A novel method for quantification of cefazolin local tissue concentration in blood, fat, synovium, and bone marrow using liquid chromatography - mass spectrometry.

To be effective, the concentration of antibiotic used must exceed the minimum inhibitory concentration (MIC) against infecting organisms at and in the surgical site. Few studies follow antibiotic levels for tissues that are manipulated during surgery. The aim of this work was to develop and validate a novel LC-MS method as well as an efficient extraction technique for the quantification of cefazolin in local tissues and whole blood. This method uses the same efficient extraction method across multiple tissue types affected by orthopedic surgery: blood, fat, synovium, and bone marrow. The ability to quantify cefazolin in these tissues will help identify surgical techniques and antibiotic dosing protocols that better protect patients from infection. The internal standard, 13C2,15N-cefazolin, co-elutes with cefazolin, and was used in calibration curves and tissue extracts as well as for cefazolin recovery and matrix effects. The protocol was rigorously tested, including measurements of reproducibility and calibration curve quality. The recovery of the extraction method ranges from 94% to 113% across all sample types. There is little to no matrix effect on cefazolin signal (98-120%). The developed method was used to determine cefazolin concentrations in tissues of 10 patients undergoing a total knee replacement. Cefazolin blood concentrations were approximately 500 times higher than in adipose, synovium, and bone marrow tissues. This clinical data shows that although the minimum inhibitory concentration is largely surpassed in blood, the concentration of cefazolin in fat, synovium, and bone marrow could be insufficient during a knee replacement. This method of cefazolin quantification will help surgeons optimize antibiotic concentrations in the local tissues during knee replacement surgery and potentially reduce serious post-surgical infections.

[Novel criteria for assessing red blood cell viability in blood banks]. / Nouveaux critères d'évaluation de la viabilité des hématies destinées à la transfusion.

UNLABELLED: In light of recent results on the mechanism of programmed cell death of human red blood cells (RBC), the aim of the present study was to solve the enigma of the rapid clearance of transfused RBCs. MATERIALS AND METHODS: We describe new criteria of RBC viability founded on the use of flow cytometry. They were applied, in association with the classical ones: ATP and hemolysis measurements, to RBCs stored in SAGM medium for 42 days. RESULTS AND CONCLUSIONS: Application of an original method of flow cytometric quantitation of in vitro erythrophagocytosis showed that an important proportion of stored RBCs were phagocytized although the following classical signals for phagocytosis were absent, i.e.: desialylation, phosphatidylserine exposure in the outer leaflet of the RBC membrane, loss of CD47 receptor, an antiphagocytosis signal. In addition, ATP was still present and hemolysis was very low. This enigma was solved by the use of scanning electron microscopy, which showed the disappearance of discocytes and the presence of an important proportion of spheroechinocytes, which are the phagocytable forms of RBCs. The mechanism of this dramatic morphological transformation remains to be elucidated.

Programmed cell death in mature erythrocytes: a model for investigating death effector pathways operating in the absence of mitochondria.

Human mature erythrocytes have been considered as unable to undergo programmed cell death (PCD), due to their lack of mitochondria, nucleus and other organelles, and to the finding that they survive two conditions that induce PCD in vitro in all human nucleated cells, treatment with staurosporine and serum deprivation. Here we report that mature erythrocytes can undergo a rapid self-destruction process sharing several features with apoptosis, including cell shrinkage, plasma membrane microvesiculation, phosphatidylserine externalization, and leading to erythrocyte disintegration, or, in the presence of macrophages, to macrophage ingestion of dying erythrocytes. This regulated form of PCD was induced by Ca(2+) influx, and prevented by cysteine protease inhibitors that allowed erythrocyte survival in vitro and in vivo. The cysteine proteinases involved seem not to be caspases, since (i) proforms of caspase 3, while present in erythrocytes, were not activated during erythrocyte death; (ii) cytochrome c, a critical component of the apoptosome, was lacking; and (iii) cell-free assays did not detect activated effectors of nuclear apoptosis in dying erythrocytes. Our findings provide the first identification that a death program can operate in the absence of mitochondria. They indicate that mature erythrocytes share with all other mammalian cell types the capacity to self-destruct in response to environmental signals, and imply that erythrocyte survival may be modulated by therapeutic intervention.

