Mercury speciation in whole blood by gas chromatography coupled to ICP-MS with a fast microwave-assisted sample preparation procedure

A simple and fast method is described for simultaneous determination of methylmercury (MeHg), ethylmercury (Et-Hg) and inorganic mercury (Ino-Hg) in blood samples by using capillary gas chromatography-inductively coupled plasma mass spectrometry (GC-ICP-MS) after derivatization and alkaline digestion. Closed-vessel microwave assisted digestion conditions with tetramethylammonium hydroxide (TMAH) have been optimized. Derivatization by using ethylation and propylation procedures have also been evaluated and compared. The absolute detection limits (using a 1 mu L injection) obtained by GC-ICP-MS with ethylation were 40 fg for MeHg and Ino-Hg, respectively, and with propylation were 50, 20 and 50 fg for MeHg, Et-Hg and Ino-Hg, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). Additional validation is provided based on the comparison of results obtained for mercury speciation in blood samples with the proposed procedure and with a previously reported LC-ICP-MS method. With the new proposed procedure no tedious clean-up steps are required and a considerable improvement of the time of analysis was achieved compared to other methods using GC separation.

Influence sexual dimorphism on the persistence of blood parasites in infected Calomys callosus

Gender has long been known to be a contributory factor in the incidence and progression of disorders associated with immune system disregulation. The aims of this experiment were to verify the influences of sexual dimorphism on the persistence of blood parasites out of the acute phase of infection. Male and female Calomys callosus were separated and infected with two strains of Trypanosoma cruzi, and let age until 120 days. Xenogiagnostic, culture of organs and blood, histopathology and lytic antibody percentages were evaluated on late chronic phase. Xenodiagnosis, hemoculture and lytic antibody percentages were positive front 45 until 120 days. For both strains in adrenal and heart, amastigote burdens were present until 45 days, scarcely found on 60 days and absent on 120 days. Steroid hormones, although having a protective role, does not enable animals to get completely rid of the infection. Even without showing apparent signs of pathological unbalance, parasite persists, hidden throughout the host`s body. (C) 2008 Elsevier Ltd. All rights reserved.

Innate immune lectins kill bacteria expressing blood group antigen

The expression of ABO(H) blood group antigens causes deletion of cells that generate self-specific antibodies to these antigens but this deletion limits adaptive immunity toward pathogens bearing cognate blood group antigens. To explore potential defense mechanisms against such pathogens, given these limitations in adaptive immunity, we screened for innate proteins that could recognize human blood group antigens. Here we report that two innate immune lectins, galectin-4 (Gal-4) and Gal-8, which are expressed in the intestinal tract, recognize and kill human blood group antigen-expressing Escherichia coli while failing to alter the viability of other E. coli strains or other Gram-negative or Gram-positive organisms both in vitro and in vivo. The killing activity of both Gal-4 and Gal-8 is mediated by their C-terminal domains, occurs rapidly and independently of complement and is accompanied by disruption of membrane integrity. These results demonstrate that innate defense lectins can provide immunity against pathogens that express blood group-like antigens on their surface.

The Ruthenium Complex cis-(Dichloro) Tetraammineruthenium(III) Chloride Presents Immune Stimulatory Activity on Human Peripheral Blood Mononuclear Cells

Ruthenium compounds in general are well suited for medicinal applications. They have been investigated as immunosuppressants, nitric oxide scavengers, antimicrobial agents, and antimalarials. The aim of this study is to evaluate the immunomodulatory activity of cis-(dichloro) tetraammineruthenium(III) chloride (cis-[RuCl(2)(NH(3))(4)]Cl) on human peripheral blood mononuclear cells (PBMC). The cytotoxic studies performed here revealed that the ruthenium( III) complex presents a cytotoxic activity towards normal human PBMC, only at very high concentration. Results also showed that cis-[ RuCl(2)(NH(3))(4)] Cl presents a dual role on PBMC stimulating proliferation and interleukin-2 (IL-2) production at low concentration and inducing cytotoxicity, inability to proliferate, and inhibiting IL-2 production at high concentration. The noncytotoxic activity of cis-[RuCl(2)(NH(3))(4)] Cl at low concentration towards PBMC, which correlates with the small number of annexin V positive cells and also the absence of DNA fragmentation, suggest that this compound does not induce apoptosis on PBMC. For the first time, we show that, at low concentration (10-100 mu g L(-1)), the cis-[ RuCl(2)(NH(3))(4)] Cl compound induces peripheral blood lymphocytes proliferation and also stimulates them to IL-2 production. These results open a new potential applicability of ruthenium(III) complexes as a possible immune regulatory compound acting as immune suppressor at high concentration and as immune stimulator at low concentration.

