A systematic electron microscopic study on the uptake of barium sulphate nano-, submicro-, microparticles by bone marrow-derived phagocytosing cells.

Nanoparticles can act as transporters for synthetic molecules and biomolecules into cells, also in immunology. Antigen-presenting cells like dendritic cells are important targets for immunotherapy in nanomedicine. Therefore, we have used primary murine bone marrow-derived phagocytosing cells (bmPCs), i.e. dendritic cells and macrophages, to study their interaction with spherical barium sulphate particles of different size (40 nm, 420 nm, and 1 µm) and to follow their uptake pathway. Barium sulphate is chemically and biologically inert (no dissolution, no catalytic effects), i.e. we can separate the particle uptake effect from potential biological reactions. The colloidal stabilization of the nanoparticles was achieved by a layer of carboxymethylcellulose (CMC) which is biologically inert and gives the particles a negative zeta potential (i.e. charge). The particles were made fluorescent by conjugating 6-aminofluoresceine to CMC. Their uptake was visualized by flow cytometry, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and correlative light and electron microscopy (CLEM). Barium sulphate particles of all sizes were readily taken up by dendritic cells and even more by macrophages, with the uptake increasing with time and particle concentration. They were mainly localized inside phagosomes, heterophagosomes, and in the case of nanoparticles also in the nearby cytosol. No particles were found in the nucleus. In nanomedicine, inorganic nanoparticles from the nanometer to the micrometer size are therefore well suited as transporters of biomolecules, including antigens, into dendritic cells and macrophages. The presented model system may also serve to describe the aseptic loosening of endoprostheses caused by abrasive wear of inert particles and the subsequent cell reaction, a question which relates to the field of nanotoxicology. STATEMENT OF SIGNIFICANCE: The interaction of particles and cells is at the heart of nanomedicine and nanotoxicology, including abrasive wear from endoprostheses. It also comprises the immunological reaction to different kinds of nanomaterials, triggered by an immune response, e.g. by antigen-presenting cells. However, it is often difficult to separate the particle effect from a chemical or biochemical reaction to particles or their cargo. We show how chemically inert barium sulphate particles with three different sizes (nano, sub-micro, and micro) interact with relevant immune cells (primary dendritic cells and macrophages). Particles of all three sizes are readily taken up into both cell types by phagocytosis, but the uptake by macrophages is significantly more prominent than that by dendritic cells. The cells take up particles until they are virtually stuffed, but without direct adverse effect. The uptake increases with time and particle concentration. Thus, we have an ideal model system to follow particles into and inside cells without the side effect of a chemical particle effect, e.g. by degradation or ion release.

DNA methylation in the pathophysiology of hyperphenylalaninemia in the PAH(enu2) mouse model of phenylketonuria.

Phenylalanine hydroxylase deficient phenylketonuria (PKU) is the paradigm for a treatable inborn error of metabolism where maintaining plasma phenylalanine (Phe) in the therapeutic range relates to improved clinical outcomes. While Phe is the presumed intoxicating analyte causal in neurologic damage, the mechanism(s) of Phe toxicity has remained elusive. Altered DNA methylation is a recognized response associated with exposure to numerous small molecule toxic agents. Paralleling this effect, we hypothesized that chronic Phe over-exposure in the brain would lead to aberrant DNA methylation with secondary influence upon gene regulation that would ultimately contribute to PKU neuropathology. The PAH(enu2) mouse models human PKU with intrinsic hyperphenylalaninemia, abnormal response to Phe challenge, and neurologic deficit. To examine this hypothesis, we assessed DNA methylation patterns in brain tissues using methylated DNA immunoprecipitation and paired end sequencing in adult PAH(enu2) animals maintained under either continuous dietary Phe restriction or chronic hyperphenylalaninemia. Heterozygous PAH(enu2/WT) litter mates served as controls for normal Phe exposure. Extensive repatterning of DNA methylation was observed in brain tissue of hyperphenylalaninemic animals while Phe restricted animals displayed an attenuated pattern of aberrant DNA methylation. Affected gene coding regions displayed aberrant hypermethylation and hypomethylation. Gene body methylation of noncoding RNA genes was observed and among these microRNA genes were prominent. Of particular note, observed only in hyperphenylalaninemic animals, was hypomethylation of miRNA genes within the imprinted Dlk1-Dio3 locus on chromosome 12. Aberrant methylation of microRNA genes influenced their expression which has secondary effects upon the expression of targeted protein coding genes. Differential hypermethylation of gene promoters was exclusive to hyperphenylalaninemic PAH(enu2) animals. Genes with synaptic involvement were targets of promoter hypermethylation that resulted in down-regulation of their expression. Gene dysregulation secondary to abnormal DNA methylation may be contributing to PKU neuropathology. These results suggest drugs that prevent or correct aberrant DNA methylation may offer a novel therapeutic option to management of neurological symptoms in PKU patients.