Improved storage of erythrocytes by prior leukodepletion: flow cytometric evaluation of stored erythrocytes.

In vivo phagocytosis of senescent red blood cells (RBCs) by macrophages occurs 120 days after their release into the circulation. It depends on two sequential signals that trigger phagocytosis: (1) desialylation of membrane glycoconjugates with the exposure of the penultimate beta-galactosyl residues and (2) exposure of phosphatidylserine in the membrane outer leaflet. Leukodepleted and nonleukodepleted RBCs were compared using flow cytometric procedures to determine whether the in vitro deterioration of RBCs during storage might be attributable to an identical mechanism of desialylation induced by leukocyte neuraminidases, resulting in exposure of beta-galactosyl and subsequently phosphatidylserine residues - signals of senescent RBCs. Without prior leukodepletion, stored RBCs showed an increased population of senescent RBCs (using light scatter measurements), extensive desialylation with the exposure of beta-galactosyl residues (using specific fluorescein isothiocyanate [FITC]-lectins), significant exposure of phosphatidylserine in the outer leaflet of the RBC membrane (using FITC-annexin V), and extensive in vitro phagocytosis (using PKH-26-labeled RBCs). There were minimal changes observed with the leukodepleted RBCs. These results lead to the conclusion that leukocyte enzymes, including neuraminidases, are definitive contributers to the desialylation of RBCs during storage and to the exposure of phosphatidylserine residues. These deleterious effects resulting from highly active leukocyte enzymes are preventable by prior leukodepletion of the stored RBCs. Previously developed flow cytometric procedures to detect in vivo "RBC senescence" have been applied and proved to be reliable criteria to monitor the viability of stored RBCs.

Cellular and molecular mechanisms of senescent erythrocyte phagocytosis by macrophages. A review.

Human red blood cells (RBCs) have a life-span of 120 days in circulation, after which they are phagocytized by resident macrophages. Extensive studies have been undertaken by many investigators in order to elucidate the cellular and molecular mechanisms of the erythrophagocytosis. The critical questions addressed by physiologists, clinicians and biochemists are: 'which of the many traumatic blemishes that appear on the erythrocyte surface as it winds its way through the circulation is the primary signal for clearance of the effete RBC from the circulation?', or 'What is the critical signal that it, and it alone, will activate the resident macrophage to adhere to and engulf it?'. Numerous, and often conflicting, hypotheses have been proposed. Each investigator focusing on but one of the many modifications that afflict the cell surface of the ageing erythrocyte, viz changes in either or both the carbohydrate or peptidic moieties of glycoproteins; abolishment of the pre-existing asymmetry in the lipid bilayer with the exposure of phosphatidylserine residues; or alterations in spectrin, to mention but a few. Many of these investigators also have invoked an intermediary role for auto-immune antibodies that recognise the change(s) on the erythrocyte surface and thereby serve as opsonins as a prelude to the erythrophagocytosis. The objective of the present review is to evaluate the data in support of the various hypotheses, and to submit some of our own recent observations involving the use of flow cytometric procedures that: i) provide evidence that the cell surface sialic acid serves as a determinant of the life-span; ii) characterise the senescent erythrocyte population that is specifically captured and phagocytized by macrophages (utilising the rapid and sensitive procedure we developed for quantification of in vitro erythrophagocytosis); and finally iii) provide evidence for the existence of an alternative pathway that is independent of immunoglobulins.

Molecular mechanisms of erythrophagocytosis: flow cytometric quantitation of in vitro erythrocyte phagocytosis by macrophages.