Isolation and Characterization of a Natriuretic Peptide from Crotalus oreganus abyssus (Grand Canyon Rattlesnake) and its Effects on Systemic Blood Pressure and Nitrite Levels

Studies on the therapeutic potential of venom peptides have significantly advanced the development of new peptide drugs. A good example is captopril, a synthetic peptide drug, which acts as an anti-hypertensive and potentiating bradykinin, inhibiting the angiotensin-converting enzyme, whose precursor was isolated from the venom of Bothrops jararacussu. The natriuretic peptide (NPs) family comprises three members, ANP (atrial natriuretic peptide), BNP (B-type natriuretic peptide) and CNP (C-type natriuretic peptide), and has an important role in blood pressure regulation and electrolyte homeostasis. In this study, we describe, for the first time, the isolation and characterization of a novel natriuretic-like peptide (Coa_NP), isolated from Crotalus Oreganus abyssus venom. The peptide has 32 amino acids and its complete sequence is SKRLSNGCFGLKLDRIGAMSGLGCWRLINESK. The Coa_NP has an average molecular mass of 3510.98 Da and its amino acid sequence presents the loop region that is characteristic of natriuretic peptides (17 amino acids, NP domain consensus; CFGXXXDRIXXXSGLGC). Coa_NP is a natriuretic peptide of the ANP/BNP-like family, since the carboxy terminal region of CNP has its own NP domain. The functional experiments showed that Coa_NP produced biological effects similar to those of the other natriuretic peptides: (1) a dose-dependent decrease in mean arterial pressure; (2) significant increases in plasma nitrite levels, and (3) vasorelaxation in thoracic aortic rings that were pre-contracted with phenylephrine. The structural and biological aspects confirm Coa_NP as a natriuretic peptide isolated from snake venom, thus expanding the diversification of venom components.

Biomarkers of selenium status in the amazonian context: Blood, urine and sequential hair segments

Selenium (Se) is an essential element and deficit or excess of dietary Se is associated with health disorders. Relatively elevated Se levels have been reported in the Brazilian Amazon, where there are also important annual variations in the availability of different foods. The present study was conducted among six riparian communities of the Tapajos River to evaluate seasonal variations in blood and sequential hair cm Se concentrations, and to examine the relationships between Se in blood and hair, and blood and urine. Two cross-sectional studies were conducted, at the descending water (DWS, n = 259) and the rising water (RWS, n = 137) seasons, with repeated measures for a subgroup (n = 112). Blood Se (B-Se), hair Se (H-Se) and urine Se (U-Se) were determined. Match-paired analyses were used for seasonal comparisons and the method of best fit was used to describe the relationships between biomarkers. B-Se levels presented a very large range (142-2447 mu g/l) with no overall seasonal variation (median 284 and 292 mu g/l, respectively). Sequential analysis of 13 cm hair strands showed significant variations over time: Se concentrations at the DWS were significantly lower compared with the rising water season (medians: 0.7 and 0.9 mu g/g; ranges: 0.2-4.3 mu g/g and 0.2-5.4 mu g/g, respectively). At both seasons, the relationships between B-Se and H-Se were linear and highly significant (r(2) = 67.9 and 63.6, respectively), while the relationship between B-Se and U-Se was best described by a sigmoid curve. Gender, age, education and smoking did not influence Se status or biomarker relationships. Variations in H-Se suggest that there may be seasonal availability of Se sources in local food. For populations presenting a large range and/or elevated Se exposure, sequential analyses of H-Se may provide a good reflection of variations in Se status.

Differences in both matrix metalloproteinase 9 concentration and zymographic profile between plasma and serum with clot activators are due to the presence of amorphous silica or silicate salts in blood collection devices

Matrix metalloproteinases (MMPs) are promising diagnostic tools, and blood sampling/handling alters MMP concentrations between plasma and serum and between serum with and without clot activators. To explain the higher MMP-9 expression in serum collected with clot accelerators relative to serum with no additives and to plasma, we analyzed the effects of increasing amounts of silica and silicates (components of clot activators) in,citrate plasma, serum, and huffy coats collected in both plastic and glass tubes from 50 healthy donors, and we analyzed the effects of silica and silicate on cultured leukemia cells. The levels of MMP-2 did not show significant changes between glass and plastic tubes, between serum and plasma, between serum with and without clot accelerators, or between silica and silicate treatments. No modification of MMP-9 expression was obtained by the addition of silica or silicate to previously separated plasma and serum. Increasing the amounts of nonsoluble silica and soluble silicate added to citrate and empty tubes prior to blood collection resulted in increasing levels of MMP-9 relative to citrate plasma and serum. Silica and silicate added to buffy coats and leukemia cells significantly induced MMP-9 release/secretion, demonstrating that both silica and silicate induce the release of pro- and complexed MMP-9 forms. We recommend limiting the misuse of serum and avoiding the interfering effects of clot activators. (c) 2007 Elsevier Inc. All rights reserved.