Methylome repatterning in a mouse model of Maternal PKU Syndrome.

Maternal PKU Syndrome (MPKU) is an embryopathy resulting from in utero phenylalanine (PHE) toxicity secondary to maternal phenylalanine hydroxylase deficient phenylketonuria (PKU). Clinical phenotypes in MPKU include mental retardation, microcephaly, in utero growth restriction, and congenital heart defects. Numerous in utero toxic exposures alter DNA methylation in the fetus. The PAH(enu2) mouse is a model of classical PKU while offspring born of hyperphenylalaninemic dams model MPKU. We investigated offspring of PAH(enu2) dams to determine if altered patterns of DNA methylation occurred in response to in utero PHE exposure. As neurologic deficit is the most prominent MPKU phenotype, methylome patterns were assessed in brain tissue using methylated DNA immunoprecipitation and paired-end sequencing. Brain tissues were assessed in E18.5-19 fetuses of PHE unrestricted PAH(enu2) dams, PHE restricted PAH(enu2) dams, and heterozygous(wt/enu2) control dams. Extensive methylome repatterning was observed in offspring of hyperphenylalaninemic dams while the offspring of PHE restricted dams displayed attenuated methylome repatterning. Methylation within coding regions was dominated by noncoding RNA genes. Differential methylation of promoters targeted protein coding genes. To assess the impact of methylome repatterning on gene expression, brain tissue in experimental and control animals were queried with microarrays assessing expression of microRNAs and protein coding genes. Altered expression of methylome-modified microRNAs and protein coding genes was extensive in offspring of hyperphenylalaninemic dams while minimal changes were observed in offspring of PHE restricted dams. Several genes displaying significantly reduced expression have roles in neurological function or genetic disease with neurological phenotypes. These data indicate in utero PHE toxicity alters DNA methylation in the brain which has downstream impact upon gene expression. Altered gene expression may contribute to pathophysiology of neurologic presentation in MPKU.

Altered global gene expression in first trimester placentas of women destined to develop preeclampsia.

BACKGROUND: Preeclampsia is a pregnancy-specific disorder that remains a leading cause of maternal, fetal and neonatal morbidity and mortality, and is associated with risk for future cardiovascular disease. There are no reliable predictors, specific preventative measures or treatments other than delivery. A widely held view is that the antecedents of preeclampsia lie with impaired placentation in early pregnancy. Accordingly, we hypothesized dysregulation of global gene expression in first trimester placentas of women who later manifested preeclampsia. METHODS: Surplus chorionic villus sampling (CVS) tissues were collected at 10-12 weeks gestation in 160 patients with singleton fetuses. Four patients developed preeclampsia, and their banked CVS specimens were matched to 8 control samples from patients with unaffected pregnancies. Affymetrix HG-U133 Plus 2.0 GeneChips were utilized for microarray analysis. Naïve Bayes prediction modeling and pathway analysis were conducted. qRT-PCR examined three of the dysregulated genes. RESULTS: Thirty-six differentially expressed genes were identified in the preeclampsia placentas. qRT-PCR verified the microarray analysis. Thirty-one genes were down-regulated. Many were related to inflammation/immunoregulation and cell motility. Decidual gene dysregulation was prominent. No evidence was found for alterations in hypoxia and oxidative stress regulated genes. CONCLUSIONS: To our knowledge, this is the first study to show dysregulation of gene expression in the early placentas of women approximately 6 months before developing preeclampsia, thereby reinforcing a placental origin of the disorder. We hypothesize that placentation in preeclampsia is compromised in the first trimester by maternal and fetal immune dysregulation, abnormal decidualization, or both, thereby impairing trophoblast invasion. Several of the genes provide potential targets for the development of clinical biomarkers in maternal blood during the first trimester. Supplementary materials are available for this article via the publisher's online edition.

Exercise-induced hypersensitivity syndromes in recreational and competitive athletes: a PRACTALL consensus report (what the general practitioner should know about sports and allergy).