A rapid, sensitive, and reproducible flow cytofluorimetric procedure is described for quantitation of erythrophagocytosis based on the use of red blood cells (RBCs) labeled with the fluorescent probe PKH-26. The procedure involves the following steps: i) incubation of PKH-26-labeled erythrocytes with macrophages, ii) removal of un-bound red blood cells, iii) lysis of membrane-bound RBCs, and iv) measurement of extent of phagocytosis by direct flow-cytometric analysis of intact macrophages. Each step was controlled by fluorescence microscopy. Use of fluorescent, instead of radio-labeled RBCs, makes the method more sensitive, rapid, and avoids radioactive hazards. Furthermore, this approach is multi-parametric and can distinguish different populations of macrophages with reference to their erythrophagocytic potential. This technology moreover, has broad applications from the initial step of contact (between effector and target cells to study the specific receptor mediated attachment) to the subsequent cascade of time-dependent changes resulting from that signal transduction.

Molecular mechanisms of erythrophagocytosis. Characterization of the senescent erythrocytes that are phagocytized by macrophages.

We have recently developed a flow cytometric assay for the quantitation of erythrophagocytosis, using PKH 26-labeled erythrocytes as the target cells. Using this assay we have shown that there is extensive phagocytosis of desialylated erythrocytes. Furthermore, we have demonstrated that it is the densest population of erythrocytes obtained on a self-forming gradient of Percoll that shows the greatest susceptibility to phagocytosis. We designate this population of erythrocytes as fraction X; it is even denser than the fraction 5 found previously. This population of erythrocytes corresponds to zone X previously seen in the dot-plot of the flow cytometric analyses of human erythrocytes. Further scrutiny of this fraction indicates that a) it shows the greatest reactivity with annexin V, which is specific for the detection of phosphatidylserine (PS) exposed on the outer leaflet of the erythrocyte membrane, b) it is the most susceptible to erythrophagocytosis by resident murine peritoneal macrophages, and c) this erythrophagocytosis of PKH 26-labeled erythrocytes can be inhibited by annexin V and by liposomes containing PS. Scanning electron microscopy of fraction X shows two populations of erythrocytes: (A) spheroechinocytes with filipodes and (B) echinocytes without filipods. After a 2-h period of phagocytosis, the cells remaining in fraction X show a decrease in population A, commensurate with a decrease in reactivity with FITC-labeled annexin V from 65.5 to 24%.

Lactoferrin-lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide.

The ability of lactoferrin (Lf), an iron-binding glycoprotein that is also called lactotransferrin, to bind lipopolysaccharide (LPS) may be relevant to some of its biological properties. A knowledge of the LPS-binding site on Lf may help to explain the mechanism of its involvement in host defence. Our report reveals the presence of two Escherichia coli 055B5 LPS-binding sites on human Lf (hLf): a high-affinity binding site (Kd 3.6 +/- 1 nM) and a low-affinity binding site (Kd 390 +/- 20 nM). Bovine Lf (bLf), which shares about 70% amino acid sequence identity with hLf, exhibits the same behaviour towards LPS. Like hLf, bLf also contains a low- and a high-affinity LPS-binding site. The Kd value (4.5 +/- 2 nM) corresponding to the high-affinity binding site is similar to that obtained for hLf. Different LPS-binding sites for human serum transferrin have been suggested, as this protein, which is known to bind bacterial endotoxin, produced only 12% inhibition of hLf-LPS interaction. Binding and competitive binding experiments performed with the N-tryptic fragment (residues 4-283), the C-tryptic fragment (residues 284-692) and the N2-glycopeptide (residues 91-255) isolated from hLf have demonstrated that the high-affinity binding site is located in the N-terminal domain I of hLf, and the low-affinity binding site is present in the C-terminal lobe. The inhibition of hLf-LPS interaction by a synthetic octadecapeptide corresponding to residues 20-37 of hLf and lactoferricin B (residues 17-41), a proteolytic fragment from bLf, revealed the importance of the 28-34 loop region of hLf and the homologous region of bLf for LPS binding. Direct evidence that this amino acid sequence is involved in the high-affinity binding to LPS was demonstrated by assays carried out with EGS-loop hLf, a recombinant hLf mutated at residues 28-34.

Specificity of the isolectins from the plant cactus Machaerocereus eruca for oligosaccharides from porcine stomach mucin.

