Natural resistance to intracellular infections: natural resistance-associated macrophage protein 1 (Nramp1) functions as a pH-dependent manganese transporter at the phagosomal membrane.

Mutations at the natural resistance-associated macrophage protein 1 (Nramp1) locus cause susceptibility to infection with antigenically unrelated intracellular pathogens. Nramp1 codes for an integral membrane protein expressed in the lysosomal compartment of macrophages, and is recruited to the membrane of phagosomes soon after the completion of phagocytosis. To define whether Nramp1 functions as a transporter at the phagosomal membrane, a divalent cation-sensitive fluorescent probe was designed and covalently attached to a porous particle. The resulting conjugate, zymosan-FF6, was ingested by macrophages and its fluorescence emission was recorded in situ after phagocytosis, using digital imaging. Quenching of the probe by Mn(2+) was used to monitor the flux of divalent cations across the phagosomal membrane in peritoneal macrophages obtained from Nramp1-expressing (+/+) and Nramp1-deficient (-/-) macrophages. Phagosomes from Nramp1(+/+) mice extrude Mn(2+) faster than their Nramp(-/-) counterparts. The difference in the rate of transport is eliminated when acidification of the phagosomal lumen is dissipated, suggesting that divalent metal transport through Nramp1 is H(+) dependent. These studies suggest that Nramp1 contributes to defense against infection by extrusion of divalent cations from the phagosomal space. Such cations are likely essential for microbial function and their removal from the phagosomal microenvironment impairs pathogenesis, resulting in enhanced bacteriostasis or bactericidal activity.

Primary red cell hydration disorders: Pathogenesis and diagnosis.

Hydration status is critical for erythrocyte survival and is mainly determined by intracellular cation content. Active pumps, passive transporters, and ion channels are the key components of volume homeostasis, whereas water passively fits ionic movements. Whenever cation content increases, erythrocyte swells, whereas it shrinks when cation content decreases. Thus, inappropriate cation leak causes erythrocyte hydration disorders, hemolytic anemia, and characteristic red cell shape abnormalities named stomatocytosis. All types of stomatocytosis either overhydrated or dehydrated are linked to inherited or de novo mutations in genes encoding ion transporters or channels. Although intracellular ion content can be assessed by experimental methods, laboratory diagnosis is guided by a combination of red blood cell parameters and deformability measurement when possible, and confirmed by sequencing of the putative genes. A better knowledge of the mechanisms underlying erythrocyte hydration imbalance will further lead to therapeutic improvements.

Cancer-testis antigen expression in bladder cancer.

PURPOSE: To evaluate the potential of cCancer-t/Testis antigens (CTAs) as targets for immunotherapy of bladder cancer, we evaluated the expression of 9 CTA genes or families of genes in normal urothelia, bladder tumours and bladder cancer human bladder tissuescell lines. As expression of most CTAs is controlled by epigenetic mechanisms, we also evaluated the effect of the DNA methylase inhibitor 5-aza-2'-deoxycytidine (5-AZA-DC), and/or theand histone deacetylase inhibitors Trichostatin A (TSA) on their expression in bladder cancer cell lines. MATERIAL AND METHODS: Expression of NY-ESO-1/LAGE-1, MAGE-A, MAGE-C1, BAGE, HOM-TES-85, SCP-1, SSX-1, SSX-2 and SSX-4 was analyzed by semi-quantitative RT-PCR and Western blotting on 10 normal urothelia, 23 24 superficial and 223 invasive tumours and on 10 cell lines treated with 5-aza-2'-deoxycytidine (5-AZA-DC) and/or Trichostatin A (TSA). RESULTS: Expression of all CTA genes could be observed in at least 1 tumour except for HOM-TES-85 for which mRNA was never detected. MAGE-A, BAGE and NY-ESO-1/LAGE-1 mRNAs were the most frequently detected, respectively in 5677%, 212% and 89% of superficial and in 6461%, 4139% and 276% of invasive tumours. With the exception of MAGE-A, CTA transcripts were rarely detected in the cell lines. However, expression of all CTA genes, except SCP-1, could be induced at various levels by the drugs and 5-AZA-DC was a much more potent inducer than TSA. CONCLUSION: These data suggest that immunotherapy of bladder cancer could target CTAs, especially those expressed at higher frequency such as MAGE-A, BAGE and NY-ESO-1/LAGE-1. Moreover, their induction by chemotherapeutic agents such as 5-AZA-DC, provides a potential pretreatment aimed at inducing the immunogenicity of the tumours.

ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders.