Blood pressure and heart rate during tonic immobility in the black tipped reef shark, Carcharhinus melanoptera

Tonic immobility was induced in black tipped reef sharks (Carcharhinus melanoptera) and heart rate and ventral aortic blood pressure recorded. Without branchial irrigation, tonic immobility was correlated with a significant depression in blood pressure and heart rate irrespective of the sharks being in air or in water. Tonic immobility with branchial irrigation resulted in a significant increase in blood pressure in sharks in air, but not in water. Heart rate was unchanged when the gills were irrigated. Intra-arterial injections of atropine abolished the bradycardia and blood pressure rise associated with tonic immobility. We conclude that, during tonic immobility, sharks are able to receive afferent information from the ventilatory system and make appropriate responses via the vagus nerve.

Methemoglobin Reduction in Crocodile Blood: Are High Levels of MetHb Typical of Healthy Reptiles?

In 82 wild-caught Crocodylus porosus, levels of NADH-MetHb reductase and GSH seem adequate to maintain hemoglobin in its reduced functional state. Studies of C. porosus erythrocytes in vitro show reduction of metHb in the presence of lactate, glucose and plasma, but not pyruvate. These findings, together with recent data which show low metHb in a variety of reptiles, cast doubt on the accepted view that high levels of MetHb are typical of healthy reptiles. One explanation for the sharp contrast between earlier and more recent data could be technical. We found low metHb in Crocodylus johnstoni, Chelodina longicollis and Sphenomorphus quoyi. However, high and variable values reminiscent of many of the earlier data were obtained by omitting final centrifugation prior to spectrophotometry. Interestingly, this step is not part of the standard clinical method but is necessary in analyses of blood with nucleated red cells. These observations suggest that high metHb may not be typical of reptiles after all.

Peptidergic control of gastrointestinal blood flow in the estuarine crocodile, Crocodylus porosus

Peptidergic mechanisms influencing the resistance of the gastrointestinal vascular bed of the estuarine crocodile, Crocodylus porosus, were investigated. The gut was perfused in situ via the mesenteric and the celiac arteries, and the effects of different neuropeptides were tested using bolus injections. Effects on vascular resistance were recorded as changes in inflow pressures. Peptides found in sensory neurons [substance P, neurokinin A, and calcitonin gene-related peptide (CGRP)] all caused significant relaxation of the celiac vascular bed, as did vasoactive intestinal polypeptide (VIP), another well-known vasodilator. Except for VIP, the peptides also induced transitory gut contractions. Somatostatin and neuropeptide Y (NPY), which coexist in adrenergic neurons of the C. porosus, induced vasoconstriction in the celiac vascular bed without affecting the gut motility. Galanin caused vasoconstriction and occasionally activated the gut wall. To elucidate direct effects on individual vessels, the different peptides were tested on isolated ring preparations of the mesenteric and celiac arteries. Only CGRP and VIP relaxed the epinephrine-precontracted celiac artery, whereas the effects on the mesenteric artery were variable. Somatostatin and NPY did not affect the resting tonus of these vessels, but somatostatin potentiated the epinephrine-induced contraction of the celiac artery. Immunohistochemistry revealed the existence and localization of the above-mentioned peptides in nerve fibers innervating vessels of different sizes in the gut region. These data support the hypothesis of an important role for neuropeptides in the control of the vascular bed of the gastrointestinal tract in C. porosus.

Relationships between blood pressure and heart rate in the saltwater crocodile Crocodylus porosus

The cardiac limb of the baroreflex loop was studied in the saltwater crocodile Crocodylus porosus, The classical pharmacological methodology using phenylephrine and sodium nitroprusside was used to trigger blood pressure changes, and the resulting alterations in heart rate were analysed quantitatively using a logistic function. Interindividual differences in resting heart rates and blood pressures were observed, but all seven animals displayed clear baroreflex responses. Atropine and sotalol greatly attenuated the response. A maximal baroreflex gain of 7.2 beats min(-1) kPa(-1) was found at a mean aortic pressure of 6.1 kPa, indicating the active role of the baroreflex in a wide pressure range encompassing hypotensive and hypertensive states. At the lowest mean aortic pressures (5.0 kPa), the synergistic role of the pulmonary-to-systemic shunt in buffering the blood pressure drop also contributes to blood pressure regulation, Pulse pressure showed a better correlation,vith heart rate and also a higher gain than mean aortic, systolic or diastolic pressures, and this is taken as an indicator of the existence of a differential control element working simultaneously with a linear proportional element.

