Erythrocytes

In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the...

Infection of red blood cells (RBC) subjects the malaria parasite to oxidative stress. Therefore, efficient antioxidant and redox systems are required to prevent damage by reactive oxygen species. Plasmodium spp. have thioredoxin and glutathione (GSH) systems that are thought to play a major role as antioxidants during blood stage infection. In this report, we analyzed a critical component of the GSH biosynthesis pathway using reverse genetics. Plasmodium berghei parasites lacking expression of gamma-glutamylcysteine synthetase (c-GCS), the rate limiting enzyme in de novo synthesis of GSH, were generated through targeted gene disruption thus demonstrating, quite unexpectedly, that c-GCS is not essential for blood stage development. Despite a significant reduction in GSH levels, blood stage forms of pbggcs 2 parasites showed only a defect in growth as compared to wild type. In contrast, a dramatic effect on development of the parasites in the mosquito was observed. Infection of mosquitoes with pbggcs 2 parasites resulted in reduced numbers of stunted oocysts that did not produce sporozoites. These results have important implications for the design of drugs aiming at interfering with the GSH redox-system in blood stages and demonstrate that de novo synthesis of GSH is pivotal for development of Plasmodium in the mosquito.

We have devised an analytical method for the determination of fatty acid composition of erythrocyte membrane sphingomyelin by chemical ionization mass spectrometry combined with capillary column gas-liquid chromatography. Fatty acid composition of erythrocyte membrane sphingomyelin from 8 patients with adrenoleukodystrophy (ALD) and 16 healthy controls were exam-

A large number of micropropagated Euphorbia millii shoots from temporary immersion bioreactor showed thick broad leaves that were translucent, wrinkled and/or curled and brittle, symptoms of hyperhydricity. The environment inside bioreactor normally used in plant micropropagation is characterised by high relative humidity, poor gaseous exchange between the internal atmosphere of the bioreactor and its surrounding environment, and the accumulation of ethylene, conditions that may induce physiological disorders. A comparison of hyperhydric shoots (HS) with normal plants shows marked increase in malondialdehyde (MDA) content in HS plants. MDA, a decomposition product of polyunsaturated fatty acids hydroperoxides, has been utilized very often as a suitable biomarker for lipid peroxidation, which is an effect of oxidative damage. This hypothesis is also confirmed by the higher lipoxygenase (LOX) activity in HS plants. The potential role of antioxidant enzymes in protecting hyperhydric shoots from oxidative injury was examined by analyzing enzyme activities and isozyme profiles of hyperhydric and non-hyperhydric leaves of E. millii. Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity were significantly higher in hyperhydric tissue as compared to non-hyperhydric normal leaf tissue. After native polyacrylamide gel electrophoresis (PAGE) analysis, seven SOD isoenzymes were detected and the increase in SOD activity observed in hyperhydric tissue seemed to be mainly due to Mn-SOD and Cu/Zn-SOD. The activity of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) was proportionally increased in HS tissue compared to normal leaves indicating a crucial role in eliminating toxic H 2 O 2 from plant cells. The depletion of GSH and total glutathione in spite of higher GR activities observed in HS tissue indicates that mechanism of antioxidant defense was by enhanced oxidation of GSH to GSSG by DHAR yielding ascorbate (AA). The antioxidant metabolism has been shown to be important in determining the ability of plants to survive in hyperhydric stress and the up regulation of these enzymes would help to reduce the build up of ROS.

Blood doping represents one main trend in doping strategies. Blood doping refers to the practice of boosting the number of red blood cells (RBCs) in the bloodstream in order to enhance athletic performance, by means of blood transfusions, administration of erythropoiesis-stimulating substances, blood substitutes, natural or artificial altitude facilities, and innovative gene therapies. While detection of recombinant EPO and homologous transfusion is already feasible through electrophoretic, mass spectrometry or flow cytometry-based approaches, no method is currently available to tackle doping strategies relying on autologous transfusions. We exploited an in vitro model of autologous transfusion through a 1:10 dilution of concentrated RBCs after 30 days of storage upon appropriate dilution in freshly withdrawn RBCs from the same donor. Western blot towards membrane Prdx2 and Percoll density gradients were exploited to assess their suitability as biomarkers of transfusion. Membrane Pr...