Exercise-induced (EI) hypersensitivity disorders are significant problems for both recreational and competitive athletes. These include EI-asthma, EI-bronchoconstriction, EI-rhinitis, EI-anaphylaxis and EI-urticaria. A group of experts from the European Academy of Allergology and Clinical Immunology and the American Academy of Allergy Asthma and Immunology met to discuss the pathogenesis of these disorders and how to diagnose and treat them, and then to develop a consensus report. Key words (exercise with asthma, bronchoconstriction, rhinitis, urticaria or anaphylaxis) were used to search Medline, the Cochrane database and related websites through February 2008 to obtain pertinent information which, along with personal reference databases and institutional experience with these disorders, were used to develop this report. The goal is to provide physicians with guidance in the diagnosis, understanding and management of EI-hypersensitivity disorders to enable their patients to safely return to exercise-related activities.

Anti-miRNA oligonucleotides (AMOs): ammunition to target miRNAs implicated in human disease?

MicroRNAs (miRNAs) are endogenous 19-25 nucleotide RNAs that have recently emerged as a novel class of important gene-regulatory molecules involved in many critical developmental and cellular functions. miRNAs have been implicated in the pathogenesis of several human diseases, such as neurodegenerative disorders, cancer, and more recently in viral and metabolic diseases. Unraveling the roles of miRNAs in cellular processes linked to human diseases will lead to novel opportunities for the regulation of protein function and will help to evaluate their potential for therapeutic intervention. Approaches to interfere with miRNA function in vitro and in vivo based on synthetic anti-miRNA oligonucleotides (AMOs) are discussed in this review.

Microbial risk indicators of early childhood caries.

The aim of this study was to use molecular identification methods, such as 16S RNA gene sequence and reverse-capture checkerboard hybridization, for identification of the bacteria associated with dental caries and with dental health in a subset of 204 twins aged 1.5 to 7 years old. A total of 448 plaque samples (118 collected from caries-free subjects and 330 from caries-active subjects) were used for analysis. We compared the bacteria found in biofilms of children exhibiting severe dental caries, with different degrees of lesion severity, with those found in biofilms of caries-free children. A panel of 82 bacterial species was selected, and a PCR-based reverse-capture checkerboard method was used for detection. A simple univariate test was used to determine the overabundance and underabundance of bacterial species in the diseased and in the healthy groups. Features identified with this univariate test were used to construct a probabilistic disease prediction model. Furthermore, a method for the analysis of global patterns of gene expression was performed to permit simultaneous analysis of the abundance of significant species by allowing cross-bacterial comparisons of abundance profiles between caries-active and caries-free subjects. Our results suggested that global patterns of microbial abundance in this population are very distinctive. The top bacterial species found to be overabundant in the caries-active group were Actinomyces sp. strain B19SC, Streptococcus mutans, and Lactobacillus spp., which exhibited an inverse relationship to beneficial bacterial species, such as Streptococcus parasanguinis, Abiotrophia defectiva, Streptococcus mitis, Streptococcus oralis, and Streptococcus sanguinis.

Inhibition of CD40-mediated endothelial cell activation with antisense oligonucleotides.

BACKGROUND: CD40-CD154 interactions play a pivotal role in the amplification of immune responses and, as such, represent an attractive target for immune intervention in a number of disease indications. We have previously shown that binding of human CD154 expressed on the Jurkat D1.1 cell line to porcine CD40 on pig aortic endothelial cells (PAECs) can lead to up-regulation of vascular cell adhesion molecule (VCAM)-1 and MHC class II. This activation can be completely inhibited by the addition of a monoclonal antibody (mAb) to human CD154. In this study, we explore an alternative approach to blocking this pathway with antisense oligonucleotides (ASOs). METHODS: Ten ASOs were generated on the basis of the porcine CD40 cDNA sequence. The ASOs that were found to reduce CD40 expression on PAECs were analyzed for their ability to reduce CD40-mediated PAEC activation. RESULTS: Four ASOs were found to significantly lower surface expression of porcine CD40 on PAECs 48 hr after transfection. Eight of the ASOs were seen to lead to mRNA cleavage products by ribonuclease protection assay. Of the four ASOs tested in the PAEC activation assay, one (ASO-9) showed a dramatic inhibition of PAEC activation (IC50 approximately 1 nM) results comparable to the use of a blocking mAb. Furthermore, we compared the effect of CD40 ASO on tumor necrosis factor alpha receptor signaling, in which we observed no effect, which confirmed ASO specificity. CONCLUSIONS: These results indicate that a CD40-dependent activation pathway can be inhibited with an ASO with high potency and specificity. ASO could be an attractive alternative therapy to the use of mAbs.