Heart rate, blood lactate and kinematic data of elite colts (under-19) rugby union players during competition

Physiological and kinematic data were collected from elite under-19 rugby union players to provide a greater understanding of the physical demands of rugby union. Heart rate, blood lactate and time-motion analysis data were collected from 24 players (mean +/- s((x) over bar): body mass 88.7 +/- 9.9 kg, height 185 +/- 7 cm, age 18.4 +/- 0.5 years) during six competitive premiership fixtures. Six players were chosen at random from each of four groups: props and locks, back row forwards, inside backs, outside backs. Heart rate records were classified based on percent time spent in four zones (>95%, 85-95%, 75-84%, <75% HRmax). Blood lactate concentration was measured periodically throughout each match, with movements being classified as standing, walking, jogging, cruising, sprinting, utility, rucking/mauling and scrummaging. The heart rate data indicated that props and locks (58.4%) and back row forwards (56.2%) spent significantly more time in high exertion (85-95% HRmax) than inside backs (40.5%) and outside backs (33.9%) (P < 0.001). Inside backs (36.5%) and outside backs (38.5%) spent significantly more time in moderate exertion (75-84% HRmax) than props and locks (22.6%) and back row forwards (19.8%) (P < 0.05). Outside backs (20.1%) spent significantly more time in low exertion (< 75% HRmax) than props and locks (5.8%) and back row forwards (5.6%) (P < 0.05). Mean blood lactate concentration did not differ significantly between groups (range: 4.67 mmol.l(-1) for outside backs to 7.22 mmol.l(-1) for back row forwards; P < 0.05). The motion analysis data indicated that outside backs (5750 m) covered a significantly greater total distance than either props and locks or back row forwards (4400 and 4080 m, respectively; P < 0.05). Inside backs and outside backs covered significantly greater distances walking (1740 and 1780 m, respectively; P < 0.001), in utility movements (417 and 475 m, respectively; P < 0.001) and sprinting (208 and 340 m, respectively; P < 0.001) than either props and locks or back row forwards (walking: 1000 and 991 m; utility movements: 106 and 154 m; sprinting: 72 and 94 m, respectively). Outside backs covered a significantly greater distance sprinting than inside backs (208 and 340 m, respectively; P < 0.001). Forwards maintained a higher level of exertion than backs, due to more constant motion and a large involvement in static high-intensity activities. A mean blood lactate concentration of 4.8-7.2 mmol.l(-1) indicated a need for 'lactate tolerance' training to improve hydrogen ion buffering and facilitate removal following high-intensity efforts. Furthermore, the large distances (4.2-5.6 km) covered during, and intermittent nature of, match-play indicated a need for sound aerobic conditioning in all groups (particularly backs) to minimize fatigue and facilitate recovery between high-intensity efforts.

Dendritic cells in rheumatoid arthritis - A model autoimmune inflammatory site

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease in which unknown arthrogenic autoantigen is presented to CD4+ T cells. The strong association of the disease with an epitope within the HLA-DR chain shared between various alleles of HLA-DR4 and DR1 emphasizes the importance of antigen presentation. This immune response predominantly occurs in the synovial tissue and fluid of the joints and autoreactive T cells are readily demonstrable in both the synovial compartment and blood. Circulating dendritic cells (DC) are phenotypically and functionally identical with normal peripheral blood (PB) DC. In the synovial tissue, fully differentiated perivascular DC are found in close association with T cells and with B cell follicles, sometimes containing follicular DC. These perivascular DC migrate across the activated endothelium from blood and receive differentiative signals within the joint from monocyte-derived cytokines and CD40-ligand+ T cells. In the SF, DC manifest an intermediate phenotype, similar to that of monocyte-derived DC in vitro. Like a delayed-type hypersensitivity response, the rheumatoid synovium represents an effector site. DC at many effector sites have a characteristic pattern of infiltration and differentiation. It is important to note that the effector response is not self-limiting in RA autoimmune inflammation. In this article, we argue that the presentation of self-antigen by DC and by autoantibody-producing B cells is critical for the perpetuation of the autoimmune response. Permanently arresting this ongoing immune response with either pharmaceutical agents or immunotherapy is a major challenge for immunology.