The distribution of blood-group A and B determinants was studied by isolating blood-group ABH-active polyglycosyl peptides from delipidated human blood-group AB erythrocyte membranes after extensive digestion with pronase followed by chromatography on Bandeiraea simplicifolia I (BsI) lectin coupled to Sepharose. 20% of the polyglycosyl peptides were bound to BsI lectin. The glycopeptides bound were further fractionated using the blood-group-A-specific lectin from Vicia cracca (Vc). Approximately half of these were bound to the Vc lectin. The glycopeptides, which were bound to the Vc column, were not bound to the blood-group-B-specific isolectin from B. simplicifolia (BsIB4) whereas the Vc-unbound glycopeptides readily bound. The results indicate that in the polyglycosyl peptides isolated from AB erythrocytes A and B determinants are located in different carbohydrate chains. The polyglycosyl peptides, which did not bind to BsI lectin, were composed on the average of 30 monosaccharide units and those that bound contained on the average 55 monosaccharide units. The sugar composition was similar in both fractions except that N-acetylgalactosamine was found only in the BsI-bound glycopeptides. The substitution patterns of the monosaccharides were quite similar in both fractions except 2,3-O-linked galactose, which was enriched 7.5-fold in the BsI-bound glycopeptides and 3,6-O-linked galactose, which also enriched in the BsI-bound glycopeptides suggesting that these have a more branched structure than the BsI-unbound glycopeptides. Glycopeptides derived from bands 3 and 4.5 were prepared from A1B-blood-group erythrocyte membranes and fractionated as above. 25% of the glycopeptides were bound to BsI-lectin from both samples. 70% of the BsI-bound material from band 3 was bound to Vc lectin and 60% from band 4.5. The results indicate heterogeneity in the glycosylation of these bands.

Hemoglobin (Hb), packed cell volume (PCV), red blood cells (RBCs), mean cell volume (MCV), and mean cell hemoglobin concentration (MCHC) were measured by a single operator in a single laboratory by means of an electronic particle counter in 942 healthy adults (49 1 females and 45 1 males) residing at five altitudes: 0, 1000, 1860, 2220, and 2670 meters above sea level. The subjects were carefully screened clinically, and subjects with low transferrin saturation (< 15%) were excluded. In both sexes there was a differential behavior as a function of altitude, of Hb and PCV on the one hand, and number of RBCs on the other. The findings suggest the presence of two sequential mechanisms of adaptation t o progressively lower atmosphere oxygen pressure: One operating from sea level to 1860 meters, which leads t o a progressively increasing number of relatively microcytic RBC; and a second onefrom 1860 to 2670 metersin which there is an increased but constant number of progressively more normocytic RBC, so that a simplistic model of equal magnitude increases in the three parameters and is seen at 2670 meters, but not at the intermediate altitudes. The middle group's comparativity, essential t o these interpretations, apparently was achieved with regard to time elapsed between sampling and testing and with regard to the people integrating the groups.

The emergence of strains of multidrug-resistant Gram-negative bacteria mandates a search for new types of antimicrobial agents. Alyteserin-2a (ILGKLLSTAAGLLSNL.NH 2) is a cationic, a-helical peptide, first isolated from skin secretions of the midwife toad, Alytes obstetricans, which displays relatively weak antimicrobial and haemolytic activities. Increasing the cationicity of alyteserin-2a while maintaining amphipathicity by the substitution Gly 11 ! Lys enhanced the potency against both Gram-negative and Gram-positive bacteria by between fourfold and 16-fold but concomitantly increased cytotoxic activity against human erythrocytes by sixfold (mean concentration of peptide producing 50% cell death; LC 50 = 24 mM). Antimicrobial potency was increased further by the additional substitution Ser 7 !Lys, but the resulting analogue remained cytotoxic to erythrocytes (LC 50 = 38 mM). However, the peptide containing D-lysine at positions 7 and 11 showed high potency against a range of Gram-negative bacteria, including multidrugresistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (minimum inhibitory concentration = 8 mM) but appreciably lower haemolytic activity (LC 50 = 185 mM) and cytotoxicity against A549 human alveolar basal epithelial cells (LC 50 = 65 mM). The analogue shows potential for treatment of nosocomial pulmonary infections caused by bacteria that have developed resistance to commonly used antibiotics.

In order to assess peripheral levels and activities of a broad spectrum of non-enzymatic and enzymatic antioxidants in elderly subjects with mild cognitive impairment (MCI) and Alzheimer's disease (AD), plasma levels of water-soluble (Vitamin C and uric acid) and of lipophilic (Vitamin A, Vitamin E and carotenoids including lutein, zeaxanthin, ␤-cryptoxanthin, lycopene, ␣and ␤-carotene) antioxidant micronutrients as well as activities of plasma and red blood cell (RBC) superoxide dismutase (SOD) and of plasma glutathione peroxidase (GPx) were measured in 25 patients with MCI, 63 AD patients and 53 controls. Peripheral levels and activities of antioxidants were similarly lower in MCI and AD patients as compared to controls. As MCI may represent a prodromal stage of AD, and oxidative damage appears to occur as one of the earliest pathophysiological events in AD, an increased intake of antioxidants in patients with MCI could be helpful in lowering the risk of conversion to dementia.

