasma samples at both preclinical and clinical stages. From these studies, we conclude that MDS could be a promising tool for the early diagnosis of TSEs from blood samples.

[Blood transfusion - the value of Research and Development programs]. / Transfusion sanguine : un modèle de questionnement en recherche et développement.

Transfusion is a mixed discipline which includes the production of blood components, applied biology aiming in particular at establishing the highest compatibility for immunological characteristics between blood components to be delivered to patients and recipients, and, finally translational medicine to evaluate the effectiveness of the transfused products and to proactively avoid hazards, at least those that are preventible and can be anticipated. The whole chain takes place with the concern of continuous improvement of quality and safety. These two principles (quality/safety) have been and still are concerns of constant progress applicable to all the transfusion chain steps; they benefit from programs of Research and Development (R&D). Fundamental research, basic but also applied and clinical research, accompanies, in a constantly joint manner, scientific and medical progresses by providing new solutions to dampen the current problems and to prepare the future.

An innovative biologic recycling process of leukoreduction filters to produce active human antimicrobial peptides.

BACKGROUND: Human neutrophil peptides (HNPs) 1 to 3 are the major antimicrobial peptides of the azurophilic granules of neutrophils. They represent an important arm of the innate immune system. Their production by chemical synthesis and recombinant technologies is expensive and limited by technical constraints due to their composition and the presence of three disulfide bonds. STUDY DESIGN AND METHODS: We have developed an original approach based on the purification of the natural human defensins HNPs 1 to 3 from neutrophils trapped on leukoreduction filters used in blood processing. The purification of HNPs 1 to 3 from these filters is performed in two steps: extraction of HNPs 1 to 3 retained in the filters followed by their immunoprecipitation. Studies were performed to determine the stability of defensins in the filters stored at room temperature. The activity of HNPs 1 to 3 obtained by our rapid protocol was validated by determining minimal inhibitory concentrations (MICs) against six reference bacterial strains and 12 clinical isolates. RESULTS: The human defensins HNPs 1 to 3 extracted from leukoreduction filters displayed high antimicrobial activity against tested strains, with MIC values between 0.12 and 1 µg/mL. Kinetics assays showed the appearance of activity 15 minutes after peptide addition. Moreover, we found that the HNPs 1 to 3 purified from leukoreduction filters that had been stored for 45 days at room temperature remained active. CONCLUSION: Leukoreduction filters provide a rich and safe source of active human defensins HNPs 1 to 3. Moreover, the stability of the peptides in filters stored at room temperature allows envisaging a large-scale development of the process.

Microconductometric immunosensor for label-free and sensitive detection of Gram-negative bacteria.

Blood safety is a global health goal. In developed countries, bacterial contamination of platelet concentrates is the highest infectious risk in transfusion despite the current preventive strategies. We aimed to develop a conductometric biosensor for the generic, rapid and sensitive detection of Gram-negative bacteria. Our strategy is based on immunosensors: addressable magnetic nanoparticles coupled with anti-LPS antibodies were used for the generic capture of Gram-negative bacteria. Bacterial capture was characterized by impedancemetric and microscopic measurements. The results obtained with conductometric measurements allowed real-time, sensitive detection of Escherichia coli or Serratia marcescens cultures from 1 to 10(3) CFU mL(-1). The ability of the immunosensor to detect Gram negative bacteria was also tested on clinically relevant strains. The conductometric immunosensor allowed the direct detection of 10-10(3) CFU mL(-1) of Pseudomonas aeruginosa and Acinetobacter baumannii strains that were undetectable using standard immunoblot methods. Results showed that the conductometric response was not inhibited in 1% serum.

Plasminogen-based capture combined with amplification technology for the detection of PrP(TSE) in the pre-clinical phase of infection.

BACKGROUND: Variant Creutzfeldt-Jakob disease (vCJD) is a neurodegenerative infectious disorder, characterized by a prominent accumulation of pathological isoforms of the prion protein (PrP(TSE)) in the brain and lymphoid tissues. Since the publication in the United Kingdom of four apparent vCJD cases following transfusion of red blood cells and one apparent case following treatment with factor VIII, the presence of vCJD infectivity in the blood seems highly probable. For effective blood testing of vCJD individuals in the preclinical or clinical phase of infection, it is considered necessary that assays detect PrP(TSE) concentrations in the femtomolar range. METHODOLOGY/PRINCIPAL FINDINGS: We have developed a three-step assay that firstly captures PrP(TSE) from infected blood using a plasminogen-coated magnetic-nanobead method prior to its serial amplification via protein misfolding cyclic amplification (PMCA) and specific PrP(TSE) detection by western blot. We achieved a PrP(TSE) capture yield of 95% from scrapie-infected material. We demonstrated the possibility of detecting PrP(TSE) in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease. The test also allowed the detection of PrP(TSE) in human plasma spiked with a 10(-8) dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrP(TSE) in asymptomatic carriers. The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples. CONCLUSION/SIGNIFICANCE: We have developed a sensitive and specific amplification assay allowing the detection of PrP(TSE) in the plasma and buffy coat fractions of blood collected at the pre-clinical phase of the disease. This assay represents a good candidate as a confirmatory assay for the presence of PrP(TSE) in blood of patients displaying positivity in large scale screening tests.

