Inhibition of collagen-induced platelet aggregation by anopheline antiplatelet protein, a saliva protein from a malaria vector mosquito

During blood feeding, mosquitoes inject saliva containing a mixture of molecules that inactivate or inhibit various components of the hemostatic response to the bite injury as well as the inflammatory reactions produced by the bite, to facilitate the ingestion of blood. However, the molecular functions of the individual saliva components remain largely unknown. Here, we describe anopheline antiplatelet protein (AAPP) isolated from the saliva of Anopheles stephensi, a human malaria vector mosquito. AAPP exhibited a strong and specific inhibitory activity toward collagen-induced platelet aggregation. The inhibitory mechanism involves direct binding of AAPP to collagen, which blocks platelet adhesion to collagen and inhibits the subsequent increase in intracellular Ca(2+) concentration ([Ca(2+)]i). The binding of AAPP to collagen effectively blocked platelet adhesion via glycoprotein VI (GPVI) and integrin alpha(2)beta(1). Cell adhesion assay showed that AAPP inhibited the binding of GPVI to collagen type I and III without direct effect on GPVI. Moreover, intravenously administered recombinant AAPP strongly inhibited collagen-induced platelet aggregation ex vivo in rats. In summary, AAPP is a malaria vector mosquito-derived specific antagonist of receptors that mediate the adhesion of platelets to collagen. Our study may provide important insights for elucidating the effects of mosquito blood feeding against host hemostasis.     

Extracts of mosquito salivary gland inhibit tumour necrosis factor alpha release from mast cells

Extracts of salivary glands of the yellow fever mosquito Aedes aegypti inhibit tumour cell-stimulated release of tumour necrosis factor alpha (TNFα) from rat mast cells, but do not inhibit antigen-induced histamine secretion. This inhibitory activity for TNFα is found in salivary glands of female but not in male mosquitoes. This inhibition is not mediated by bacterial contamination (LPS), by calcitonin gene related peptide (CGRP), nerve growth factor (NGF), epidermal growth factor (EGF) or transforming growth factor β (TGFβ). The factor(s) has a molecular weight > 10 kDa and is neutralized by boiling for 10 min or heating at 56°C for 30 min. The modulation of this proinflammatory mediator, TNFα, produced by mast cells in sites of blood feeding may facilitate completion of the blood meal, and as reported for certain vector-transmitted parasites, may enhance infectivity.     

Comparison of common platelet receptors between the chacma baboon (Papio ursinus) and human for use in pre-clinical human-targeted anti-platelet studies

Anti-platelet agents play a central part in the treatment and prevention of acute thrombotic events. Discriminating animal models are needed for the development of novel agents. The chacma baboon has been extensively used as a model to evaluate anti-platelet agents. However, limited data exist to prove the translatability of this species to humans. We aimed to determine the suitability of the chacma baboon in preclinical human targeted GPIIb/IIIa, GPIbα and P2Y12 studies. Light-transmission platelet aggregometry (LTA), whole blood impedance aggregometry, receptor number quantification and genomic DNA sequencing were performed. Baboon ADP and arachidonic acid-induced LTA aggregation results differed significantly from human values, even at increased concentrations. LTA ristocetin-induced agglutination was comparable between species, but baboon platelets needed twice the concentration of ristocetin to elicit a similar response. Citrated baboon blood had significantly less aggregation than humans when evaluated with impedance aggregometry. However, hirudinised baboon whole blood gave similar aggregation as humans at the same agonist concentrations. GPIIb, GPIIIa and GPIbα numbers were significantly more on the baboon platelets. None of the amino acids deemed vital for receptor function, ligand binding or receptor inhibition, were radically different between the species. However, a conservative change in a calcium-binding region of GPIIb may render the baboon platelets more sensitive to calcium-binding agents. The chacma baboon may be used for the evaluation of human-targeted GPIIb/IIIa-, GPIbα- and P2Y12-inhibiting agents. However, the best anticoagulant, optimal agonist concentrations, increase in receptor number and sequence differences must be considered for any future studies.     

