Platelets are dispensable for antibody-mediated transfusion-related acute lung injury in the mouse.

UNLABELLED: Essentials Role of platelets in immunological transfusion-related acute lung injury (TRALI) is debated. Immunological TRALI was tested in mice exhibiting severe thrombocytopenia or platelet dysfunction. Platelets are required to prevent lung hemorrhage but not edema formation and respiratory distress. Platelets are dispensable for the initiation and development of TRALI. SUMMARY: Background Transfusion-related acute lung injury (TRALI) is a serious transfusion-related complication. Previous conflicting studies have indicated that platelets are either crucial or dispensable for TRALI. Objectives To evaluate the role of platelets in major histocompatibility complex (MHC) I-induced-TRALI. Methods Antibody-mediated TRALI was experimentally induced in mice by lipopolysaccharide priming followed by the administration of an anti-MHC I mAb. Results TRALI was tested in the context of severe thrombocytopenia provoked by the administration of diphtheria toxin (DT) in transgenic iDTR mice selectively expressing DT receptor in megakaryocytes. The pathologic responses occurring within the first 10 min following the injection of the anti-MHC I mAb, i.e. the severity of lung edema and the drop in aortic blood oxygenation, were similar in severely thrombocytopenic DT-iDTR and control mice. At later times, mortality was nevertheless increased in DT-iDTR mice, owing to lung hemorrhages. When less severe thrombocytopenia was induced with an antiplatelet mAb, TRALI started and developed similarly as in control mice, but hemorrhages were absent. Furthermore, when platelet functions were defective because of administration of aspirin or clopidogrel, or because of glycoprotein (GP)IIbIIIa deficiency, TRALI still developed but no lung hemorrhages were observed. In contrast, when GPVI was immunodepleted, TRALI still occurred, but was occasionally accompanied by hemorrhages. Conclusions Platelets are dispensable for the initiation and development of MHC I-induced TRALI. Although they do not protect against the disruption of the vascular endothelial cell barrier and the subsequent plasma leakage and edema formation, platelets are essential to prevent more serious damage resulting in hemorrhages in alveoli.

Immature myeloid dendritic cells capture and remove activated platelets from preformed aggregates.

BACKGROUND: Immature dendritic cells (DCs) patrol the circulation, where they can uptake antigens. It has been reported that mature monocyte-derived DCs have the ability to interact with an activated platelet monolayer under high shear conditions (1500s(-1) ). OBJECTIVES: In this study, we investigated whether platelets can recruit immature myeloid DCs (CD1c(+) ) directly isolated from blood (BDCs) and if so, which receptors are involved. RESULTS: Using flow cytometry and electron microscopy, we showed that BDCs interact with activated but not resting platelets in suspension. Interaction was also observed after perfusing BDCs under low flow conditions (150 s(-1) ) through collagen-coated microcapillaries in which platelets had adhered and formed aggregates. No such interaction could be detected at higher shear rates. Whereas initial transient attachment required the exposure of P-selectin on activated platelets and PSGL-1 on BDCs, subsequent stationary adhesion was dependent on α(IIb) ß(3) and α(M) ß(2) integrins on platelets and BDCs, respectively. Moreover, during their transient interaction, BDCs preferentially removed platelets located at the outer margin of the thrombus in a P-selectin- and integrin-dependent manner. CONCLUSION: This study provides evidence for an interaction between activated platelets and immature myeloid DCs only under low shear conditions. This could be of importance for BDC maturation and antigen uptake during normal hemostasis or in the context of atherothrombosis at sites of reduced blood flow.

Characterization of the biofouling and cleaning efficiency of nanofiltration membranes.