Impact of leucocyte depletion and prion reduction filters on TSE blood borne transmission.

The identification in the UK of 4 v-CJD infected patients thought to be due to the use of transfused Red Blood Cell units prepared from blood of donors incubating v-CJD raised major concerns in transfusion medicine. The demonstration of leucocyte associated infectivity using various animal models of TSE infection led to the implementation of systematic leuco-depletion (LD) of Red Blood cells concentrates (RBCs) in a number of countries. In the same models, plasma also demonstrated a significant level of infectivity which raised questions on the impact of LD on the v-CJD transmission risk. The recent development of filters combining LD and the capture of non-leucocyte associated prion infectivity meant a comparison of the benefits of LD alone versus LD/prion-reduction filters (LD/PR) on blood-borne TSE transmission could be made. Due to the similarity of blood/plasma volumes to human transfusion medicine an experimental TSE sheep model was used to characterize the abilities of whole blood, RBCs, plasma and buffy-coat to transmit the disease through the transfusion route. The impact of a standard RBCs LD filter and of two different RBCs LD/PR prototype filters on the disease transmission was then measured. Homologous recipients transfused with whole-blood, buffy-coat and RBCs developed the disease with 100% efficiency. Conversely, plasma, when intravenously administered resulted in an inconstant infection of the recipients and no disease transmission was observed in sheep that received cryo-precipitated fraction or supernatant obtained from infectious plasma. Despite their high efficacy, LD and LD/PR filtration of the Red Blood Cells concentrate did not provide absolute protection from infection. These results support the view that leuco-depletion strongly mitigates the v-CJD blood borne transmission risk and provide information about the relative benefits of prion reduction filters.

Preclinical sporadic Creutzfeldt-Jakob disease in French blood donors: an epidemiologic model-based study.

BACKGROUND: A recent case-control study showed that transfusion recipients were at an increased risk of developing sporadic Creutzfeldt-Jakob disease (sCJD), suggesting that blood donors with silent preclinical sCJD could transmit the sCJD agent. We therefore estimated the annual number of French blood donors expected to have preclinical sCJD at the time of donation. STUDY DESIGN AND METHODS: We developed a mathematical model to estimate the number of blood donors who would subsequently develop sCJD, under various assumptions about how long their blood might be infective before clinical onset. The model used distributions by age group and sex for sCJD cases, blood donor population, French general population, and mortality in the general population. RESULTS: Using 1999 to 2008 data, modeling showed that, each year, a mean of 1.1 (standard deviation [SD], 0.3) donors were within 1 year of sCJD onset at the time of blood donation, 6.9 (SD, 0.5) donors were within 5 years, 18.0 (SD, 0.6) were within 10 years, and 33.4 (SD, 1.1) were within 15 years. CONCLUSION: Few donors are expected to be in the late preclinical stage of sCJD at the time of blood donation. This result and that of the worldwide absence of any epidemic increase in sCJD over the years indicate that this risk of transfusion-transmitted sCJD, if any, is likely to be very low.

Detection of blood-transmissible agents: can screening be miniaturized?

Transfusion safety relating to blood-transmissible agents is a major public health concern, particularly when faced with the continuing emergence of new infectious agents. These include new viruses appearing alongside other known reemerging viruses (West Nile virus, Chikungunya) as well as new strains of bacteria and parasites (Plasmodium falciparum, Trypanosoma cruzi) and finally pathologic prion protein (variant Creutzfeldt-Jakob disease). Genomic mutations of known viruses (hepatitis B virus, hepatitis C virus, human immunodeficiency virus) can also be at the origin of variants susceptible to escaping detection by diagnostic tests. New technologies that would allow the simultaneous detection of several blood-transmissible agents are now needed for the development and improvement of screening strategies. DNA microarrays have been developed for use in immunohematology laboratories for blood group genotyping. Their application in the detection of infectious agents, however, has been hindered by additional technological hurdles. For instance, the variability among and within genomes of interest complicate target amplification and multiplex analysis. Advances in biosensor technologies based on alternative detection strategies have offered new perspectives on pathogen detection; however, whether they are adaptable to diagnostic applications testing biologic fluids is under debate. Elsewhere, current nanotechnologies now offer new tools to improve the sample preparation, target capture, and detection steps. Second-generation devices combining micro- and nanotechnologies have brought us one step closer to the potential development of innovative and multiplexed approaches applicable to the screening of blood for transmissible agents.