New approaches towards fluorescence labelling of messenger RNA transcripts.

Fluorescence labelling of nucleic acids is being used for a wide range of biological applications. The performance of these techniques is dependent on fluorochrome labels with a high sensitivity and high resistance to photobleaching. Indo-cyanine dyes such as Cy3, Cy5 or Cy7 have been found to fulfill these requirements. This study describes several different RNA labelling techniques allowing for a Cy5 based detection of mRNA transcripts.

A report of a rare immediate reaction after ingestion of acetaminophen.

BACKGROUND: Acetaminophen hypersensitivity is rare and, when seen, is usually in association with sensitivity to nonsteroidal anti-inflammatory drugs. METHODS: This is a case report of an immediate reaction to acetaminophen, confirmed by a drug challenge, in a subject who tolerated ibuprofen. RESULTS: After an oral challenge with 50 mg of acetaminophen, the subject had generalized pruritus, urticaria, and dyspnea. CONCLUSIONS: This patient demonstrates a rare but potentially severe reaction to acetaminophen that may occur in patients who are not otherwise sensitive to other nonsteroidal anti-inflammatory drugs.

Expression of C1 esterase inhibitor by the baculovirus expression vector system: preparation, purification, and characterization.

C1 esterase inhibitor (C1INH) is an important regulator of the classical complement pathway. Hereditary deficiency of C1INH causes angioedema of the skin, gut, and respiratory tissues that may be fatal. C1INH replacement therapy may be lifesaving for patients with this disorder. The objective of this study was to evaluate the use of the baculovirus expression vector system for mass producing biologically active human recombinant (rC1INH). A recombinant baculovirus was constructed coding the human native (nC1INH) sequence under control of the polyhedrin promoter. Spodoptera frugiperda Sf-9 insect cells were infected with this recombinant baculovirus in a medium-scale (10-L) bioreactor to produce rC1INH with a specific activity of 45 U/mg. Purification of rC1INH from the culture harvested at 60 h postinfection yielded 5.9 microg rC1INH/mL supernatant of a 75-kDa product with a specific activity of 31,000 U/mg purified rC1INH compared to 71,000 U/mg purified nC1INH from human serum using the same procedure. This rC1INH was about 25 kDa smaller than nC1INH, suggesting that Sf-9 cells express underglycosylated rC1INH. Glycan analysis showed that both N-glycan and O-glycan chains were present in rC1INH. The N-glycan chains, released using PNGaseF and fluorescently labeled, were analyzed using exoglycosidase treatment and capillary electrophoresis. Their high-mannose structure was consistent with the known failure of the insect cell glycosylation pathway to afford the fully elaborated biantennary structures found on human native nC1INH.

Certain high molecular weight heparin chains have high affinity for vitronectin.

Vitronectin is a 70-kDa protein that is found in both the extracellular matrix as well as serum. Vitronectin is one of the few proteins that regulates both the complement and the coagulation systems. Heparin is known to bind to vitronectin. Review of the literature reveals apparently conflicting outcomes of the interaction of heparin, vitronectin, and the complement system. Previous studies demonstrated that heparin diminishes vitronectin inhibition of complement activity. Numerous studies have also demonstrated that heparin exerts a net inhibitory effect on complement. We used two dimensional affinity resolution electrophoresis (2DARE) to examine this apparent paradox. 2DARE allowed simultaneous determination of binding affinity of heparin for vitronectin as well as the M(r) of the heparin species. In the 2DARE experiment, the interaction of heparin with vitronectin caused retardation of the movement of the heparin through the tube gel in the first dimension. The degree of the retardation of movement was used to calculate the approximate K(d) of that interaction. The heparin from the tube gel was then subjected to a second dimension electrophoresis to determine the M(r) of the heparin. 2DARE analysis of the interaction of heparin with vitronectin clearly demonstrated that a sub-population of heparin chains with M(r) > 8000 bound vitronectin with high affinity whereas most high M(r) chains and all lower M(r) chains showed little to no affinity for vitronectin. Our findings are consistent with the hypothesis that a unique binding domain exists in certain heparin chains for vitronectin.