Background: 1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH) 3 , in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained in vivo from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the in vivo to a bioreactor-based method.

Type 1 fimbriae are surface organelles on Escherichia coli, which mediate specific binding to D-mannosecontaining structures. These fimbriae are heteropolymers composed of a major building element, the FimA protein, and small amounts of the FimF, FimG and FJmH proteins. The FimH protein is uniquely responsible for the D-mannose receptor binding. In this work data are presented which indicate that the major subunit of type 1 fimbriae is dispensable for D-mannose-specific binding. A recombinant strain was studied which harboured an insertional deletion in the fimA gene, and was thereby unable to produce type 1 fimbriae; however, it was still able to express a D-mannose-binding phenotype. However, the deletion resulted in a 25-fold reduction of the adhesive potential, as measured by binding to D-mannosecoated Sepharose beads. Serological and specific receptor binding evidence is presented that suggests that the FimH adhesin is capable of being exposed on the bacterial surface without being an integral part of the fimbriae. . 'For correspondence. Tel. 42 884066; Fax 45 932809.

In this study, the total phenolic and flavonoid contents, the 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging ability and the ferric reducing power (FRAP) of Aloe vera were measured to determine the antioxidant activity of this species. The in vivo antidiabetic effects of the plant were also investigated using streptozotocin-induced type 2 diabetic model rats that were divided into five groups based on the treatment received:

The aim of this study was to demonstrate changes in acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities, tyrosine aminotransferase activity (TAT) and plasma corticosterone level, neuroexcitability and behavior following 24 hours and 4 weeks of soman sublethal inhalation exposure at low level. AChE activity in erythrocytes and BuChE activity in plasma was decreased (dependent on the concentration of soman) 24 h and 4 weeks after the exposure. Similar decrease in AChE activity in different brain parts was observed. One of stressogenic parameters (TAT) was changed after 24 h exposure only. 4 weeks after the exposure, these parameters (corticosterone and TAT) were in the range of normal values. Behaviour of experimental animals was changed 24 h after the exposure persisting 4 weeks after the exposure as well as neuroexcitability.

Reduction of nitrite (NO 2 Ϫ ) provides a major source of nitric oxide (NO) in the circulation, especially in hypoxemic conditions. Our previous studies suggest that xanthine oxidoreductase (XOR) is an important nitrite reductase in the heart and kidney. Herein, we have demonstrated that conversion of nitrite to NO by blood vessels and RBCs was enhanced in the presence of the XOR substrate xanthine (10 mol/L) and attenuated by the XOR inhibitor allopurinol (100 mol/L) in acidic and hypoxic conditions only. Whereas endothelial nitric oxide synthase (eNOS) inhibition had no effect on vascular nitrite reductase activity, in RBCs L-NAME, L-NMMA, and L-arginine inhibited nitrite-derived NO production by Ͼ50% (PϽ0.01) at pH 7.4 and 6.8 under hypoxic conditions. Western blot and immunohistochemical analysis of RBC membranes confirmed the presence of eNOS and abundant XOR on whole RBCs. Thus, XOR and eNOS are ideally situated on the membranes of RBCs and blood vessels to generate intravascular vasodilator NO from nitrite during ischemic episodes. In addition to the proposed role of deoxyhemoglobin, our findings suggest that the nitrite reductase activity within the circulation, under hypoxic conditions (at physiological pH), is mediated by eNOS; however, as acidosis develops, a substantial role for XOR becomes evident. (Circ Res. 2008;103:957-964.)

Human erythrocyte sialoglycoproteins, or glycophorins, were chromatographed by lectin-affinity and metal-interaction chromatography on high-performance liquid chromatographic columns. Glycophorins A, B and C were separated from other proteins and from glycophorin E by using a column containing wheat germ agglutinin, immobilized on a microparticulate silica support. The glycophorins were adsorbed on the lectin column from a mobile phase containing 0.25 A4 sodium chloride and recovered by stepwise desorption with 0.2 M N-acetylglucosamine solution. Glycophorins A, B and C were separated into the individual components on a silica-bound iminodiacetic acid stationary phase in the copper(H) chelate form. The separation of the glycophorins by metal-interaction chromatography was accomplished by decreasing salt gradient elution. Retention times and resolution of the individual glycophorins were sensitive to the initial sodium chloride concentration and the pH of the eluent. Addition of methanol to the eluent increased the resolution. The effects of linear, decreasing gradients of pH and methanol in 25 rnM phosphate buffer on the resolution of glycophorins were also investigated. In both types of chromatography the mobile phases contained 0.05% (w/v) sodium dodecyl sulfate. With octylglycoside or CHAPS in the eluent glycophorins A and C could not be eluted. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze all the chromatographic results.