Sugar specificity of the Machaerocereus eruca isolectins, MeAI and MeAII, has been determined by comparing the capacity of glycans with well defined structures to inhibit their haemagglutinating activity. Both are galactose-specific isolectins with high affinity for O-glycans. However, the two M. eruca isolectins recognize different oligosaccharidic sequences belonging to O-glycosidically linked glycans from porcine stomach mucin. The minimal structure recognized by MeAI on the porcine mucin glycans is the O-glycan core Gal beta 1,3GalNAc-ol, whereas MeAII has a more extended site and interacts with a biantennary O-glycan possessing the terminal trisaccharide Fuc alpha 1,2 (GalNAc alpha 1,3) Gal beta 1,4.

Flow cytofluorimetric analysis of young and senescent human erythrocytes probed with lectins. Evidence that sialic acids control their life span.

Comparing the properties of 'young' and senescent ('aged') O+ erythrocytes isolated by applying ultracentrifugation in a self-forming Percoll gradient, we demonstrate that the sialic acids of membrane glycoconjugates control the life span of erythrocytes and that the desialylation of glycans is responsible for the clearance of the aged erythrocytes. This capture is mediated by a beta-galactolectin present in the membrane of macrophages. The evidence supporting these conclusions is as follows: (1) Analysis by flow cytofluorimetry of the binding of fluorescein isothiocyanate labelled lectins specific for sialic acids shows that the aged erythrocytes bind less WGA, LPA, SNA and MAA than young erythrocytes. The binding of DSA and LCA is not modified. On the contrary, the number of binding sites of UEA-I specific for O antigen and of AAA decreases significantly. PNA and GNA do not bind to erythrocytes. (2) RCA120 as well as Erythrina cristagalli and Erythrina corallodendron lectins specific for terminal beta-galactose residues lead to unexpected and unexplained results with a decrease in the number of lectin binding sites associated with increasing desialylation. (3) The glycoconjugates from the old erythrocytes incorporate more sialic acid than the young cells. This observation results from the determination of the rate of transfer by alpha-2,6-sialyltransferase of fluorescent or radioactive N-acetylneuraminic acid, using as donors CMP-9-fluoresceinyl-NeuAc and CMP-[14C]-NeuAc, respectively. (4) Microscopy shows that the old erythrocytes are captured preferentially by the macrophages relative to the young ones. Fixation of erythrocytes by the macrophage membrane is inhibited by lactose, thus demonstrating the involvement of a terminal beta-galactose specific macrophage lectin. (5) Comparative study of the binding of WGA, LPA, SNA and MAA to the aged erythrocytes and to the in vitro enzymatically desialylated erythrocytes shows that the desialylation rate of aged cells is low but sufficient to lead to their capture by the macrophages.

Fine sugar specificity of the mistletoe (Viscum album) lectin I.

The behaviour of N-acetyllactosamine-type oligosaccharides and glycopeptides on a column of mistletoe lectin I (MLI) immobilized on Sepharose 4B was examined. The immobilized lectin does not show any affinity for asialo-N-glycosylpeptides and related oligosaccharides, which possess one to four unmasked N-acetyllactosamine sequences. However, substitution of at least one of the N-acetyllactosamine sequences by sialic acid residues, either at O-3 or O-6 of galactose, slightly enhances the affinity of the lectin. Such sialylated N-glycosylpeptides or oligosaccharides are eluted from the lectin column by the starting buffer as retarded fractions. Surprisingly, the affinity of the immobilized MLI is higher for P1 antigen-containing glycopeptide isolated from turtle-dove ovomucoid and for glycopeptides from bovine thyroglobulin containing terminal non-reducing Gal alpha 1-3Gal sequences. These structures are strongly bound on the lectin column and their elution is obtained with 0.15 M galactose in the starting buffer.

Fast purification of Phaseolus vulgaris isolectins.

Phytohemagglutinin from red kidney bean has been purified by affinity chromatography on a human alpha 1-acid glycoprotein Sepharose 4B column. Further purification of the hemagglutinin's five isolectins was achieved on a Mono S column with an 86% protein recovery. Each sequentially eluted isolectin from the ion exchange column displayed either hemagglutinating or mitogenic activity. The main activity of each fraction was the result of the combination of varying proportions of the L and E subunits.