Specific inhibition of the rat ligand-gated ion channel P2X3 function via methoxyethoxy-modified phosphorothioated antisense oligonucleotides.

P2X3 is one receptor of a family of seven ligand-gated ion channels responding to purines. Increasing evidence indicates its involvement in neuronal signaling and in pain. However, there is currently no selective inhibitor known for this subtype. In order to obtain such a specific inhibitor, a variety of antisense oligonucleotides (ASO) against rat P2X3 was tested, and dose-dependent, sequence-specific downregulation of the rat P2X3 receptor (expressed in a Chinese hamster ovary cell line [CHO-K1]) on the mRNA, protein, and functional levels was observed. Using real-time quantitative PCR, a dose-dependent downregulation of P2X3 mRNA by ASO, as compared with untreated and mismatch controls, was demonstrated. Subsequently, downregulation by the two most potent ASO was confirmed at the protein level by Western blot. Sequence specificity was shown by titration of mismatches to the original selected oligonucleotide, and this correlated with progressive loss of P2X3 inhibition. The functional response of the P2X3 receptor was examined using whole-cell voltage clamping. Upon application of 10 microM of a nonspecific agonist, alpha,beta-methylene-ATP (alphabeta meATP), pretreatment with increasing amounts of the most active ASO 5037 correlated with a decrease in depolarization. The ability to specifically downregulate the P2X3 receptor by ASO treatment will allow investigation of the biologic role of this receptor in neuronal tissues and eventually in in vivo models of chronic pain.

A review of the reported defects in the human C1 esterase inhibitor gene producing hereditary angioedema including four new mutations.

C1 esterase inhibitor (C1INH) is an important regulatory protein of the classical pathway of complement. Mutations in the gene for this protein cause the autosomal dominant disorder hereditary angioedema (HAE). Approximately 85% of patients with HAE have a Type I defect, characterized by a diminished level of antigenic and functional C1INH. Patients with Type II defects have sufficient protein, but one allele produces dysfunctional protein. We have sequenced the DNA from HAE patients and have discovered four previously unreported mutations. The first mutation is a splice site error at nucleotide 8721, which changes the 3' acceptor splice site AG to GG at the end of intron 5 at nucleotide 8721-8722. The second mutation is a single base insertion in exon 3 between nucleotides 2467 and 2468. The third mutation is a missense error present in the eighth exon of the C1INH; at nucleotide 16867 (amino acid 470), a T to A mutation transforms a Met to a Lys. The fourth mutation closely resembles the third mutation in that it is a missense error occurring in exon 8 in the distal hinge region; a T16827C substitution changes the Phe at amino acid 457 to Leu. This report compiles a list of 97 distinct defects in the C1INH gene that cause hereditary angioedema.

Independent and combined analyses of sequences from all three genomic compartments converge on the root of flowering plant phylogeny.

Plant phylogenetic estimates are most likely to be reliable when congruent evidence is obtained independently from the mitochondrial, plastid, and nuclear genomes with all methods of analysis. Here, results are presented from separate and combined genomic analyses of new and previously published data, including six and nine genes (8, 911 bp and 12,010 bp, respectively) for different subsets of taxa that suggest Amborella + Nymphaeales (water lilies) are the first-branching angiosperm lineage. Before and after tree-independent noise reduction, most individual genomic compartments and methods of analysis estimated the Amborella + Nymphaeales basal topology with high support. Previous phylogenetic estimates placing Amborella alone as the first extant angiosperm branch may have been misled because of a series of specific problems with paralogy, suboptimal outgroups, long-branch taxa, and method dependence. Ancestral character state reconstructions differ between the two topologies and affect inferences about the features of early angiosperms.

Effect of IGFBP-derived peptides on incorporation of(35)SO(4)into proteoglycans.

18 amino acid peptides from the C-terminal region of IGFBP-3, -5 (P3, P5), increased the incorporation of(35)SO(4)into proteoglycans in endothelial cells with greater stimulation in large vessel than microvessel cells. The homologous region of IGFBP-6 (P6) also stimulated sulfate uptake, but less potently than P3 and P5. P6 variants were synthesized with one or two amino acids changed to the basic amino acid in the equivalent position of P3. The P6 variants with one additional basic amino acid behaved similarly to P6. The P6 mutant with two altered amino acids was equipotent to P3. P3F, a scrambled version of P3 was less effective than P3. P3, P5, P6, P3F and all P6 variants all stimulated glucose uptake, which occurred only in microvessel cells. P1, P2, P4, and equimolar intact IGFBP-3 stimulated neither glucose uptake nor sulfate incorporation. Thus, C-terminal basic portions of IGFBP-3, -5 and -6 alter two specific functions of endothelial cells with sufficient differences to suggest mediation by distinct mechanisms.