INTRODUCTION: Hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary stomatocytosis (HSt) are inherited red cell disorders caused by defects in various membrane proteins. The heterogeneous clinical presentation, biochemical and genetic abnormalities in HS and HE have been well documented. The need to raise the awareness of HSt, albeit its much lower prevalence than HS, is due to the undesirable outcome of splenectomy in these patients. METHODS: The scope of this guideline is to identify the characteristic clinical features, the red cell parameters (including red cell morphology) for these red cell disorders associated, respectively, with defective cytoskeleton (HS and HE) and abnormal cation permeability in the lipid bilayer (HSt) of the red cell. The current screening tests for HS are described, and their limitations are highlighted. RESULTS: An appropriate diagnosis can often be made when the screening test result(s) is reviewed together with the patient's clinical/family history, blood count results, reticulocyte count, red cell morphology, and chemistry results. SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins, monovalent cation flux measurement, and molecular analysis of membrane protein genes are specialist tests for further investigation. CONCLUSION: Specialist tests provide additional evidence in supporting the diagnosis and that will facilitate the management of the patient. In the case of a patient's clinical phenotype being more severe than the affected members within the immediate family, molecular testing of all family members is useful for confirming the diagnosis and allows an insight into the molecular basis of the abnormality such as a recessive mode of inheritance or a de novo mutation.

Human thrombin variable region 1, including E39, is involved in interactions with alpha 1-antitrypsin M358R and protein C.

We used an antithrombin autoantibody (IgG D), the epitope of which encompasses ABE1 and amino acids located within variable region 1, to study thrombin interactions with R358 alpha 1-AT and protein C. IgG D inhibited the thrombin interaction with R358 alpha 1-AT, while hirugen had no effect, indicating that the interaction of R358 alpha 1-AT with thrombin may involve the VR1 subsite. We also obtained evidence that VR1 may be involved in the activation of protein C by thrombin in the absence of thrombomodulin. Moreover, IgG D attenuated the inhibitory effect of calcium ions during protein C activation by thrombin, probably by masking E39 within the VR1 site.

Hemodynamic changes during laparoscopic gastroplasty in morbidly obese patients.

BACKGROUND: In nonobese patients, peritoneal insufflation has consistently been shown to influence parameters of preload and afterload as well as cardiac output. Obese patients have an abnormal and particular cardiovascular status. The aim of this study was to investigate the hemodynamic changes induced by an increase of intra-abdominal pressure in morbidly obese patients (MOP). METHODS: Standard general anesthesia was administered to 15 informed MOP (body mass index > 40 kg/m2) scheduled for laparoscopic gastroplasty. Hemodynamic parameters were measured by thermodilution through a pulmonary artery catheter and through invasive blood pressure monitoring. RESULTS: CO2 insufflation with an intra-abdominal pressure of 17 mmHg caused a significant increase of mean arterial pressure (MAP) (33%, P = 0.005), mean pulmonary arterial pressure (MPAP) (40%, P = 0.001), pulmonary capillary wedge pressure (PCWP) (41%, P = 0.001), and central venous pressure (CVP) (55%, P = 0.001). The increase in diastolic filling pressures could be due to an increase in the filling volume or to a decrease in diastolic compliance. Ventricular volumes were not measured but we speculate that the rise in CVP, PCWP and MPAP is due to an increase in intrathoracic pressure as judged by the increase of pulmonary airway pressure. Stroke volume fell slightly (11%, P = 0.008), because of a reduction in transmural pressure and a fall in effective preload. Cardiac output rose slightly (16%, P = 0.005) because of an increase in heart rate (15%, P = 0.014) probably induced by sympathetic stimulation, which only became fully operative after 15 minutes. CONCLUSIONS: When compared to nonobese patients our obese patients tolerated the pneumoperitoneum surprisingly well, without experiencing fall in cardiac output. The hemodynamic consequences of peritoneal insufflation seem to be different in obese and nonobese patients.

Clinical and laboratory characteristics of paediatric and adolescent index cases with venous thromboembolism and antithrombin deficiency. An observational multicentre cohort study.

