An iron stable isotope comparison between human erythrocytes and plasma

We present precise iron stable isotope ratios measured by multicollector-ICP mass spectrometry (MC-ICP-MS) of human red blood cells (erythrocytes) and blood plasma from 12 healthy male adults taken during a clinical study. The accurate determination of stable isotope ratios in plasma first required substantial method development work, as minor iron amounts in plasma had to be separated from a large organic matrix prior to mass-spectrometric analysis to avoid spectroscopic interferences and shifts in the mass spectrometer's mass-bias. The 56Fe/54Fe ratio in erythrocytes, expressed as permil difference from the “IRMM-014” iron reference standard (δ56/54Fe), ranges from −3.1‰ to −2.2‰, a range typical for male Caucasian adults. The individual subject erythrocyte iron isotope composition can be regarded as uniform over the 21 days investigated, as variations (±0.059 to ±0.15‰) are mostly within the analytical precision of reference materials. In plasma, δ56/54Fe values measured in two different laboratories range from −3.0‰ to −2.0‰, and are on average 0.24‰ higher than those in erythrocytes. However, this difference is barely resolvable within one standard deviation of the differences (0.22‰). Taking into account the possible contamination due to hemolysis (iron concentrations are only 0.4 to 2 ppm in plasma compared to approx. 480 ppm in erythrocytes), we model the pure plasma δ56/54Fe to be on average 0.4‰ higher than that in erythrocytes. Hence, the plasma iron isotope signature lies between that of the liver and that of erythrocytes. This difference can be explained by redox processes involved during cycling of iron between transferrin and ferritin.

The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence.

Glucocorticoid alterations in the human type-1/type-2 cytokine balance and the role of CD28/B7 costimulation

We have recently reported that psychological stress is associated with a shift in the human type-1/type-2 cytokine balance toward a type-2 cytokine response. The mechanisms of these cytokine alterations are unknown, but likely involve glucocorticoid (GC) modulation of cytokine production. Therefore we sought to characterize the effects of GC on the in vitro human type-1/type-2 cytokine balance. We hypothesized that GC induce a type-2 cytokine shift through modulation of critical regulatory cytokines and alterations in the CD28/B7 costimulatory pathway. ^ We first sought to characterize the effect of the GC, dexamethasone (DEX), on type-1 (IFN-γ, IL-12) and type-2 (IL-4, IL-10) cytokine production by human peripheral blood mononuclear blood cells (pBMC) stimulated with a variety of T-lymphocyte and monocyte stimuli. DEX, at concentrations mimicking stress and supraphysiologic levels of cortisol, decreased IFN-γ and IL-12 production and increased IL-4 and IL-10 production, indicating a shift in the type-1/type-2 cytokine balance toward a type-2 response. Furthermore, both CD4+ and CD8+ T-lymphocytes were susceptible to the cytokine modulating effects of DEX. Furthermore, in the absence of the monocyte, the DEX-induced alterations in T-lymphocyte cytokine production were reduced, indicating that the interaction between the monocyte and T-lymphocyte plays a significant role. ^ We next determined the role of regulatory cytokines, known to modulate the type-1/type-2 cytokine balance, in the DEX-induced cytokine alterations. The addition of the recombinant IL-12p70 and IFN-γ, but not the neutralization of IL-4, IL-10 or IL-13 using monoclonal antibodies, attenuated the DEX-induced type-1/type-2 cytokine alterations. These data suggest that the DEX-induced cytokine alterations are mediated, at least in part, through the initial inhibition type-1 cytokines. Lastly, we investigated the role of the CD28/B7 costimulatory pathway in these cytokine alterations. DEX decreased the expression of CD80 and CD86 on THP-1 cells, a monocyte cell line, and the expression of CD28 and CTLA-4 on PHA-stimulated pBMC. The DEX-induced decrease in CD28 and CTLA-4 expression was attenuated by rhIL-12. Finally, CD28 activation attenuated the DEX-induced decrease in IFN-γ production, suggesting that modulation of the CD28/B7 costimulatory pathway may contribute to the DEX-induced type-1/type-2 cytokine alterations. ^

Data quality in trauma transfusion studies and the impact of missing data on predicting massive transfusion