Nanotechnologies for pathogen detection: Future alternatives?

The development of multiplex and flexible tests allowing the simultaneous analysis of pathogens presenting a transfusional risk is a real challenge. Current miniaturized platforms have been particularly marked by microarrays. These microsystems allow the optical detection of hundreds of individual targets simultaneously. However, they suffer from a low sensitivity and their combination with a preliminary target amplification step such as PCR is necessary. The variable level of expression of the infectious genomes of interest and their large diversity complicate multiplex amplification. Finally simultaneous analysis of multiple blood-transmitted agents poses numerous difficulties in diagnosis that remain unresolved by currently available technologies. Until recently, scientific and technological advances for pathogen detection have focused on target amplification and optical detection steps. Today, sample preparation is recognized as a critical area to improve. Nanotechnologies can reach the single-cell or molecular scale and consequently overcome several current technological obstacles. They offer new technological tools for improving sample preparation but also for avoiding target amplification and the current fluorescent labeling. The combination of nano-objects and nano-systems in current technologies offers new possibilities for potential applications in the detection of infectious agents.

Feasibility study of a screening assay that identifies the abnormal prion protein PrPTSE in plasma: initial results with 20,000 samples.

BACKGROUND: It is likely that transmission of variant Creutzfeldt-Jakob disease (vCJD) occurs by transfusion and that the candidate infectious agent (PrP(TSE)) is present in small concentrations in the blood of infected donors in the asymptomatic phase of the disease. A new blood screening assay has been developed to detect PrP(TSE) in citrated plasma samples. STUDY DESIGN AND METHODS: Three regional Blood Transfusion Establishments (ETS) in France (ETS Alsace, ETS Bourgogne Franche-Comté, and ETS Pyrénées-Méditerranée) will screen 60,000 plasma samples (20,000 in each ETS) over a time period of approximately 9 to 12 months. RESULTS: Results provided in this report are those of the first testing site in Strasbourg, Alsace. The preliminary results have demonstrated an initial specificity of 97.60%. Upon repeat testing the specificity rate achieved 99.90% (20 repeat-positive samples). Based on the known epidemiology of vCJD in France, it is likely that the repeat-reactive samples are not true-positives. CONCLUSION: The screening assay was studied in terms of specificity and practicality and was found to be suitable for use in routine testing of blood donations. However, throughput must be enhanced by automation of the assay, and traceability would be improved if automated systems were used to distribute and identify samples.

Prion protein expression and processing in human mononuclear cells: the impact of the codon 129 prion gene polymorphism.

BACKGROUND: So far, all clinical cases of new variant Creutzfeldt-Jakob disease (vCJD), thought to result from the Bovine Spongiform Encephalopathy (BSE) prion agent, have shown Methionine-Methionine (M/M) homozygosity at the M129V polymorphism of the PRNP gene. Although established, this relationship is still not understood. In both vCJD and experimental BSE models prion agents do reach the bloodstream, raising concerns regarding disease transmission through blood transfusion. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the impact of the M129V polymorphism on the expression and processing of the prion protein in human peripheral blood mononuclear cells (PBMCs) from three blood donor populations with Methionine-Methionine (M/M), Valine-Valine (V/V) and M/V genotypes. Using real-time PCR, ELISA and immunoblot assays we were unable to find differences in prion protein expression and processing relating to the M129V polymorphism. CONCLUSIONS/SIGNIFICANCE: These results suggest that in PBMCs, the M129V PrP polymorphism has no significant impact on PrP expression, processing and the apparent glycoform distribution. Prion propagation should be investigated further in other cell types or tissues.

Serum-derived hepatitis C virus infection of primary human hepatocytes is tetraspanin CD81 dependent.