Long-chain polyunsaturated fatty acids (LCPUFA) in breastmilk, specifically docosahexaenoic acid (DHA), are important for infant brain development. Accretion of DHA in the infant brain is dependent on DHA-status, intake and metabolism. The aim of this study was to describe changes in maternal and infant erythrocyte (RBC) DHA-status during the first four months of lactation. We examined 17 mothers and their term infants at 1, 2 and 4 months of age. Milk samples and RBC from the mothers and infants were obtained and analysed for fatty acid composition. Comparative analysis of the results showed that the content of DHA in maternal RBC-phosphatidylcholine (PE) decreased over the four month period and this was not accompanied by a decrease in DHA in infant RBC-PE ( P = 0.005). The ratio of n-6 PUFA to n-3 PUFA increased over time in maternal RBC-PE, but not in infant RBC-PE ( P < 0.001). The level of 22:5n-6 and the ratio of LCPUFA to precursor PUFAs in infant RBC was higher than in maternal RBC phospholipids. ( P = and P < 0.001 respectively). We found a decrease in the level of LCPUFA in milk, specifically AA. However, we did not observe a significant decrease in milk DHA, which may have been due to two outliers. These results indicate better DHA-status and a higher n-3/n-6 PUFA in RBC of infants than in mothers. Whether these differences reflect preferential n-3 PUFA transfer via breastmilk or differences in PUFA-metabolism and utilization remains to be shown.

In 13 subjects affected by 8-thalassaexnia major, in three subjects affected by 8-tlialassaemia minor and in five normal healthy persons haemoglobin synthesis and the survival of red cells transfused into normal, group compatible, healthy recipients has been studied. The existence of an excess of newly synthesized a-chains and of a negative correlation between the excess a-chain and the red-cell survival has been demonstrated. The harmful role of the a-chain excess on the erythrocyte and the implications of this finding are discussed.

This study assessed the effect of vitamin B6 status on immune responses in mechanically ventilated, critically ill patients and compared the results with those of healthy controls. Methods: This was designed as a cross-sectional observational study. Forty patients in the intensive care unit successfully completed this study. Vitamin B6 intake was recorded for 8 d. Severity of illness (Second Acute Physiology and Chronic Health Evaluation score) was recorded. Thirty-eighty healthy controls were recruited from the physical check unit of Taichung Veterans General Hospital (Taichung, Taiwan). All control subjects were given instruction on how to complete a 24-d diet recall. Vitamin B6 status was assessed by direct measures (plasma pyridoxal 5=-phosphate [PLP] and 4-pyridoxic acid) and indirect measures (erythrocyte alanine and aspartate aminotransferase activity coefficients). Levels of serum albumin, hemoglobin, hematocrit, high-sensitivity C-reactive protein, and immune responses (white blood cell, neutrophil, total lymphocytes, T lymphocytes [CD3], B lymphocytes [CD19], T-helper cells [CD4], and suppressor cells [CD8]) were determined. Results: Critically ill patients had sufficient vitamin B6 intake but showed marginal PLP deficiency (20.9 Ϯ 1.5 nmol/L). In addition, critically ill patients had significantly lower and abnormal immune responses than did healthy controls. There was no significant correlation of vitamin B6 intake and erythrocyte alanine and aspartate aminotransaminase activity coefficients with immune indices. Plasma PLP concentration was strongly negatively correlated with high-sensitivity C-reactive protein level. However, plasma PLP was significantly associated with immune responses after adjustment for age, sex, high-sensitivity C-reactive protein, and the other four vitamin B6 indicators. Conclusions: Plasma PLP is a significant indicator of immune responses in human subjects. Further research is warranted to study whether vitamin B6 supplementation in critically ill patients improves their immune responses.