Maximizing data quality may be especially difficult in trauma-related clinical research. Strategies are needed to improve data quality and assess the impact of data quality on clinical predictive models. This study had two objectives. The first was to compare missing data between two multi-center trauma transfusion studies: a retrospective study (RS) using medical chart data with minimal data quality review and the PRospective Observational Multi-center Major Trauma Transfusion (PROMMTT) study with standardized quality assurance. The second objective was to assess the impact of missing data on clinical prediction algorithms by evaluating blood transfusion prediction models using PROMMTT data. RS (2005-06) and PROMMTT (2009-10) investigated trauma patients receiving ≥ 1 unit of red blood cells (RBC) from ten Level I trauma centers. Missing data were compared for 33 variables collected in both studies using mixed effects logistic regression (including random intercepts for study site). Massive transfusion (MT) patients received ≥ 10 RBC units within 24h of admission. Correct classification percentages for three MT prediction models were evaluated using complete case analysis and multiple imputation based on the multivariate normal distribution. A sensitivity analysis for missing data was conducted to estimate the upper and lower bounds of correct classification using assumptions about missing data under best and worst case scenarios. Most variables (17/33=52%) had <1% missing data in RS and PROMMTT. Of the remaining variables, 50% demonstrated less missingness in PROMMTT, 25% had less missingness in RS, and 25% were similar between studies. Missing percentages for MT prediction variables in PROMMTT ranged from 2.2% (heart rate) to 45% (respiratory rate). For variables missing >1%, study site was associated with missingness (all p≤0.021). Survival time predicted missingness for 50% of RS and 60% of PROMMTT variables. MT models complete case proportions ranged from 41% to 88%. Complete case analysis and multiple imputation demonstrated similar correct classification results. Sensitivity analysis upper-lower bound ranges for the three MT models were 59-63%, 36-46%, and 46-58%. Prospective collection of ten-fold more variables with data quality assurance reduced overall missing data. Study site and patient survival were associated with missingness, suggesting that data were not missing completely at random, and complete case analysis may lead to biased results. Evaluating clinical prediction model accuracy may be misleading in the presence of missing data, especially with many predictor variables. The proposed sensitivity analysis estimating correct classification under upper (best case scenario)/lower (worst case scenario) bounds may be more informative than multiple imputation, which provided results similar to complete case analysis.^

Requirement of the Jak2 tyrosine kinase in Bcr -Abl oncogenic transformation

Philadelphia chromosome (Ph)-positive chronic myeloid leukemia is caused by a clonal myeloproliferative expansion of malignant primitive hematopoietic progenitor cells. The Ph results from the reciprocal translocation of the ends of chromosome 9 and 22, which generate Bcr-Abl fusion proteins. The Bcr-Abl proteins possess a constitutively activated Abl tyrosine kinase, which is the driving force responsible for causing leukemia. The activated Bcr-Abl tyrosine kinase stimulates multiple signal transduction pathway affecting growth, differentiation and survival of cells. It is known that the Bcr-Abl tyrosine kinase activates several signaling proteins including Stat5, which is a member of the Jak/Stat pathway that is activated by cytokines that control the growth and differentiation of normal hematopoietic cells. Our laboratory was the first one to report that Jak2 tyrosine kinase is activated in a human Bcr-Abl positive hematopoietic cell line. In this thesis, we further investigated the activation of Jak2 by Bcr-Abl. We found that Jak2 is activated not only in cultured Bcr-abl positive cell lines but also in blood cells from CML blast crisis patients. We also demonstrated that SH2 domain of Bcr-Abl is required for efficient activation Jak2. We further showed that Jak2 binds to the C-terminal domain of Bcr-Abl; tyrosine residue 1007, which is critical for Jak2 activation, is phosphorylated by Bcr-Abl. We searched downstream targets of Jak2 in Bcr-Abl positive cells. We treated Bcr-Abl positive cells with a Jak2 kinase inhibitor AG490 and found that c-Myc protein expression is inhibited by AG490. We further demonstrated that Jak2 inhibitor AG490 not only inhibit C-MYC transcription but also protect c-Myc protein from proteasome-dependent degradation. We also showed that AG490 did not affect Bcr-Abl kinase activity and Stat5 activation and its downstream target Bcl-xL expression. AG490 also induced apoptosis of Bcr-Abl positive cells, similar to Bcr-Abl kinase inhibitor STI571 (also termed Gliveec, a very effective drug for CML), but unlike STI571 the apoptosis effects induced by AG490 can not be rescued by IL-3 containing WEHI conditioned medium. We further established several Bcr-Abl positive clones that express a kinase-inactive Jak2 and found that these clones had reduced tumor formation in nude mice assays. Taken together, these results establish that Jak2 is activated in Bcr-Abl positive CML cells and it is required for c-Myc induction and the oncogenic effects of Bcr-Abl. Furthermore, Jak2 and Stat5 are two independent targets of Bcr-Abl. ^