斑须按蚊唾液腺中疟原虫配子体激活因子的活性动态

目的　探讨斑须按蚊生长、发育过程唾液腺中疟原虫配子体激活因子的活性动态。方法　应用体外雄配子体出丝分析方法 ,检测雌性斑须按蚊羽化后、吸血及产卵前后唾液腺中疟原虫配子体激活因子对柏氏疟原虫雄配子体出丝诱导活性的变化。结果　羽化后未吸血组斑须按蚊唾液腺配子体激活因子活性与按蚊生长、发育呈同步变化 ;吸血后未产卵组按蚊唾液腺抽提物的配子体激活因子活性在吸血后显著下降 ,吸血后第 8天恢复到吸血前水平 ;而吸血后产卵组按蚊唾液腺抽提物的配子体激活因子活性在吸血后第 4天恢复到吸血前水平。结论　吸血后斑须按蚊唾液腺配子体激活因子的消长与按蚊产卵相关。     

Molecular and phylogenetic analysis of a novel salivary defensin cDNA from malaria vector Anopheles stephensi

Manipulating the endogenous immune responses of the mosquito such as temporal and spatial expression of antimicrobial peptides may help in the development of a refractory mosquito, unable to transmit malaria. In mosquito several small antimicrobial peptides are activated locally in the midgut and salivary glands upon Plasmodium infection. Anopheles stephensi, the major urban malaria vector in India, has been considered as an important insect model to study vector-parasite interactions; however, so far no reports are available on the antimicrobial peptides from this mosquito species. In the present study, we report identification and molecular characterization of a novel cDNA encoding defensin like peptide, isolated from the salivary gland subtractive hybridization cDNA library of mosquito A. stephensi. Defensin cDNA is 396 base pair long, bearing an open reading frame of 96 amino acids. Deduced amino acid sequence of A. stephensi defensin (Astp_def) contains a signal peptide sequence of 24 amino acids followed by 32-amino acids long putative propeptide domain and a 40-amino acid mature peptide domain carrying 23-amino acid long consensuses sequence signature of insect defensin. Mature peptide of Astp_def carries six conserved cysteine residues, with a predicted molecular weight of 4.20kDa, and isoelectric point of 8.30, characteristic features of cationic defensins. Amino acid sequence similarity and phylogenetic analysis indicated a higher variation in the pre-propeptide region, as compared to the mature defensin peptide, assuring the presence of finely tuned immune responses to counter pathogens.     

One Injection of DsRed Followed by Bites from Transgenic Mosquitoes Producing DsRed in the Saliva Elicits a High Titer of Antibody in Mice

It has been proposed that transgenic mosquitoes can be used as a “flying syringe” for infectious disease control. We succeeded in generating a transgenic (TG) mosquito, Anopheles stephensi, excreting and discharging DsRed in saliva. DsRed was deposited on the membrane where the TG mosquito probed with its proboscis. Repeated feeding by the TG mosquitoes induced anti-DeRed as well as anti-SG antibodies in mice. This indicates that the TG mosquitoes can immunize the animal. Moreover, in this report, we employed a pre-immunization method before exposing mice to the TG mosquitoes. We injected DsRed to mice to prepare memory B cells and exposed the mice to bites by the TG mosquitoes excreting DsRed. The mice produced a higher titer of antibody to DsRed, suggesting that the bites from TG mosquitoes act as a booster and that primary immunization with a vaccine protein and exposure to TG mosquitoes excreting the vaccine protein in the saliva produces a synergistic effect.     