Change in glycosylation of chicken transferrin glycans biosynthesized during embryogenesis and primary culture of embryo hepatocytes.

Transferrins were isolated by immunoaffinity chromatography from chicken serum, chicken embryo serum and from the culture medium of chicken embryo hepatocytes in primary culture. The glycovariants of these three transferrins were separated by ion-exchange chromatography using a fast protein liquid chromatography (FPLC) system. The structures of the oligosaccharide-alditols released by hydrazinolysis from the glycovariants were compared after analysis by a combination of methanolysis, methylation analysis and 1H-NMR spectroscopy. In the three transferrins analysed, the oligosaccharides were of the biantennary N-acetyllactosaminic type, having several prominent features. In particular, the embryo serum transferrin glycan differed from that of chicken serum transferrin by the presence of a bisecting N-acetylglucosamine, suggesting a developmental change in glycosylation. The glycan structure of the transferrin secreted by the embryo hepatocytes in primary culture was marked by the presence of fucose (alpha 1-6) linked to the core N-acetylglucosamine, suggesting that expression of the fucosyltransferase activity is dependent on cell culture conditions. Moreover, comparative analysis of chicken serum transferrin and ovotransferrin glycans reinforces the idea that the glycosylation of two identical polypeptide chains is organ specific.

Interscalene block for pain relief after shoulder surgery. A prospective randomized study.

A prospective randomized study undertaken in 30 patients who underwent outpatient decompressive acromioplasty demonstrated the efficacy and safety of interscalene block post-operatively. Interscalene block improved the postoperative condition and well being of these patients. Their use decreased the hospitalization rate. There were no complications or side effects.

Structures of a legume lectin complexed with the human lactotransferrin N2 fragment, and with an isolated biantennary glycopeptide: role of the fucose moiety.

BACKGROUND: Lectins mediate cell-cell interactions by specifically recognizing oligosaccharide chains. Legume lectins serve as mediators for the symbiotic interactions between plants and nitrogen-fixing microorganisms, an important process in the nitrogen cycle. Lectins from the Viciae tribe have a high affinity for the fucosylated biantennary N-acetyllactosamine-type glycans which are to be found in the majority of N-glycosylproteins. While the structures of several lectins complexed with incomplete oligosaccharides have been solved, no previous structure has included the complete glycoprotein. RESULTS: We have determined the crystal structures of Lathyrus ochrus isolectin II complexed with the N2 monoglycosylated fragment of human lactotransferrin (18 kDa) and an isolated glycopeptide (2.1 kDa) fragment of human lactotransferrin (at 3.3 A and 2.8 A resolution, respectively). Comparison between the two structures showed that the protein part of the glycoprotein has little influence on either the stabilization of the complex or the sugar conformation. In both cases the oligosaccharide adopts the same extended conformation. Besides the essential mannose moiety of the monosaccharide-binding site, the fucose-1' of the core has a large surface of interaction with the lectin. This oligosaccharide conformation differs substantially from that seen in the previously determined isolectin I-octasaccharide complex. Comparison of our structure with that of concanavalin A (ConA) suggests that the ConA binding site cannot accommodate this fucose. CONCLUSIONS: Our results explain the observation that Viciae lectins have a higher affinity for fucosylated oligosaccharides than for unfucosylated ones, whereas the affinity of ConA for these types of oligosaccharides is similar. This explanation is testable by mutagenesis experiments. Our structure shows a large complementary surface area between the oligosaccharide and the lectin, in contrast with the recently determined structure of a complex between the carbohydrate recognition domain of a C-type mammalian lectin and an oligomannoside, where only the non-reducing terminal mannose residue interacts with the lectin.

Study on the binding of lactotransferrin (lactoferrin) to human PHA-activated lymphocytes and non-activated platelets. Localisation and description of the receptor-binding site.