The effects of omega -3 fatty acid -enriched fish on C -reactive protein levels in postmenopausal women

Coronary heart disease (CHD) is the leading cause of death in women and rates markedly increase among women after 65 years of age. C-reactive protein (CRP) is a new clinical indicator of atherosclerotic-related inflammation with a direct pathogenic role. Studies show lifestyle factors can modulate CRP. Omega-3 fatty acids have anti-inflammatory properties and studies suggest that eating fish high in omega-3 fatty acids may lower CHD risk in women. This study sought to assess the possible role of omega-3 fatty acids in the reduction of CHD-related inflammation by investigating the effect of fish consumption on CRP levels. Methods. Twenty-four healthy postmenopausal women were randomly assigned to a fish group (usual diet plus two servings per week of enriched fish) or control group (usual diet with no fatty fish) for eight weeks. Omega-3 fatty acid-enriched fish developed by the West Virginia University Aquaculture Division was used. Serum CRP, serum interleukin-6 (IL-6), and the fatty acid content of red blood cells (RBC) were measured before and after the study. Women also completed food records. RESULTS: Baseline levels of CRP were low (85% of the fish group had normal levels) and few changes in CRP risk category were observed. Mean IL-6 levels were reduced by 27% and 35% in the fish and control groups, respectively (p for between-group difference = 0.60). Changes in RBC fatty acid composition were not statistically significant. Compared to control women, women in the fish group had greater reductions in mean triglycerides (p = 0.08), total cholesterol (P = 0.04), and LDL cholesterol levels (p = 0.06). Baseline dietary intake of total and monounsaturated fatty acids tended to be positively associated with baseline CRP, while vitamin E intake was inversely related. Saturated fat intake tended to have a positive association with IL-6. Conclusions. Findings regarding the effect of two servings of fish on CRP and IL-6 levels are inconclusive due to low baseline levels of CRP and IL-6. However, results indicate two servings of fatty fish have favorable effects on blood lipids. The relationship of dietary components with CRP and IL-6 is complex and further research is needed to determine the varying roles of diet on the inflammatory process. ^