Blockade of the human platelet GPIIb/IIIa receptor by a murine monoclonal antibody Fab fragment (7E3): Potent dose-dependent inhibition of platelet function

Summary The platelet glycoprotein (GP) IIb/IIIa receptor can bind fibrinogen, von Willebrand factor, and other adhesive ligands; this binding is the final common pathway mediating platelet aggregation. The purpose of this study was to evaluate the safety and platelet inhibitory characteristics of the Fab fragment of the murine monoclonal anti-GPIIb/IIIa 7E3 antibody (m7E3 Fab) when administered intravenously as a single bolus dose, as a single and repeat bolus dose, and as a single bolus dose followed by continuous infusions of varying duration. Various dosage regimens of m7E3 Fab were studied in 74 patients with stable angina. Dosage regimens included single doses of m7E3 Fab from 0.1 to 0.3 mg/kg, a single dose of 0.20–0.30 mg/kg, and a repeat dose of 0.05 mg/kg, or a loading dose followed by a continuous infusion of m7E3 Fab for up to 36 hours. To assess the effect of m7E3 Fab on platelet function, quantitative blockade of GPIIb/IIIa receptors, inhibition of ex vivo platelet aggregation, and template bleeding time were measured in all patients. Dose-dependent inhibition of platelet function was evident in response to escalating bolus doses of m7E3 Fab, with maximum inhibition observed at 0.25–0.30 mg/kg body weight; at the 0.30 mg/kg dose, mean (±SE) GPIIb/IIIa receptor blockade was 81±3%, ex vivo platelet aggregation in response to 20 µM ADP was 14±6% of baseline, and the median bleeding time was >20 minutes. Although platelet function gradually recovered following a single bolus injection, platelet inhibition could be sustained by continuous, low-dose infusion of the antibody. Platelet inhibition occurred within minutes, but m7E3 Fab that did not bind to platelets cleared rapidly from circulation. Sixteen percent of the m7E3 Fab-injected subjects exhibited low titer, human anti-murine antibody responses. No significant bleeding or allergic reactions were observed in any patients. One of the 74 patients developed transient thrombocytopenia soon after receiving m7E3 Fab. These studies establish that m7E3 Fab can be administered safely at doses that cause profound inhibition of platelet function.     

Differences in the platelet proaggregatory activity of immune complexes isolated from patients with myocardial infarction or pulmonary cancer

Immune complexes were isolated immediately after the onset of the symptoms of myocardial infarction on an anti-Clq affinity column. The platelet proaggreagatory effects of these immune complexes were compared with those isolated from patients suffering from pulmonary cancer. A markedly increased proaggregatory effect of immune complexes derived from patients with myocardial infarction and changes in the sensitivity of platelets from healthy volunteers to PGI₂ (prostacyclin) and PGD₂ were found. In contrast, immune complexes from patients with pulmonary cancer did not show any significant effect. The antigenic part of immune complexes is probably relevant in the induction of platelet aggregation.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Radiation with reticulation marks the origin of a major malaria vector

Advances in genomics have led to an appreciation that introgression is common, but its evolutionary consequences are poorly understood. In recent species radiations the sharing of genetic variation across porous species boundaries can facilitate adaptation to new environments and generate novel phenotypes, which may contribute to further diversification. Most Anopheles mosquito species that are of major importance as human malaria vectors have evolved within recent and rapid radiations of largely nonvector species. Here, we focus on one of the most medically important yet understudied anopheline radiations, the Afrotropical Anopheles funestus complex (AFC), to investigate the role of introgression in its diversification and the possible link between introgression and vector potential. The AFC comprises at least seven morphologically similar species, yet only An. funestus sensu stricto is a highly efficient malaria vector with a pan-African distribution. Based on de novo genome assemblies and additional whole-genome resequencing, we use phylogenomic and population genomic analyses to establish species relationships. We show that extensive interspecific gene flow involving multiple species pairs has shaped the evolutionary history of the AFC since its diversification. The most recent introgression event involved a massive and asymmetrical movement of genes from a distantly related AFC lineage into An. funestus, an event that predated and plausibly facilitated its subsequent dramatic geographic range expansion across most of tropical Africa. We propose that introgression may be a common mechanism facilitating adaptation to new environments and enhancing vectorial capacity in Anopheles mosquitoes.