Hepatitis C virus-positive serum (HCVser, genotypes 1a to 3a) or HCV cell culture (JFH1/HCVcc) infection of primary normal human hepatocytes was assessed by measuring intracellular HCV RNA strands. Anti-CD81 antibodies and siRNA-CD81 silencing markedly inhibited (>90%) HCVser infection irrespective of HCV genotype, viral load, or liver donor, while hCD81-large intracellular loop (LEL) had no effect. However, JFH1/HCVcc infection of hepatocytes was modestly inhibited (40 to 60%) by both hCD81-LEL and anti-CD81 antibodies. In conclusion, CD81 is involved in HCVser infection of human hepatocytes, and comparative studies of HCVser versus JFH1/HCVcc infection of human hepatocytes and Huh-7.5 cells revealed that the cell-virion combination is determinant of the entry process.

Identification of apolipoprotein C-III as a potential plasmatic biomarker associated with the resolution of hepatitis C virus infection.

Understanding the virus-host interactions that lead to approximately 20% of patients with acute Hepatitis C Virus (HCV) infection to viral clearance is probably a key towards the development of more effective treatment and prevention strategies. Acute hepatitis C infection is usually asymptomatic and therefore rarely diagnosed. Nevertheless, HCV nucleic acid testing carried out on all blood donations detects donors who have resolved their HCV infection after seroconversion. Here we have used SELDI-TOF-MS technology to compare, at a proteomic level, plasma samples respectively from donors with HCV clearance, from donors with chronic HCV infection and from unexposed healthy donors (n = 15 per group). A candidate marker of about 9.4 kDa was detected as differentially expressed in the three groups. After purification we identified by nanoLC-Q-TOF-MS/MS this candidate marker as Apolipoprotein C-III (ApoC-III). The identification was confirmed by western blot analysis. Levels of ApoC-III were then determined in the 45 plasma samples by immunoturbidimetric assay. ApoC-III was found to be higher in donors who had resolved their HCV infection than in donors with chronic infection, results which were consistent with SELDI-TOF-MS data. ApoC-III is the first reported candidate biomarker in plasma associated with the spontaneous resolution of HCV infection.

Label-free detection of bacteria by electrochemical impedance spectroscopy: comparison to surface plasmon resonance.

The low but known risk of bacterial contamination has emerged as the greatest residual threat of transfusion-transmitted diseases. Label-free detection of a bacterial model, Escherichia coli, is performed using nonfaradic electrochemical impedance spectroscopy (EIS). Biotinylated polyclonal anti-E. coli is linked to a mixed self-assembled monolayer (SAM) on a gold electrode through a strong biotin-neutravidin interaction. The binding of one antibody molecule for 3.6 neutravidin molecules is determined using the surface plasmon resonance (SPR). The detection limit of E. coli found by SPR is 10(7) cfu/mL. After modeling the impedance Nyquist plot of E. coli/anti-E. coli/mixed SAM/gold electrode for increasing concentrations of E. coli (whole bacteria or lysed bacteria), the main parameter that is modified is the polarization resistance RP. A sigmoid variation of RP is observed when the log concentration of bacteria (whole or lysed) increases. A concentration of 10 cfu/mL whole bacteria is detected by EIS measurements while 103 cfu/mL is detected for lysed E. coli.

The low-density lipoprotein receptor plays a role in the infection of primary human hepatocytes by hepatitis C virus.

BACKGROUND/AIMS: The direct implication of low-density lipoprotein receptor (LDLR) in hepatitis C virus (HCV) infection of human hepatocyte has not been demonstrated. Normal primary human hepatocytes infected by serum HCV were used to document this point. METHODS: Expression and activity of LDLR were assessed by RT-PCR and LDL entry, in the absence or presence of squalestatin or 25-hydroxycholesterol that up- or down-regulates LDLR expression, respectively. Infection was performed in the absence or presence of LDL, HDL, recombinant soluble LDLR peptides encompassing full-length (r-shLDLR4-292) or truncated (r-shLDLR4-166) LDL-binding domain, monoclonal antibodies against r-shLDLR4-292, squalestatin or 25-hydroxycholesterol. Intracellular amounts of replicative and genomic HCV RNA strands used as end point of infection were assessed by RT-PCR. RESULTS: r-shLDLR4-292, antibodies against r-shLDLR4-292 and LDL inhibited viral RNA accumulation, irrespective of genotype, viral load or liver donor. Inhibition was greatest when r-shLDLR4-292 was present at the time of inoculation and gradually decreased as the delay between inoculation and r-shLDLR4-292 treatment increased. In hepatocytes pre-treated with squalestatin or 25-hydroxycholesterol before infection, viral RNA accumulation increased or decreased in parallel with LDLR mRNA expression and LDL entry. CONCLUSIONS: LDLR is involved at an early stage in infection of normal human hepatocytes by serum-derived HCV virions.