Background: Low folate status is associated with poor cognitive function and dementia in the elderly. Since 1998, grain products in the United States have been fortified with folic acid, which has reduced the prevalence of folate deficiency and hyperhomocysteinemia. Objective: We investigated whether folate status is associated with cognitive function and dementia in a cohort of elderly Latinos (aged ͧ 60 y; n ҃ 1789) exposed to folic acid fortification. Design: Global cognitive function was assessed by the Modified Mini-Mental State Examination (3MSE) and specific cognitive functions by cross-culturally validated neuropsychological tests. Dementia was diagnosed according to the American Psychiatric Association Diagnostic and Statistical Manual of Mental Disorders, 3rd edition revised, and California Alzheimer Disease Diagnostic and Treatment criteria. Red blood cell (RBC) folate was measured by automated chemiluminescence and total plasma homocysteine by HPLC. Results: The prevalence of folate deficiency (RBC folate ͨ 160 ng/mL) was 1%. After control for confounding by homocysteine, vitamin B-12, creatinine, demographic variables, and depressive symptom score, RBC folate was directly associated with 3MSE (P ҃ 0.005) and delayed recall (P ҃ 0.007) scores. In addition, adjusted odds ratios for low 3MSE score (ͨ78) and dementia diagnosis per unit increase in RBC folate were significantly below unity (P ͨ 0.008), which indicated that the relative risks of cognitive impairment and dementia decreased with increasing RBC folate concentration. In contrast, adjusted odds ratios for low 3MSE score and dementia diagnosis per unit increase in homocysteine were not significant. Conclusion: RBC folate is directly associated with cognitive function scores and is inversely associated with dementia in elderly Latinos despite folic acid fortification.

The phospholipid organization in monkey erythrocytes upon Plasmodium knowlesi infection has been studied. Parasitized and nonparasitized erythrocytes from malaria-infected blood were separated and pure erythrocyte membranes from parasitized cells were isolated using Affi-Gei beads. In this way, the phospholipid content and composition of (i) the membrane of nonparasitized cells, (ii) the erythrocyte membrane of parasitized cells and (iii) the parasite could be determined. The phospholipid content and composition of the erythrocyte membranes of nonparasitized and parasitized cells and erythrocytes from chloroquine-treated monkeys cured from malaria, were the same as in normal erythrocytes. The phnspholipid content of the parasite increased during its development, but its composition remained unchanged. Three independent techniques, i.e., treatment of intact cells with phospholipase A 2 and sphingomyelinase C, fluorescamine labeling of aminophospholipids and a phosphatidylcholine-transfer protein-mediated exchange procedure have been applied to assess the disposition of phnspholipids in: (i) erythrocytes from healthy monkeys, (ii) nonparasitized and parasitized erythrocytes from monkeys infected with Plasmodium knowlesi, and (iii) erythrocytes from monkeys that had been cured from malaria by chloroquine treatment. The results obtained by these experiments do not show any abnormality in phospholipid asymmetry in the erythrocyte from malaria-infected (splenectomized) monkeys, neither in the nonparasitized cells, nor in the parasitized cells at any stage of parasite development. Nevertheless, a considerable degree of lipid bilayer destabilization in the membrane of the parasitized cells is apparent from the enhanced exchangeability of the PC from those cells, as well as from their increased permeability towards fluorescamine.

Background Both cigarette smoke and ultraviolet (UV) radiation are known to cause changes of the skin which can be regarded as premature ageing. Objectives To assess the theory that the effects of these two exposures could be linked by a phototoxic action of cigarette smoke. Methods A photohaemolysis test was used, in which human erythrocytes were incubated with cigarette smoke condensate, followed by UV irradiation and measurement of exposure-dependent haemolysis.

FP-A and FP-B are LDPs produced by the plant pathogen Pseudomonas fuscovaginae. As expected from their primary structure, they shared a similar mechanism of action with the better characterized SPs, synthesized by strains of Pseudomonas syringae pv. syringae. Indeed, they displayed hemolytic activity on human erythrocytes and were able to induce calcein release from LUVs: the effect was dependent on the concentration of the FPs and the lipid composition of the liposome and, in particular, it increased with the SM content of the membrane. The permeabilizing activity was further investigated on PLMs. FPs were able to open pores on pure POPC membranes. Pore opening was strongly voltage dependent: by switching the potential from negative to positive values, an increase in the absolute amplitude of transmembrane current was induced with simultaneous closure of pores. In 0.1 M KCl both FPs' pores had a conductance of 4 and 9 pS at − 140 mV and + 140 mV, respectively. Studies on ion selectivity indicated that FPs formed cation-selective channels. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.

Introduction: Severe malaria has been attributed partly to the sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the microvasculature of vital host organs. Identification of P. falciparum cytoadherence phenotypes that are associated with severe malaria may lead to the development of novel strategies against life-threatening malaria.