Once considered a rare anthropogenic aberration in animals, interspecific hybridization is now recognized to be both taxonomically widespread and pervasive, particularly in rapidly diversifying groups (1–3). Moreover, mounting genome-scale evidence suggests that introgression, the genetic exchange between species through hybridization and backcrossing, is also prevalent and may be consequential for evolution. Examples from fish, birds, mammals, and insects—including Anopheles mosquitoes—have shown that introgressed variation favored by natural selection can facilitate adaptation, enhance fitness, and drive evolutionary innovation and diversification (4–7). It has been postulated that introgressive hybridization is most prevalent in species-rich and rapidly diversifying radiations (2, 3, 8). Introgression in these groups may solely be opportunistic, given the multiplicity of young species in geographic proximity, but the process may also favor adaptive radiation through the generation of completely novel phenotypes (6, 9, 10).There are three to four dozen Anopheles mosquito species that are of major importance as human malaria vectors, and all have evolved within recent and rapid radiations of morphologically cryptic species (informally classified as species complexes) (11, 12). Most members of these species complexes play no or very minor roles in disease transmission. The repeated de novo origin of major malaria vectors across these independent species radiations therefore holds clues about the nature of key evolutionary innovations that confer the ability to transmit disease widely and efficiently. However, most Anopheles species complexes are understudied. This is especially true of the secondary or nonvector species for which genomic resources are lacking, and basic knowledge of distribution, ecology, and behavior is scant.Until now, the single best-studied group has been the Anopheles gambiae complex, composed of at least eight morphologically indistinguishable species that diversified rapidly and recently, likely within the last half-million years (7, 13, 14). Phylogenomic analysis revealed widespread genealogical discordance (7). Some discordance was due to incomplete lineage sorting as a result of both rapid radiation and large effective population sizes (7), but the majority was caused by massive introgression between the main vector species, involving both the autosomes and the centromere-proximal region of the X chromosome. So extensive was its impact that the inferred species branching order was evident in only 2% of the genome—mostly on the distal portion of the X chromosome, which is protected from introgression by a succession of fixed chromosomal inversion differences.One of the most medically important of the understudied Anopheles species complexes is the Afrotropical Anopheles funestus complex (AFC). The AFC comprises at least seven morphologically similar species (15–18), yet only An. funestus sensu stricto (hereafter, An. funestus) is a highly efficient malaria vector, rivaled in importance solely by An. gambiae and its sister species Anopheles coluzzii in the An. gambiae complex (19–22). Comparative genomics of these two complexes may therefore be instructive with regard to malaria vectorial capacity. Both groups diversified in sub-Saharan Africa and may have experienced common geographic, ecoclimatic, and anthropogenic forces that shaped their history. In addition, the primary vector An. funestus broadly shares several characteristics with primary vectors in the An. gambiae complex: a geographic range that encompasses most of tropical Africa (Fig. 1A), high levels of chromosomal inversion polymorphism (23–25), large effective population size, and little population genetic structure across the continent (26, 27). Furthermore, the discovery of two very distantly related mitochondrial DNA (mtDNA) haplotypes (clades 1 and 2) segregating in An. funestus (27) raises the prospect of historical introgression analogous to that documented for An. gambiae, prompting an intriguing question: Can introgression be a source of evolutionary novelty leading to augmented vectoral capacity?Open in a separate windowFig. 1.Distribution and genetic variation in the AFC. Color coding of species is consistent across panels. (A) Location and distribution of sampled species, adapted from ref. 21. Approximate sample locations for An. funestus are indicated by a black star. For full sample information, see SI Appendix, Table S1. (B) Phylogeny of complete mtDNA genomes constructed using BEAST2 indicating divergent clades of An. funestus (red shading) and An. funestus-like (green shading) (see SI Appendix, Fig. S12 for phylogeny with outgroup). (C) Neighbor-joining phylogeny averaged over the complete nuclear genome. (D) Summary evolutionary history displaying three introgression events as inferred by the methods described in the main text. Introgression events shown as green horizontal arrows between pairs of species indicate the majority direction of introgression. Median divergence and introgression times are displayed in millions of years ago (Mya). See SI Appendix, Table S11 for details. An. funestus (Fun), An. funestus-like (Lik), An. longipalpis C (Lon), An. parensis (Par), and An. vaneedeni (Van), An. rivulorum (Riv).Here, we examine the role of introgression in the evolution of the AFC, using recent methods of phylogenetic network reconstruction that allow for divergence and reticulation to be inferred jointly. We use a combination of phylogenomic and population genomic analyses, based on de novo genome assemblies and additional whole genome resequencing, to: 1) establish species relationships, 2) determine the direction, extent, and genomic architecture of introgression across the complex, and 3) assess the role of introgression in the evolution of the primary vector An. funestus. We show that extensive interspecific gene flow involving multiple species pairs has shaped the evolutionary history of the AFC since its diversification ∼216 thousand years ago (Kya). The most recent introgression event ∼13 Kya involved a massive and asymmetrical movement of genes from a distantly related AFC lineage into An. funestus, an event that predated and plausibly facilitated its subsequent dramatic geographic range expansion across most of tropical Africa. We propose that introgression may be a common mechanism facilitating adaptation to new environments and enhancing vectorial capacity in Anopheles mosquitoes.     