Asthma in United States olympic athletes who participated in the 1998 olympic winter games.

BACKGROUND: About one of every 5 athletes who participated in the 1996 Summer Olympic Games in Atlanta had a past history of asthma, had symptoms that suggested asthma, or took asthma medications. No previous study has determined the prevalence of asthma in all US athletes who participated in an Olympic Winter Games. OBJECTIVES: We sought to determine how many US athletes who participated in the 1998 Olympic Winter Games had a past history of asthma, had symptoms that suggested asthma, or indicated taking a medication used to treat asthma. METHODS: We evaluated responses to questions that asked about allergic and respiratory diseases in the United States Olympic Committee Medical History Questionnaire that was completed by all 196 athletes who represented the United States at the 1998 Olympic Winter Games in Nagano, Japan. RESULTS: Forty-three (21.9%) of the 196 athletes had a previous diagnosis of asthma, and 36 (18. 4%) recorded use of an asthma medication at some time in the past. Forty-four (22.4%) reported use of an asthma medication, a diagnosis of asthma, or both (our basis for the diagnosis of asthma). Thirty-four (17.4%) of the athletes were currently taking an asthma medication at the time that they completed the questionnaire or indicated that they took these medications on a permanent or semipermanent basis and were considered to have active asthma. Athletes who participated in Nordic combined, cross-country, and short track events had the highest prevalence of having been told that they had asthma or had taken an asthma medication in the past (60.7%) in contrast with only one (2.8%) of the 36 athletes who participated in bobsled, biathlon, luge, and ski jumping. Eighteen (24%) of 75 athletes who participated in alpine, long track, figure skating, snow boarding, and curling had a previous diagnosis of asthma or recorded use of an asthma medication. CONCLUSIONS: We conclude that asthma appeared to have been more common in athletes who participated in the 1998 Winter Games than in athletes who participated in either the 1996 or 1984 Summer Games. Clearly, asthma rates vary widely among sports. This suggests that the environment in which exercise is performed is important in leading to a decrease in the amount of exercise required to trigger asthma and perhaps in causing injury to the airways.

[Quality of whole blood as a result of storage and preparation (inline-leukocyte depletion). Evidence for autologous predeposit]. / Die Qualität von Vollblut in Abhängigkeit von Herstellungsverfahren (Leukozytendepletion) und Lagerungsdauer. Bedeutung für die operationsvorbereitende Eigenblutspende.

AIM OF THE STUDY: Investigation of various laboratory parameters in stored whole blood, with respect to duration of storage and kind of product. METHODS: Whole blood was donated by 12 healthy volunteers using CPDA1 stabilisator. Six units were filtered with the Leukotrap A1-System (PALL Comp., Dreieich, Germany) for leukocyte depletion. The twelve units were stored for 49 days. Several hematological, biochemical and coagulatory parameters were analysed during storage. RESULTS: There was an adequate reduction of lycocytes by filtration (< 3 x 10(6) white cells per unit). ATP decreased during storage to 45% of initial value at the 49th day, without any influence of the kind of preparation. The course of other parameters such as lactate and free haemoglobin (increase), PH-value (decrease), antithrombin III (decrease), prothrombin, protein C, thrombin-antithrombin-complex, alpha-2-Anti-plasmin (decrease or indifferent) did not show any influence of the kind of preparation. Coagulation factors V and VIII decreased in both preparations, which was significantly less pronounced in whole blood with leukocyte depletion. In contrast parameters of activated coagulation such as D-Dimere and fibrinmonomeric did not change during storage after leukocyte reduction but increased at the end of storage time in CPDA1-blood. CONCLUSIONS: Several parameters indicating quality of stored blood were constant in whole blood independent of the kind of preparation during a storage of 49 days. This is in contrast to the main part of specific scientific communications. A beneficial influence of leukocyte depletion was observed for some coagulation parameters whereas increasing characteristics of activated coagulation in CPDA1-stored whole blood at the end of storage time had to be observed. The preparation of whole CPDA1-blood is recommended for autologous predonation, if storage time does not exceed 30 days. Storage time of > 40 days seems to be possible for autologous whole blood after filtration for leucocyte depletion.