2-Fluoro-ara-AMP (fludarabine phosphate) is a purine analogue with anti-neoplastic activity in lymphoproliferative malignancies. Fludarabine phosphate activity and toxicity is schedule-dependent; multiple daily administrations (for five days) are more effective than single dose. We have encapsulated fludarabine phosphate in human erythrocytes and found that it is slowly released as fludarabine for more than four days. Encapsulated fludarabine phosphate does not affect erythrocyte metabolism and is rapidly converted by erythrocyte enzymes both to fludarabine with a K m of 0.4 mM and a Vma x of 20 nmol/min per g hemoglobin and to fludarabine diphosphate and triphosphate. The apparent K m for fludarabine monophosphate in the phosphorylation reaction was 0.4 mM and the Vma x 40 nmol/min per g hemoglobin. In the phosphorylation of 2-fluoro-ara-AMP to the di-and triphosphate derivatives, ATP was the phosphate donor with apparent K m of 0.12 and 1.0 mM, respectively. During incubations of 2-fluoro-ara-AMP-loaded erythrocytes at 37°C fludarabine was found in equilibrium between the erythrocyte and the culture medium suggesting that permeation of the erythrocyte membrane is not rate-limiting. Thus, fludarabine phosphate-loaded erythrocytes might be used as a slow-delivery system for fludarabine administration in the treatment of lymphoid malignancies.

To accurately phenotype red cells from patients with a positive direct antiglobulin test (DAT), nonlytic elution procedures were assessed for their ability to dissociate IgG from antibody-coated red cells without altering red cell antigen expression. STUDY DESIGN AND METHODS: Antibodies coating red cells that were sensitized in vivo (warm-reactive autoantibodies: 8 patients) or in vitro were eluted by using glycine-HCI and EDTA (acid/ EDTA), heat (56"C, 10 min), or chloroquine method. RESULTS: Acid/EDTA elution gave the best results, reducing DAT positivity to microscopic levels or rendering the DAT negative in 48 of 50 instances, whereas 4 samples remained resistant to heat elution and 24 to chloroquine. Standard DAT agglutination scores demonstrated that both acid/EDTA and heat elution were superior to the chloroquine method (p<O.OOOl). With the gel low-ionic-strength saline indirect antiglobulin test, acid/ EDTA was superior to heat (p<O.OOl). Overall, acid/ EDTA elution dissociated more antibodies than heat (p<O.OOOl), especially for Kell system (K, k, Kpa, Kpb) alloantibodies. Common red cell antigens, other than Kell system antigens, were unaffected by acid/EDTA elution. In contrast, the expression of most blood group antigens was diminished after heat elution. However, it was possible to type red cell antigens by using gel low-ionicstrength saline indirect antiglobulin tests or tube agglutination methods.

Imaging was done in 80 patients with gastrointestinal hemorrhage using red blood cells labeled with 99mTc by a modified in vivo technique. Bleeding was detected in 65% of 40 patients with bright red blood per rectum, 71% of 35 patients with melena, and in none of the five patients with occult bleeding and chronic anemia. No clinical evidence of further

Previously symptomatic and permanently asymptomatic carriers of a gene mutation for acute intermittent porphyria as well as matched controls were screened with regard to a series of variables of possible relevance to the development of porphyric symptoms. The basis for the study was a concept of acute porphyria as a condition of a permanent system overload of oxidative stress, with long term effects on hepatic and renal tissue, and with instances of periodic overload of free radicals giving rise to acute neurologic involvement.

To examine the development and tracking of long-term vitamin B-6 status from infancy to early adolescence, measurements of erythrocyte pyridoxal 5&#39;-phosphate concentration (EPLP), the erythrocyte aspartate transaminase (EAST) stimulation test including measurements of basal activity (EASTo) and activation coefficient (alpha EAST), were made in a follow-up study of healthy children aged 2 (n = 139), 4 (n = 147), 6 (n = 157), 9 (n = 159) and 12 mo (n = 188) and 5 y (n = 148). The EAST stimulation test was repeated at 11 y (n = 153). Vitamin B-6 status, high during infancy, reached the adult level by 5 y of age. The 10th to 90th percentile ranges for EPLP values were 61-201 nmol/L at 4 mo, 49-101 nmol/L at 12 mo and 27-59 nmol/L at 5 y. The respective ranges for Easto were 16-24 microkat/L at 4 mo, 13-19 microkat/L at 12 mo, 9-14 microkat/L at 5 y and 25-39 microkat/L at 11 y of age. For alpha EAST values were 1.29-1.54 at 4 mo, 1.48-1.77 at 12 mo, 1.70-2.07 at 5 y and 2.00-2.57 at...