Methodology and outcomes of platelet aggregation testing in Australia, New Zealand and the Asia-Pacific region: results of a survey from the Royal College of Pathologists of Australasia Haematology Quality Assurance Program

Platelet aggregometry is widely used to investigate platelet function but its performance is poorly standardized between laboratories. The aim of this work was to document the platelet aggregation methods used in specialist laboratories enrolled in the Haematology Quality Assurance Program of the Royal College of Pathologists of Australasia. A questionnaire requesting many details of methodology was distributed and from the responses, we determined a consensus view. Consensus was defined here as >70% agreement among respondents in answer to a question and this was seen for a number of aspects of the preanalytical, analytical and interpretive phases. However, for many questions there was a wide variation in responses. Sixteen laboratories provided a breakdown of the types of abnormal results typically seen in a 12-month period. In these laboratories a total of 1400 patients were tested and 390 (27%) had abnormal platelet function. Although it was common to diagnose the cause as aspirin or an aspirin-like defect or a release/storage pool disorder, the range of experience was wide and other rare defects were reported. We conclude that whilst there are a number of points of agreement between laboratories in platelet function testing, standardization could be improved.     

应用rDNA ITS2区段基因序列分析对浙江省传疟媒介的鉴定

目的 对浙江省传疟媒介种类进行鉴定，以指导疟疾防治工作。方法 针对媒介按蚊核糖体核酸内转录间隔2（rDNAITS2）区段基因特征，应用PCR基因鉴别技术，对浙江省现场捕捉的按蚊与嗜人按蚊、八代按蚊、中华按蚊、雷氏按蚊实验室标本进行基因鉴别和比较。结果 浙江省现场捕捉的按蚊DNA样本的PCR扩增产物均为250bp，与实验室中华按蚊标本一致。结论 中华按蚊目前仍是浙江省的主要传疟媒介，现场调查未发现嗜人按蚊。     

Deltamethrin-impregnated bednets as an operational tool for malaria control in a hyper-endemic region of Burundi: impact on vector population and malaria morbidity

Within the framework of the National Malaria Control Programme Burundi, impregnated bednets were promoted through health care facilities, schools and local administration in Nyanza Lac district. The decision to buy a bednet was left to the inhabitants and, as a result, coverage rates between 6 and 65% were observed at sub-district level. Three intervention regions were specified based on the intervention start date. From November 1992 until March 1995, bi-monthly parasitological and entomological surveys were carried out in two areas each of Region 1 and Region 2. After introduction of impregnated bednets in Region 1 the proportions of children under 5 with high parasitaemia were reduced by 42 and 53% in the 2 parasitological survey areas, where the average bednet coverages were 55 and 44% respectively. In the survey areas of Region 2 (control) no significant change occurred during the same period. During the second part of the intervention from September 1994, when intervention was also operational in Region 2, significant decreases in the proportion of high parasitaemia (63 and 42%) among children under 5 years were obtained in both parasitological survey areas of Region 2 (average coverages of 51 and 29%). The positive output of the intervention was maintained and even reinforced in the survey areas of Region 1. Bednets as a tool for malaria control entail specific problems such as coverage, daily use, reimpregnation, and renewal of old and torn nets. Further evaluation has to point out the possible shift of the clinical spectrum and the age-specific admission of malaria cases to assess the long-term benefit of this control method.     