Venous thromboembolism [TE] is a multifactorial disease and antithrombin deficiency [ATD] constitutes a major risk factor. In the present study the prevalence of ATD and the clinical presentation at TE onset in a cohort of paediatric index cases are reported. In 319 unselected paediatric patients (0.1-18 years) from 313 families, recruited between July 1996 and December 2013, a comprehensive thrombophilia screening was performed along with recording of anamnestic data. 21 of 319 paediatric patients (6.6%), corresponding to 16 of 313 families (5.1%), were AT-deficient with confirmed underlying AT gene mutations. Mean age at first TE onset was 14 years (range 0.1 to 17). Thrombotic locations were renal veins (n=2), cerebral veins (n=5), deep veins (DVT) of the leg (n=9), DVT & pulmonary embolism (n=4) and pelvic veins (n=1). ATD co-occurred with the factor-V-Leiden mutation in one and the prothrombin G20210A mutation in two children. In 57.2% of patients a concomitant risk factor for TE was identified, whereas 42.8% of patients developed TE spontaneously. A second TE event within primarily healthy siblings occurred in three of 313 families and a third event among siblings was observed in one family. In an unselected cohort of paediatric patients with symptomatic TE, the prevalence of ATD adjusted for family status was 5.1%. Given its clinical implication for patients and family members, thrombophilia testing should be performed and the benefit of medical or educational interventions should be evaluated in this high risk population.

A chemically-modified inactive antithrombin as a potent antagonist of fondaparinux and heparin anticoagulant activity.

BACKGROUND: Heparin and its analogs, mediating their anticoagulant activity through antithrombin (AT) activation, remain largely used for the preventive and curative treatment of thrombosis. The major adverse reaction of these drugs is the bleeding risk associated with overdose. Unfractionnated heparin (UFH) can be efficiently and rapidly neutralized by protamine sulfate, but this reversal partially neutralizes low-molecular-weight heparin (LMWH) and is inefficient in reversing fondaparinux. To secure administration of AT-mediated anticoagulants and counteract bleeding disorders, we previously designed a recombinant inactive AT as an antidote to heparin derivatives. OBJECTIVES: To get around the limited production level of recombinant AT, we propose in this study an alternative strategy to produce a chemically modified inactive AT, exhibiting increased heparin affinity, as an antagonist of heparin analogs. METHODS: Plasma-derived AT was chemically modified with 2,3 butanedione, a diketone known to specifically react with the arginine side chain. The chemical reaction was conducted in the presence of heparin to preserve basic residues within the heparin binding site from modifications. RESULTS: AT treated by butanedione and selected for its high heparin affinity (AT-BD) was indeed modified on reactive Arg393 and thus exhibited decreased anticoagulant activity and increased heparin affinity. AT-BD was able to neutralize anticoagulant activity of heparin derivatives in vitro and in vivo and was devoid of intrinsic anticoagulant activity, as assessed by activated partial thromboplastin time assay. CONCLUSIONS: AT-BD appears to be as efficient as protamine to neutralize UFH in vivo but could be more largely used because it also reverses fondaparinux and LMWH.

Comprehensive characterization of the photodissociation pathways of protonated tryptophan.

The photofragmentation of protonated tryptophan has been investigated in a unique experimental setup, in which ion and neutral issued from the photofragmentation are detected in coincidence, in time and in position. From these data are extracted the kinetic energy, the number of neutral fragments associated with an ion, their masses, and the order of the fragmentation steps. Moreover, the fragmentation time scale ranging from tens of nanoseconds to milliseconds is obtained. From all these data, a comprehensive fragmentation mechanism is proposed.

Comparative evaluation of five agglutination techniques and a new miniaturized system for rapid identification of methicillin-resistant strains of Staphylococcus aureus.

The speciation of methicillin-resistant Staphylococcus aureus (MRSA) poses a significant diagnostic problem when rapid identification methods such as slide agglutination tests, are used, because of the high proportion of false-negative reactions. 150 perfectly identified MRSA strains were tested on 5 commonly used agglutination reagents ("Bacto staph latex test", "Monostaph", "Pastorex staph", "Staphaurex", and "Staphyslide test") in comparison with a new micromethod ("RAPIDEC staph") which detects a type of staphylocoagulase within 2 hours by a fluorescence test. The "RAPIDEC staph" reagent enabled identification of all the MRSA while the agglutination tests gave poorer results: "Monostaph" correctly identified 64.6% of strains, "Staphyslide", 59.3%, "Bacto staph latex test", 44.6%, "Pastorex staph", 38.6% and "Staphaurex", 28.6%. These results show that agglutination slide tests are not reliable enough for the identification of MRSA which are more and more encountered in hospital wards. The authors recommend not to use slide agglutination methods. They suggest the tube test for coagulase which is the reference technique, although it is time-consuming and not well standardized. The results of this evaluation encourage the use of the "RAPIDEC staph" reagent since it is an easy-to-use, reliable technique for the rapid identification of Staphylococcus aureus.