Evaluation of the enhanced bacterial detection system for screening of contaminated platelets.

BACKGROUND: The Pall third-generation enhanced bacterial detection system (eBDS) was recently approved for detection of bacterial contamination in leukoreduced platelets (PLTs). The method is based on the measurement of the oxygen content as a marker for bacteria. eBDS incorporates major modifications including removal of the sample-set filter, modification of the culture medium, and incubation with agitation of the sample pouch. STUDY DESIGN AND METHODS: Ten whole blood-derived random-donor PLT units collected on Day 1 after donation and 10 single-donor apheresis PLT units were spiked with low levels of bacteria in three different blood transfusion centers. Inoculation was performed at a final concentration of 5 to 50 colony-forming units per mL with reference strains of five organisms involved in severe transfusion-associated infections. PLT units were stored at 22 degrees C for 24 hours before sampling. Six sample sets were then sterile-connected to each unit and placed on a horizontal agitator at 35 degrees C for 18 or 24 hours of incubation. RESULTS: No false-positive results were obtained, indicating a 100 percent specificity of the assay. Of 126 spiked sample pouches tested, 61 of 63 (96.82%) and 63 of 63 (100%) were detected positive after 18 or 24 hours of incubation, respectively. In the two missed cases that failed to detect Bacillus cereus, the measured oxygen was slightly above the detection threshold but was markedly different from the negative samples. CONCLUSION: The eBDS method allows definitive testing of PLTs as soon as 42 hours after collection and offers an alternative culture method to the BacT/ALERT system.

The seroepidemiology of human T-lymphotropic viruses: types I and II in Europe: a prospective study of pregnant women.

BACKGROUND: Up to 20 million persons are infected with the human retroviruses human T-lymphotropic virus (HTLV)-I and HTLV-II globally. Most data on the seroprevalence of HTLV-I and HTLV-II in Europe are from studies of low-risk blood donors or high-risk injection drug users (IDUs). Little is known about the general population. METHODS: A prospective anonymous study of HTLV-I and HTLV-II seroprevalence among 234,078 pregnant women in Belgium, France, Germany, Italy, Portugal, Spain, and the United Kingdom was conducted. Maternal antibody status was determined by standard methods using sera obtained for routine antenatal infection screens or eluted from infant heel prick dried blood spots obtained for routine neonatal metabolic screens. RESULTS: Anti-HTLV-I/II antibodies were detected and confirmed in 96 pregnant women (4.4 per 10,000, 95% confidence interval [CI]: 3.5-5.2). Of these, 73 were anti-HTLV-I, 17 were anti-HTLV-II, and 6 were specifically anti-HTLV but untyped. The seroprevalence ranged from 0.7 per 10,000 in Germany to 11.5 per 10,000 in France. CONCLUSIONS: Pregnant women better reflect the general population than blood donors or IDUs. The seroprevalence of HTLV-I and HTLV-II in Western Europe is 6-fold higher among pregnant women (4.4 per 10,000) than among blood donors (0.07 per 10,000). These data provide a robust baseline against which changes in HTLV-I and HTLV-II seroprevalence in Europe can be measured.

Use of serial analysis of gene expression (SAGE) technology reveals new granulocytic markers.

Because of its short life span in blood, its low RNA content and its highly condensed nucleus, the granulocyte was initially considered as a terminally differentiated cell unable to express novel genes. However, mature granulocytes still contain a variety of mRNAs and may respond to external stimuli by rapid and complex changes in gene expression. The present work was undertaken to provide a wider view of the gene expression profile in unstimulated circulating PMNs. We used serial analysis of gene expression (SAGE) adapted to downsized extracts (SADE) to cope with their small mRNA content. As cell samples of the highest degree of purity were crucial for this project, we adapted a magnetic cell sorting method to reach the required high levels of purity (99.55 +/- 0.19%) together with low activation rates (1.37 +/- 0.28). We analyzed 20,787 tags identifying 8547 different transcripts, of which 47.8% were unknown, 34.6% were transcripts of known genes, and 13.8% matched with expressed sequence tags (EST). Highly expressed genes were those involved in cell mobility, diapedesis, cell signaling and destruction of micro-organisms. In addition, this method led to the identification of genes which had not previously been reported in granulocytes. These results could provide new molecular markers and a reliable reference for the investigation of pathologies involving alteration of the granulocyte gene expression profile.