Proceedings of the Twenty-Fifth Annual Biochemical Engineering Symposium

The Annual Biochemical Engineering Symposium Series started in 1970 when Professors Larry E. Erickson (Kansas State University) and Peter J. Reilly (then with University of Nebraska-Lincoln) got together in Manhattan, KS along with their students for a half-day powwow and technical presentation by their students. Ever since then, it has been a forum for Biochemical Engineering students in the heartland of USA to present their research to their colleagues in the form of talks and posters. The institutions actively involved with this annual symposium include Colorado State University, Kansas State University, Iowa State University, University of Colorado, University of Kansas, University of Missouri-Columbia, and University of Oklahoma. The University of lowa and University of Nebraska-Lincoln have also participated in the conference in recent years. The host institutions for the different symposia have been: Kansas State University (1, 3, 5, 9, 12, 16, 20), Iowa State University (6, 7, 10, 13, 17, 22), University of Missouri-Columbia (8, 14, 19, 25), Colorado State University (II, 15, 21), University of Colorado (18, 24), University of Nebraska-Lincoln (2, 4), University of Oklahoma (23). The next symposium will be held at Kansas State University. Proceedings of the Symposium are edited by faculty of the host institution and include manuscripts written and submitted by the presenters (students). These often include works-in-progress and final publication usually takes place in refereed journals. ContentsPatrick C. Gilcrease and Vincent G. Murphy, Colorado State University. Use of 2,4,6-Trinitrotoluene (TNT) As A Nitrogen Source By A Pseudomonas florescens Species Under Aerobic Conditions. Marulidharan Narayanan, Lawrence C. Davis, and Larry E. Erickson, Kansas State University. Biodegradation Studies of Chlorinated Organic Pollutants in a Chamber in the Presence of Alfalfa Plants. S.K. Santharam, L.E. Erickson, and L.T. Fan, Kansas State University.Surfactant-Enhanced Remediation of a Non-Aqueous Phase Contaminant in Soil. Barry Vant-Hull, Larry Gold, and Robert H. Davis, University of Colorado.The Binding of T7 RNA Polymerase to Double-Stranded RNA. Jeffrey A. Kern and Robert H. Davis, University of Colorado.Improvement of RNA Transcription Yield Using a Fed-Batch Enzyme Reactor. G. Szakacs, M. Pecs, J. Sipocz, I. Kaszas, S.R. Deecker, J.C. Linden, R.P. Tengerdy, Colorado State University.Bioprocessing of Sweet Sorghum With In Situ Produced Enzymes. Brad Forlow and Matthias Nollert, University of Oklahoma.The Effect of Shear Stress ad P-selectin Site Density on the Rolling Velocity of White Blood Cells. Martin C. Heller and Theodore W. Randolph, University of Colorado.The Effects of Plyethylene Glycol and Dextran on the Lyophilization of Human Hemoglobin. LaToya S. Jones and Theodore W. Randolph, University of Colorado.Purification of Recombinant Hepatitis B Vaccine: Effect of Virus/Surfactant Interactions. Ching-Yuan Lee, Michael G. Sportiello, Stephen Cape, Sean Ferree, Paul Todd, Craig E. Kundrot, and Cindy Barnes, University of Colorado.Application of Osmotic Dewatering to the Crystallization of Oligonucleotides for Crystallography. Xueou Deng, L.E. Erickson, and D.Y.C. Fung, Kansas State University.Production of Protein-Rich Beverages from Cheese Whey and Soybean by rapid Hydration Hydrothermal Cooking. Pedro M. Coutinho, Michael K. Dowd, and Peter J. Reilly, Iowa State University.Automated Docking of Glucoamylase Substrates and Inhibitors. J. Johansson and R.K. Bajpai, University of Missouri.Adsorption of Albumin on Polymeric Microporous Membranes.

(Table 1) Body condition and plasma POP concentrations of glaucous gulls from Bjørnøya, Svalbard archipelago

Behavioural field observations are increasingly being used in ecotoxicological research to identify potential adverse effects of exposure to persistent organic pollutants (POPs). We investigated thermal conditions inside the nest and parental behaviour of glaucous gulls, Larus hyperboreus, breeding in the Norwegian Arctic in relation to the concentrations of major classes of POPs (organochlorines, brominated flame retardants and metabolically derived products) accumulated in their blood. Most notably, nest temperature was negatively correlated with the concentrations of the sum of DDT, sum of PCB and several quantitatively minor POP classes within the incubating parent. To investigate the relationship between incubation ability and parental POP exposure further, we experimentally increased the costs of incubation by artificially increasing the clutch size from two to four eggs. Clutch enlargement was followed by a decrease in nest temperature, but this drop in temperature was not associated with POP concentrations within the incubating parent. However, males, which had higher POP concentrations and lower white blood cell counts than females, seemed less able to maintain nest temperature. There was virtually no evidence to suggest that the sum of PCB or DDT were associated with changes in the time a bird spent incubating. However, there was some indication that nest site attendance by nonincubating males was negatively related to the sum of DDT, suggesting that nest protection may have been compromised. The results suggest that adverse effects of parental POP exposure may occur through suboptimal thermal conditions for embryo development and possibly increased egg predation risk.

Seawater carbonate chemistry during experiments with Mytilus edulis, 2008

The effects of medium term (32 d) hypercapnia on the immune response of Mytilus edulis were investigated in mussels exposed to acidified (using CO2) sea water (pH 7.7, 7.5 or 6.7; control: pH 7.8). Levels of phagocytosis increased significantly during the exposure period, suggesting an immune response induced by the experimental set-up. However, this induced stress response was suppressed when mussels were exposed to acidified sea water. Acidified sea water did not have any significant effects on other immuno-surveillance parameters measured (superoxide anion production, total and differential cell counts). These results suggest that ocean acidification may impact the physiological condition and functionality of the haemocytes and could have a significant effect on cellular signalling pathways, particularly those pathways that rely on specific concentrations of calcium, and so may be disrupted by calcium carbonate shell dissolution.