Quality of recovery in children: sevoflurane versus propofol.

BACKGROUND: Sevoflurane, with its low pungency and low blood and tissue solubility, is an attractive anaesthetic in paediatric outpatient surgery. Propofol-anaesthesia is recognised for its rapid and clear-headed emergence. This study was designed to compare emergence and recovery characteristics of sevoflurane and propofol anaesthesia for tonsillectomy in children. METHODS: Children aged 3-10 years, undergoing elective tonsillectomy, were randomly assigned to receive propofol (n=25, induction with 3 mg x kg(-1), maintenance with 100-250 microg x kg(-1) min(-1)) or sevoflurane anaesthesia (n=25, induction 7 vol.%, maintenance 2-3 vol.%). Tracheal intubation was performed with alfentanil 20 microg x kg(-1) and atracurium 0.5 mg x kg(-1). Ventilation was controlled to maintain normocapnia and all patients received N2O/O2 (60:40 vol.%) for induction and maintenance of anaesthesia. At the end of surgery infiltration of the operative sites with bupivacaine 2 mg x kg(-1) was provided for postoperative analgesia. Emergence, recovery, discharge times, and incidence of side effects were compared between the two groups. RESULTS: Time to extubation (14 vs 15 min), time to response to simple verbal command (21 vs 21 min) and time to discharge from the recovery room (45 vs 50 min) were similar in the sevoflurane and propofol groups, respectively. There was a significantly greater incidence of postoperative agitation in the sevoflurane group (46%) compared with the propofol group (9%) (P=0.008). This did not, however, delay discharge from the recovery room. The incidence of nausea and vomiting was not significantly different (8% vs 0%; P=0.49). CONCLUSION: In children, recovery from anaesthesia with sevoflurane results in a higher incidence of agitation compared with propofol.

Partial glycosylation of antithrombin III asparagine-135 is caused by the serine in the third position of its N-glycosylation consensus sequence and is responsible for production of the beta-antithrombin III isoform with enhanced heparin affinity.

Two antithrombin III (ATIII) isoforms occur naturally in human plasma. The alpha-ATIII isoform has four N-linked oligosaccharides attached to asparagines 96, 135, 155, and 192. The beta-ATIII isoform lacks carbohydrate on asparagine-135 (N135), which is near the heparin binding site, and binds heparin with higher affinity than does alpha-ATIII. Two isoforms are also produced when the normal human ATIII cDNA sequence is expressed in baculovirus-infected insect cells, and the recombinant beta' isoform similarly binds heparin with higher affinity than the recombinant alpha' isoform. Consensus sequences (CSs) of the ATIII N-glycosylation sites are N-X-S for 135 and N-X-T for 96, 155, and 192. On the basis of database and in vitro glycosylation studies suggesting that N-X-S CSs are utilized less efficiently than N-X-T CSs, we hypothesized that the beta-ATIII isoform might result from inefficient core glycosylation of the N135 N-X-S CS due to the presence of a serine, rather than a threonine, in the third position. ATIIIs with N-X-S, N-X-T, and N-X-A consensus sequences were expressed in baculovirus-infected insect cells. In contrast to the N-X-S sequence, which expressed a mixture of alpha' and beta' molecules, the N-X-T variant produced alpha' exclusively, while the N-X-A variant produced beta' exclusively. Thus, serine in the third position of the N135 CS is responsible for its "partial" glycosylation and leads to production of beta-ATIII.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation-induced exposure of the thrombin anion-binding exosite. Interactions of recombinant mutant prothrombins with thrombomodulin and a thrombin exosite-specific antibody.

The activation of serine protease zymogens involves conformational changes that increase the affinity of substrate binding and the activity of the catalytic center. The activation of prothrombin is particularly complex and requires several cleavages in the proenzyme region in addition to the conserved activation cleavage after Arg320. To understand how these cleavages lead to the exposure of the thrombin anion-binding exosite, a major macromolecular recognition site, interactions of recombinant human prothrombin derivatives with thrombomodulin, and an exosite-specific antibody were studied by competition binding and immunoprecipitation. By either method, the anion-binding exosite is not functional on prethrombin 2, which is cleaved after Arg271 and lacks fragment 1.2, nor on meizothrombin, which is cleaved only after Arg320. In contrast, the exosite is fully exposed on meizothrombin des-F1, which is cleaved after both Arg320 and Arg155 and therefore lacks amino-terminal fragment 1 (F1). Thus, two events are required to create the exosite. First, cleavage after Arg320 causes conformational changes that are much more extensive than those accompanying the activation of trypsinogen. Second, removal of amino-terminal F1 is necessary, perhaps to relieve steric hindrance. These results indicate that the F1 fragment regulates access to the thrombin exosite. The properties of meizothrombin des-F1 suggest that this prothrombin derivative could have a biological function.