Identification of the control region for tissue-specific imprinting of the stimulatory G protein alpha-subunit

Gnas is a complex gene with multiple imprinted promoters. The upstream Nesp and Nespas/Gnasxl promoters are paternally and maternally methylated, respectively. The downstream promoter for the stimulatory G protein alpha-subunit (G(s)alpha) is unmethylated, although in some tissues (e.g., renal proximal tubules), G(s)alpha is poorly expressed from the paternal allele. Just upstream of the G(s)alpha promoter is a primary imprint mark (1A region) where maternal-specific methylation is established during oogenesis. Pseudohypoparathyroidism type 1B, a disorder of renal parathyroid hormone resistance, is associated with loss of 1A methylation. Analysis of embryos of Dnmt3L(-/-) mothers (which cannot methylate maternal imprint marks) showed that Nesp, Nespas/Gnasxl, and 1A imprinting depend on one or more maternal primary imprint marks. We generated mice with deletion of the 1A differentially methylated region. These mice had normal Nesp-Nespas/Gnasxl imprinting, indicating that the Gnas locus contains two independent imprinting domains (Nespas-Nespas/Gnasxl and 1A-G(s)alpha) controlled by distinct maternal primary imprint marks. Paternal, but not maternal, 1A deletion resulted in G(s)alpha overexpression in proximal tubules and evidence for increased parathyroid hormone sensitivity but had no effect on G(s)alpha expression in other tissues where G(s)alpha is normally not imprinted. The 1A region is a maternal imprint mark that contains one or more methylation-sensitive cis-acting elements that suppress G(s)alpha expression from the paternal allele in a tissue-specific manner.     

Identification of a region within the cytoplasmic domain of the subtype B Vpu protein of human immunodeficiency virus type 1 (HIV-1) that is responsible for retention in the golgi complex and its absence in the Vpu protein from a subtype C HIV-1

The structure of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) is composed of a short Nterminal domain (NTD), a transmembrane domain (TM), and a cytoplasmic domain (CD). Previous studies have shown that the Vpu protein from subtype B HIV-1 is transported predominantly to the rough endoplasmic reticulum (RER)/Golgi complex compartments of the cell and is not incorporated into virions. Using a previously described VpuEGFP reporter system in which the Vpu protein was fused to the gene for enhanced green fluorescent protein (EGFP), we showed that the subtype B Vpu fusion protein was localized to the RER/Golgi region of the cell, similar to the native protein. In the present study, we show that fusion of the subtype C Vpu to EGFP results in a fusion protein that is transported to the cell surface. Using this reporter system, chimeric Vpu proteins in which the CD of the subtype B and C proteins were exchanged showed that the CD was sufficient for targeting the subtype B protein to the Golgi complex of the cell. Following identification of the cytoplasmic domain as being responsible for intracellular targeting, we then generated a series of mutants in which 13, 23, 31, 38, 51, and 56 amino acids were deleted from the cytoplasmic domain of subtype B Vpu. These deletion mutants were analyzed by SDS-PAGE for size, for membrane localization, and intracellular localization by confocal fluorescence microscopy. Our results indicate that the mutant with the carboxyl-terminal 13 amino acids deleted was still localized to the Golgi complex but mutants with 23, 31, 38, 51, and 56 amino acids from the carboxyl-terminus of the subtype B Vpu were transported to the cell surface. These results suggest that a signal for the retention of the subtype B Vpu within the Golgi complex resides in the second alpha-helical domain.     

A Leu55 to Pro substitution in the integrin alphaIIb is responsible for a case of Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) is a hereditary bleeding disorder caused by a quantitative or qualitative defect in the integrin alphaIIbbeta3. A new mutation, a T to C substitution at base 258 in the alphaIIb gene, leading to the replacement of Leu55 with Pro, was found by sequence analysis of a patient's alphaIIb cDNA. In transfection experiments using COS7 cells, the cells co-transfected with the mutated alphaIIb cDNA containing C258 and wild-type beta3 cDNA scarcely expressed the alphaIIbbeta3 complex. The Leu55 to Pro substitution in the alphaIIb gene was found to be responsible for this case of Glanzmann's thrombasthenia.     