The nanofiltration (NF) drinking water production unit of the Mery-sur-Oise plant (Val d'Oise, France) consists of eight identical filtration trains composed of three stages positioned in steps for a production capacity of 140,000 m(3) day(-1). To gain a better understanding of the irreversible fouling of the NF membranes, spiral wound modules in operation for 8 years from each of the three stages of the plant were autopsied before and after chemical cleaning and analysis by Attenuated Total Reflection Fourier Transform Infrared spectroscopy, Inductive Coupled Plasma-Atomic Emission Spectrometry, contact angles, adenosine triphosphate (ATP) content measurements, and rheometry. The fouled membranes from the three stages had similar contact angles of approximately 60 degrees . Relative infrared signals typical of biofilms were classified in descending order from stage 1 to stage 3. The foulant matter of stages 1 and 2 contained similar but weaker ATP concentrations than stage 3. During rheometry experiments, rotation and oscillation analyses demonstrated that the biofilm of stage 3 was less viscous and less elastic than the biofilms of stages 1 and 2. After cleaning, all the parameters analyzed demonstrated a quantitative decrease in the fouling matter at the NF membrane surface, but a biofilm with intact viscoelastic properties (unchanged G' and G'' values) remained at the membrane surface for the three stages. The persistence of biofilm material with intact mechanical properties at the NF membrane surface after chemical cleaning may result in permanent permeability decreases.

Rheology of biofilms formed at the surface of NF membranes in a drinking water production unit.

In this study, the mechanical properties of biofilms formed at the surface of nano-filtration (NF) membranes from a drinking water plant were analysed. Confocal laser scanning microscopy observations revealed that the NF biofilms formed a dense and heterogeneous structure at the membrane surface, with a mean thickness of 32.5 +/- 17.7 mum. The biofilms were scraped from the membrane surface and analysed in rotation and oscillation experiments with a RheoStress 150 rotating disk rheometer. During rotation analyses, a viscosity decrease with speed of shearing characteristic of rheofluidification was observed (eta = 300 Pa s for ý = 0.3 s(-1)). In the oscillation analyses with a sweeping of frequency (1-100 Hz), elasticity (G') ranged from 3000 to 3500 Pa and viscosity (G'') from 800 to 1200 Pa. Creep curves obtained with an application of a shear stress of 30 Pa were viscoelastic in nature. The G(0) and eta values were, respectively, 1.4 +/- 0.3 x 10(3) Pa and 3.3 +/- 0.65 x 10(6) Pa s. The relationship between the characteristics of NF biofilms and the flow conditions encountered during NF is discussed.

Assessing chemical cleaning of nanofiltration membranes in a drinking water production plant: a combination of chemical composition analysis and fluorescence microscopy.

The efficiency of cleaning procedures to remove the fouling deposit from the surface of NF membranes operating in the drinking water plant of Méry sur Oise (Val d'Oise, France) was assessed by a combination of chemical analysis and fluorescence microscopy. The ATR-FTIR spectra of the fouled membranes revealed the presence of biological matter at the membrane surface, mainly composed of polysaccharides, nucleic acids and proteins. IR bands corresponding to the membrane material were detected for stage 1 but not for stage 3. Confocal laser scanning microscopy (CLSM) observations confirmed the microbial origin of the fouling deposit. After chemical cleaning, the analysis of the inorganic foulants revealed a significant decrease of the inorganic content. Moreover, ATR-FTIR spectra of the fouled membranes were modified, mainly in a broad complex region corresponding to polysaccharides and nucleic acids. The amide bands were also altered for stage 1, and some peaks corresponding to the clean membrane appeared for stage 3 after cleaning. CLSM observations revealed a general decrease of the lectin staining for the two stages with some variations between lectins. A decrease of the DAPI staining indicative of the removal of some microbial cells was also observed for stage 1. In conclusion, cleaning of the NF fouled membranes decreased significantly the inorganic foulants but only partially removed the organic fouling deposit characteristic of a microbial biofilm.

The 5' part of the mouse immunoglobulin kappa locus.