The moderate heat treatment of amphotericin B (AmB) in its micellar form (M-AmB) results in superaggregates (H-AmB) that present a substantially lower toxicity and similar activity. The aim of this work was to evaluate the H-AmB behavior after a freeze-drying process. H-AmB and M-AmB micelles were evaluated before and after freeze-drying concerning their physicochemical and biological properties by spectrophotometry and activity/toxicity assay, respectively. Four concentrations of M-AmB and H-AmB were studied aiming to correlate their aggregation state and the respective biological behavior: 50 mg L −1 , 5 mg L −1 , 0.5 mg L −1 , and 0.05 mg L −1. Then, potassium leakage and hemoglobin leakage from red blood cells were used to evaluate the acute and chronic toxicity, respectively. The efficacy of M-AmB and H-AmB formulations was assessed by potassium leakage from Candida albicans and by the broth microdilution method. After heating, in addition to an evident turbidity, a slight blueshift from 327 to 323 nm was also observed at the concentrations of 50 and 5 mg L −1 for H-AmB. Additionally, an increase in the absorbance at 323 nm at the concentration of 0.5 mg L −1 was detected. Concerning the toxicity, H-AmB caused significantly lower hemoglobin leakage than M-AmB. These results were observed for H-AmB before and after freeze-drying. However, there was no difference between H-AmB and M-AmB concerning their activity. Accordingly, the freeze-drying cycle did not show any influence on the behavior of heated formulations, highlighting the suitability of such a method to produce a new AmB product with a long shelf life and with both greater efficiency and less toxicity.

There is a growing body of evidences that near infrared (NIR) light exerts beneficial effects on cells. Its usefulness in the treatment of cancer, acute brain injuries, strokes and neurodegenerative disorders have been proposed. The mechanism of NIR action is probably of photochemical nature, however it has not been fully understood. Here, using relatively simple biological model, human red blood cells (RBCs), and polychromatic non-polarized light source, we investigate the impact of NIR radiation on the oxygen carrier, hemoglobin (Hb) and anion exchanger (AE1, Band 3). Exposition of intact RBCs on NIR causes quaternary transitions of Hb, dehydration of proteins and decreases the amount of physiologically inactive methemoglobin, as detected by Raman spectroscopy. These effects are accompanied by lowering of intracellular pHi and changes of the cell membrane topography, documented by atomic force microscopy (AFM). All those changes are in the line with our previous studies where alterations of membrane fluidity and membrane potential were attributed to NIR action on the RBCs. The rate of the above listed changes depends strictly on the dose of NIR the cells receive, nonetheless it should not be considered as a thermal effect.

T cell subpopulations (Ty and T-y cells) were examined in the peripheral blood from fourteen patients with mycosis fungoides and S&ary syndrome. One patient with Sezary syndrome having low lymphocyte count had higher proportions of Tr cells when compared to controls while the other with high lymphocyte count (75'5 SCzary cells) lacked Tr cells and had normal proportions of TEL cells. T cells from a third patient with SCzary syndrome having high lymphocyte count (95% SCzary cells) lacked almost completely both Tp and Tr cells. Three of eleven patients with mycosis fungoides had a high proportion of Tr cells and one had a high proportion of Tp cells. Study of T cells in the peripheral blood, lymph nodes, and bone marrow from two patients with mycosis fungoides demonstrated that the quantitative abnormality of TEL and Ty cells is shared by the peripheral blood and bone marrow and not by the lymph nodes. Heterogeneity of T cells subsets in mycosis fungoides appears to be in non-malignant T cells. However, in SCzary syndrome malignant Sezary T cells demonstrate heterogeneity with regard to receptors for IgM (Tr) and IgG (Tr).