Use of remifentanil in a patient with chronic hepatic failure.

We describe a 73-yr-old woman anaesthetized for a laminectomy. She suffered from hepatic failure with mild encephalopathy complicated by several exacerbations associated with sedative and opioid therapy. The challenge for anaesthesia management was to provide adequate analgesia and avoid causing hepatic encephalopathy during and after the surgery. We used remifentanil to provide intraoperative and postoperative analgesia, because it has a short duration of action and does not require hepatic metabolism. We closely monitored the respiratory and the neurological status throughout the administration and conclude that remifentanil can provide perioperative analgesia in patients at risk of developing hepatic encephalopathy.

Nramp 2 (DCT1/DMT1) expressed at the plasma membrane transports iron and other divalent cations into a calcein-accessible cytoplasmic pool.

Nramp2, also known as DMT1 and DCT1, is a 12-transmembrane (TM) domain protein responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. Nramp2/DMT1 produces by alternative splicing two isoforms differing at their C terminus (isoforms I and II). The subcellular localization, mechanism of action, and destination of divalent cations transported by the two Nramp2 isoforms are not completely understood. Stable CHO transfectants expressing Nramp2 isoform II modified by addition of a hemaglutinin epitope in the loop defined by the TM7-TM8 interval were generated. Immunofluorescence with permeabilized and intact cells established that Nramp2 isoform II is expressed at the plasma membrane and demonstrated the predicted extracytoplasmic location of the TM7-TM8 loop. Using the fluorescent, metal-sensitive dye calcein, and a combination of membrane-permeant and -impermeant iron chelators, Nramp2 transport was measured and quantitated with respect to kinetic parameters and at steady state. Iron transport at the plasma membrane was time- and pH-dependent, saturable, and proportional to the amount of Nramp2 expression. Iron uptake by Nramp2 at the plasma membrane was into the nonferritin-bound, calcein-accessible so-called "labile iron pool." Ion selectivity experiments show that Nramp2 isoform II can also transport Co(2+) and Cd(2+) but not Mg(2+) into the calcein-accessible pool. Parallel experiments with transfectants expressing the lysosomal Nramp1 homolog do not show any divalent cation transport activity, establishing major functional differences between Nramp1 and Nramp2. Monitoring the effect of Nramp2 on the calcein-sensisitve labile iron pool allows a simple, rapid, and nonisotopic approach to the functional study of this protein.

Perturbation of rat brain serotonergic systems results in an inverse relation between substance P and serotonin concentrations measured in discrete nuclei.

Since substance P (SP) has been demonstrated to coexist with serotonin (5-HT) in the same population of neurons in the descending raphe system, we have studied the possibility of interactions between these neurotransmitters in other brain areas. Brain nuclei were punched from frozen 300-micron slices of rat brain and extracted with 0.1 M HCIO4 or 2 M acetic acid prior to assay, respectively, of 5-HT content by HPLC with electrochemical detection or SP content by specific radioimmunoassay. Ten days after injection of rats with the 5-HT neurotoxin P-chloroamphetamine (PCA, 10 mg/kg, B.W., i.p.) or 3 days after 5-HT synthesis blockade with p-chlorophenylalanine (PCPA, 300 mg/kg, B.W., i.p.), the 5-HT content of all brain nuclei studied was reduced by means of, respectively, 50% and 81%. In PCA-treated animals, the SP content of the periaqueductal grey matter was significantly increased; PCPA treatment caused, in addition, large increases in the SP content of five other brain nuclei. Blockade of 5-HT receptors by methysergide (15 mg/kg for 5 days) did not significantly change 5-HT levels or turnover, but resulted in 50-200% increases in the SP content of 10 of the 28 brain nuclei studied. Significant decreases in the SP content of numerous areas were seen following treatments (pargyline 30 mg/kg, alone or in combination with 5-hydroxytryptophan, 60 mg/kg) that simultaneously increased 5-HT levels. These results illustrate the modulation of distinct SP-containing systems of the rat brain by perturbation of central serotoninergic pathways and indicate a reciprocal relationship between the SP and 5-HT concentrations of numerous brain nuclei, in particular n. striae terminalis, n. raphe dorsalis, n. accumbens, n. septi, substantia grisea centralis, and n. raphes medianus.