The 5' region of the mouse kappa locus comprises 63 Vkappa genes in six contigs of together 1.5 Mb, including one which links the region to the central part of the locus. The structures of the contigs were established by detailed restriction mapping of cosmid clones prepared from libraries of mouse C57BL/6 DNA and of yeast and bacterial artificial chromosomes (YACs, BACs with mouse DNA inserts). Pulsed-field gel electrophoresis of yeast artificial chromosome digests indicated that the gaps between the contigs were 10 to 60 kb, comprising together about 160 kb. The region of the kappa locus described here contains Vkappa1, Vkappa2, Vkappa9/10, Vkappa11, Vkappa12/13, Vkappa20, Vkappa24, Vkappa32, Vkappa33/34 and Vkappa38C genes as well as the VkappaRF gene and, towards the center of the locus, a number of Vkappa4/5 genes. Near the 5' end of the locus interspersed alpha-tubulin gene-like sequences were found. At its 3' side the region borders on the Vkappa4/5 contigs of the central region of the locus which is described in the accompanying report (Eur. J. Immunol. 1999. 29: 2057-2064). Structural details are to be found in the Internet at http://www.med.uni-muenchen.de/biochemie/zach au/kappa.htm. In a concluding section the main features of the structure of the mouse kappa locus are summarized.

The variable genes and gene families of the mouse immunoglobulin kappa locus.

In this report 118 mouse Vkappa genes are described which, together with the 22 Vkappa genes reported previously (T. Kirschbaum et al., Eur. J. Immunol. 1998. 28: 1458-1466) amount to 140 genes that had been cloned and sequenced in our laboratory. For 73 of them cDNAs are known, i. e. they have to be considered functional genes, although 10 genes of this group have 1-bp deviations from the canonical promoter, splice site or heptanucleotide recombination signal sequences. Twenty Vkappa genes have been defined as only potentially functional since they do not contain any defect, but no cDNAs have been found (yet) for them. Of the 140 Vkappa genes 47 are pseudogenes. There are indications that two to five Vkappa genes or pseudogenes exist in the kappa locus which we have not yet been able to clone. The 140 Vkappa genes and pseudogenes were assigned to 18 gene families, 4 of them being one-member families. This differs from previous enumerations of the families only by the combination of the Vkappa9 and Vkappa10 families and by the addition of the Vkappa dv gene as a new separate family. Sequence identity usually was 80% or above within the gene families and 55-80% between genes of different families. Many of the mouse Vkappa gene families show significant homologies to the human ones, indicating that in evolution Vkappa gene diversification predated the divergence of the primate and rodent clades.

The 3' part of the immunoglobulin kappa locus of the mouse.

A detailed restriction map of a 430-kb contig comprising the single Ckappa, the 5 Jkappa and the adjoining 22 Vkappa gene segments is presented. The first 12 Vkappa genes following the JkappaCkappa region belong to the Vkappa21 family, the subsequent ones to the closely related families Vkappa8 and Vkappal 9/ 28. Previous difficulties in cloning all Vkappa21 genes can now be explained by the presence of a duplicated region in this part of the locus. The structure was established by analysis of yeast artificial chromosome, bacterial artificial chromosome and cosmid clones and by the so-called long template PCR technique. The distance between Ckappa and the proximal Vkappa21 gene is 22 kb and the average distances between the Vkappa genes are about 20 kb. Of the 12 Vkappa21 genes 5 were sequenced for the first time and 8 of the 12 genes were found to be expressed. Of the 10 Vkappa8 and Vkappa19/28 germline genes 9 are new; expression products of 8 of the 10 genes were known. The known 5', 3' polarities allow to specify for the 22 Vkappa genes whether they are rearranged to the JkappaCkappa element by a deletion or an inversion mechanism. Also the formation of interesting rearrangement products in classical cell lines as MPC11, MOPC41 and PC 7043 can be explained now. The non-Vkappa sequence L10 whose rearrangement by inversion has been described earlier (Hoechtl and Zachau, Nature 1983. 302: 260-263) was now localized downstream of JkappaCkappa.