Identification of miltirone as active ingredient of Salvia miltiorrhiza responsible for the reducing effect of root extracts on alcohol intake in rats

BACKGROUND: Previous work found that extracts from the roots of Salvia miltiorrhiza, a Chinese medicinal herb, reduced alcohol intake in selectively bred Sardinian alcohol-preferring (sP) rats. The present study was designed to evaluate whether miltirone, one of the possible active constituents of S. miltiorrhiza, might be responsible for the reducing effect of the extracts on alcohol intake. METHODS: An initial experiment assessed the effect of 100 mg/kg (intragastric, i.g.) of 4 extracts of S. miltiorrhiza, differing in miltirone content (0, 2, 3, and 7%, respectively), on alcohol intake in alcohol-experienced sP rats exposed to the 2-bottle "alcohol (10%, volume in volume) versus water" choice regimen. Subsequently, the effect of pure miltirone (2.5-10 mg/kg, i.g., i.e., a dose range comparable to its content in the effective doses of the active extracts) on acquisition and maintenance of alcohol-drinking behavior was evaluated in alcohol-naive and alcohol-experienced sP rats exposed to the 2-bottle choice regimen. The effect of miltirone (10 mg/kg, i.g.) on blood alcohol levels was assessed after the i.g. and intraperitoneal (i.p.) administration of alcohol. Finally, the effect of miltirone (30-100 mg/kg, i.g.) on the severity of alcohol withdrawal syndrome was evaluated in Wistar rats made physically dependent on alcohol by the repeated administration of intoxicating doses of alcohol. RESULTS: The reducing effect of 4 different extracts of S. miltiorrhiza on alcohol intake was positively and significantly correlated with their miltirone content. Pure miltirone reduced alcohol intake in alcohol-experienced rats and delayed acquisition of alcohol-drinking behavior in alcohol-naive rats. Similar to S. miltiorrhiza extracts, miltirone markedly reduced blood alcohol levels when alcohol was administered i.g. but not i.p., suggesting that miltirone hampered alcohol absorption from the gastrointestinal system. Finally, miltirone failed to affect the severity of alcohol withdrawal syndrome in alcohol-dependent rats. CONCLUSIONS: The results of the present study suggest that miltirone is the likely active constituent of S. miltiorrhiza responsible for the reducing effect of its extracts on alcohol intake in different experimental models of excessive alcohol consumption.     

Multicentre standardisation of a clinical grade procedure for the preparation of allogeneic platelet concentrates from umbilical cord blood

BackgroundIn addition to a largely prevalent use for bleeding prophylaxis, platelet concentrates from adult blood have also been used for many years to prepare platelet gels for the repair of topical skin ulcers. Platelet gel can be obtained by activation of fresh, cryopreserved, autologous or allogeneic platelet concentrates with calcium gluconate, thrombin and/or batroxobin. The high content of tissue regenerative factors in cord blood platelets and the widespread availability of allogeneic cord blood units generously donated for haematopoietic transplant but unsuitable for this use solely because of low haematopoietic stem cell content prompted us to develop a national programme to standardise the production of allogeneic cryopreserved cord blood platelet concentrates (CBPC) suitable for later preparation of clinical-grade cord blood platelet gel.ResultsDuring 14 months, 13 banks produced 1,080 CBPC with mean (± standard deviation) volume of 11.4±4.4 mL and platelet concentration of 1,003±229×10⁹/L. Total platelet count per CBPC was 11.3±4.9×10⁹. Platelet recovery from cord blood was 47.7±17.8%. About one-third of cord blood units donated for haematopoietic transplant could meet the requirements for preparation of CBPC. The cost of preparation was € 160.92/CBPC. About 2 hours were needed for one technician to prepare four CBPC.DiscussionThis study yielded valuable scientific and operational information regarding the development of clinical trials using allogeneic CBPC.