Lesion, and haematologic and serum characteristics of Weddell seals (Leptonychotes weddellii) sampled in McMurdo Sound, Antarctica

We examined and collected biomedical samples from Weddell seals (Leptonychotes weddellii) during studies of post-breeding-season foraging behaviour of adults and movements of weaned pups as a complement to ongoing studies on the ecology and population dynamics of the McMurdo seals (Stewart et al. 2000, 2003). Here we report on Weddell seal health assessments conducted during the 1996/97, 1997/98 and 1998/99 breeding seasons at the Delbridge Islands (77.68°S, 166.50°E), McMurdo Sound, Antarctica. Our objectives were to compile baseline biomedical data for Weddell seals in McMurdo Sound, and to identify infectious and non-infectious diseases affecting the population. Development of such a database, including information on normal background morbidity and mortality, is an important first step in evaluating natural versus anthropogenic impacts on population health (Geraci et al. 1999; Reddy et al. 2001). These data will be integral to international studies of southern ocean pinnipeds that seek to evaluate the influence of biotic and abiotic factors on the ecology of these apex predators.

A Recombinant Sal k 1 Isoform as an Alternative to the Polymorphic Allergen from Salsola kali Pollen for Allergy Diagnosis

The incidence of Amaranthaceae pollen allergy has increased due to the desertification occurring in many countries. In some regions of Spain, Salsola kali is the main cause of pollinosis, at almost the same level as olive and grass pollen. Sal k 1 - the sensitization marker of S. kali pollinosis - is used in clinical diagnosis, but is purified at a low yield from pollen. We aimed to produce a recombinant (r)Sal k 1 able to span the structural and immunological properties of the natural isoforms from pollen, and validate its potential use for diagnosis. METHODS: Specific cDNA was amplified by PCR, cloned into the pET41b vector and used to transform BL21 (DE3) Escherichia coli cells. Immunoblotting, ELISA, basophil activation and skin-prick tests were used to validate the recombinant protein against Sal k 1 isolated from pollen. Sera and blood cells from S. kali pollen-sensitized patients and specific monoclonal and polyclonal antisera were used. RESULTS: rSal k 1 was produced in bacteria with a yield of 7.5 mg/l of cell culture. The protein was purified to homogeneity and structural and immunologically validated against the natural form. rSal k 1 exhibited a higher IgE cross-reactivity with plant-derived food extracts such as peanut, almond or tomato than with pollen sources such as Platanus acerifolia and Oleaceae members. CONCLUSIONS: rSal k 1 expressed in bacteria retains intact structural and immunological properties in comparison to the pollen-derived allergen. It spans the immunological properties of most of the isoforms found in pollen, and it might substitute natural Sal k 1 in clinical diagnosis.

Plasmodium falciparum domain mediating adhesion to chondroitin sulfate A: A receptor for human placental infection

Malaria during the first pregnancy causes a high rate of fetal and neonatal death. The decreasing susceptibility during subsequent pregnancies correlates with acquisition of antibodies that block binding of infected red cells to chondroitin sulfate A (CSA), a receptor for parasites in the placenta. Here we identify a domain within a particular Plasmodium falciparum erythrocyte membrane protein 1 that binds CSA. We cloned a var gene expressed in CSA-binding parasitized red blood cells (PRBCs). The gene had eight receptor-like domains, each of which was expressed on the surface of Chinese hamster ovary cells and was tested for CSA binding. CSA linked to biotin used as a probe demonstrated that two Duffy-binding-like (DBL) domains (DBL3 and DBL7) bound CSA. DBL7, but not DBL3, also bound chondroitin sulfate C (CSC) linked to biotin, a negatively charged sugar that does not support PRBC adhesion. Furthermore, CSA, but not CSC, blocked the interaction with DBL3; both CSA and CSC blocked binding to DBL7. Thus, only the DBL3 domain displays the same binding specificity as PRBCs. Because protective antibodies present after pregnancy block binding to CSA of parasites from different parts of the world, DBL-3, although variant, may induce cross-reactive immunity that will protect pregnant women and their fetuses.