Fluorescein isothiocyanate derivatization of human lactotransferrin on Lys-264 as well as covalent addition of sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'- dithiopropionate (SASD)* on Lys-74 inhibits the binding of the glycoprotein to both human PHA-activated lymphocytes and non-activated platelets. This suggests that the cell binding site of lactotransferrin is located in the vicinity of the lysine residues 74 & 264 and does not occur either through electrostatic or lectin interactions. In contrast, the derivatization of lactotransferrin using sulfosuccinimidyl 6-(4'-azido-2'-nitrophenyl-amino) hexanoate (sulfo-SANPAH), on Lys-281 does not modify the binding parameters of lactotransferrin to the cells. Molecular modeling showed the position of SASD, sulfo-SANPAH and fluorescein molecules at the surface of the protein and suggested that SASD and fluorescein could mask the two loop-containing regions of human lactotransferrin (residues 28-34 and 38-45). Elsewhere, a 6 kDa peptide covering the peptide chain from residues 4 to 52 was isolated and its inhibitory effect on the binding of lactotransferrin to both human PHA-activated lymphocytes and non-activated platelets was demonstrated. Inhibition of ADP-induced platelet aggregation by lactotransferrin (50% inhibition = 10 nM) was also found with the N-t fragment of lactotransferrin (residues 3-281; 50% inhibition = 2 microM) and with two synthetic peptides: KRDS tetrapeptide (50% inhibition = 350 microM) and CFQWQRNMRKVRGPPVSC octodecapeptide (50% inhibition = 20 microM) corresponding to the lactotransferrin amino acid sequence 39-42 and 20-37, respectively.

Characterization of two kinds of lactotransferrin (lactoferrin) receptors on different target cells.

Lactotransferrin (Lf), an iron-binding glycoprotein present as a major component in the specific granules of human neutrophilic granulocytes is released in the blood during the acute phase of infection and participates in the regulation of the host-defence mechanisms. Our previous observations (Mazurier et al., 1989) showing i) that the activation by PHA of T-lymphocytes induces the appearance at the cell surface of Lf-receptors which are absent from the membrane of resting lymphocytes and ii) that Lf becomes a growth factor for the activated lymphocytes, led us to undertake a series of researches on the presence of Lf receptors at the surface of different blood cells. Characterization of Lf receptors was performed by flow cytofluorimetry using either Lf labelled on its glycan moiety with fluorescein or purified anti-lymphocyte Lf receptor antibodies. High affinity receptors for Lf were characterized only at the surface of human activated lymphocytes and of non-activated platelets. These two receptors possess common physicochemical properties and antigenic epitopes. Low affinity receptors for Lf were characterized on monocytes, eosinophils and neutrophils. These receptors are immunologically different from those found on activated lymphocytes and on non-activated platelets. Cell-lines of human lymphocyte T and megakaryocyte possess lactotransferrin receptors whose properties are similar to those found on peripheral blood cells. The soluble form of the receptor identified in the lymphocytes T culture medium possesses a molecular mass close to that of the membrane receptor suggesting that the cytoplasmic tail of the receptor should be very short.

Primary and three-dimensional structure of lactotransferrin (lactoferrin) glycans.

In order to establish relationships between glycan structure and biological activity, the authors undertook a comparative study of the glycan primary structure of different transferrins from several species. By associating permethylation-mass spectrometry and 1H-NMR spectroscopy, the primary structure of the human, bovine, caprine, murine and porcine lactotransferrin glycans were determined. Using the same methods, the glycan structure of 9 serotransferrins was determined. The results obtained led to the conclusion that glycans are specific for each transferrin and, for a given transferrin, specific to the species. No relationship could be established between primary structure and function of transferrin glycans. Glycan molecular modelling, molecular dynamics simulations and X-ray diffraction studies of free glycans confirm the mobility in space of antennae. In contrast, the glycan associated with a protein is immobilized into only one conformation, as in the case of glycan-lectin associations or of "internal" glycan-protein interactions, like in rabbit serotransferrin, in which the glycan forms a bridge between the two lobes of the peptide chain, and maintains the protein in a biologically active conformation. In the case of human sero- and lactotransferrins, the glycans are in an external position on the molecules and could play a role of recognition signals.