It is generally assumed that bumetanide possesses some selectivity for the renal Na-K-Cl cotransporter NKCC2, although the results are scarce in the literature and comparisons were done with extra-renal NKCC1 at its basal, almost silent state. Here we investigated NKCC2/NKCC1 selectivity of loop diuretic drugs (bumetanide, piretanide and furosemide) as a function of the NKCC1 activated state (NKCC1 was activated by hypertonic media). NKCC2 activity was measured in isolated rat medullary thick ascending limb (mTAL) and NKCC1 in rat thymocytes and erythrocytes. When NKCC2 was compared with NKCC1at its activated state, all three diuretic drugs inhibited NKCC2 and NKCC1 with the same potency (bumetanide pIC 50 =6.48, 6.48 and 6.47; piretanide pIC 50 =5.97, 5.99 and 6.29; and furosemide pIC 50 =5.15, 5.04 and 5.21 for mTAL NKCC2, erythrocyte NKCC1 and thymocyte NKCC1, respectively). Basal NKCC1 exhibited a lower diuretic sensitivity, although with marked differences depending on the diuretic drug and the cell type in consideration and with the notable exception of furosemide in erythrocytes. Molecular modelling showed that bumetanide and piretanide possess four potentially active groups, of which three are shared with furosemide at similar intergroup distances. Of these three common groups, one should not bind to basal NKCC1 in thymocytes. The fourth (phenoxy) group (absent in furosemide) confers higher lipophilicity and should not bind to basal NKCC1 in erythrocytes. In conclusion, loop diuretics had no NKCC2/NKCC1 selectivity, when NKCC1 is measured at its activated state. Basal NKCC1 has a reduced diuretic sensitivity, of very different magnitude depending on the diuretic drug and cell type in consideration.

Single-chain variable fragment antibodies (scFv) retain antigen specificity and offer advantages over intact antibodies as therapeutic agents. We cloned the cDNA of the V H and V n regions from a mouse hybridoma (HB-9645) directed against human CD20. In addition to the basic scFv construct (V n-L-V H), we genetically engineered a secretory signal, six histidine residues, and a dFluT tag to facilitate secretion, purification, and detection. A glycosyl-phosphatidylinositol (GPI) modification signal was added at the C terminus. The GPI-tagged and the non-tagged scFvs were expressed in high yields on the surface of stably transfected insect cells. The CD20-binding properties of purified non-GPI tagged scFv were examined using flow cytometry and immunocytochemistry. The non-GPI-tagged scFv selectively recognizes CD20-positive cells in a concentration-dependent manner. Double-flow cytometry analysis using fresh peripheral blood lymphocytes and WSU-FSCCL cells revealed that our scFv resolves the B-cell population better than the intact antibody. The GPI-tagged scFv was loaded onto the surface of sheep erythrocytes to form rosettes with CD20-positive cells. The genetically engineered anti-CD20 scFv and GPI-tagged derivative have binding specificity for the CD20 antigen. The scFvs described here has potential

This study aimed to compare the trace element status of patients with type 2 diabetes (n = 53) with those of nondiabetic healthy controls (n = 50). The concentrations of seven trace elements were determined in the whole blood, blood plasma, erythrocytes, and lymphocytes of the study subjects. Vanadium and iron levels in lymphocytes were significantly higher in diabetic patients as compared to controls (p < 0.05 for iron and p < 0.01 for vanadium). In contrast, lower manganese (p < 0.01) and selenium (p < 0.01) concentrations were detected in lymphocytes derived from patients with type 2 diabetes versus healthy subjects. Furthermore, significantly lower chromium levels (p < 0.05) were found in the plasma of diabetic individuals as compared to controls. Trace element concentrations were not dependent on the degree of glucose control as determined by correlation analysis between HBA 1c versus metal levels in the four blood fractions. In summary, this study primarily demonstrated that trace element levels in lymphocytes of patients with type 2 diabetes could deviate significantly

Gallic acid-based indanone derivatives have been synthesised. Some of the indanones showed very good anticancer activity in MTT assay. Compounds 10, 11, 12 and 14 possessed potent anticancer activity against various human cancer cell lines. The most potent indanone (10, IC 50 = 2.2 lM), against MCF-7, that is, hormone-dependent breast cancer cell line, showed no toxicity to human erythrocytes even at higher concentrations (100 lg/ml, 258 lM). While, indanones 11, 12 and 14 showed toxicities to erythrocytes at higher concentrations.

A series of C-(3-aminomethyl-cyclohexyl)-methylamine derivatives were synthesized and evaluated for their antitubercular activity. Some of the compounds exhibited potent activity against Mycobacterium tuberculosis H37Rv. One of the compound having t-butyl at para position of the benzene ring showed excellent activity even better than the standard drug ethambutol with MIC value 1.1 ± 0.2 lM. The time-kill kinetics study of two most active compounds showed rapid killing of the M. tuberculosis within 4 days. Additionally atom-based quantitative structure-activity relationship (QSAR) model was developed that gave a statistically satisfying result (R 2 ) = 0.92, Q 2 = 0.75, Pearson-R = 0.96 and effectively predicts the anti-tuberculosis activity of training and test set compounds.