Adhesion of human platelets to albumin is synergistically increased by lysophosphatidic acid and adrenaline in a donor-dependent fashion.

Lysophosphatidic acid (LPA) and adrenaline are weak platelet activators considered important for thrombus formation, and were previously shown to synergistically increase platelet aggregation. Here we investigate synergistic activation by LPA and adrenaline when measuring platelet adhesion. Platelet-rich plasma from healthy blood donors together with adrenaline and/or LPA were added to protein-coated microplates. Platelets were allowed to adhere and the amount of adhesion detected enzymatically. The LPA and adrenaline combination induced a synergistic increase of platelet adhesion to a normally non-adhesive albumin surface. The degree of synergy varied markedly between individuals; these variations could not be explained by age, gender, blood type or different amounts of platelets, oxidized low-density lipoprotein, insulin or glucose in plasma. There was a trend indicating increased synergistic effect for platelets sensitive to adrenaline stimulation. The synergistic effect was blocked by the alpha2-adrenoceptor antagonist yohimbine and inhibited by the ADP scavenger system creatine phosphate/creatine phosphokinase and antibodies against alphaIIbbeta3. Furthermore, platelets adhering to albumin after adrenaline and LPA treatment expressed P-selectin. In conclusion, LPA and adrenaline act synergistically to increase alphaIIbbeta3-mediated platelet adhesion to albumin, dependent on alpha2-adrenoceptor signalling and platelet secretion. We also confirm that synergistic platelet activation achieved with LPA and adrenaline is highly donor dependent.     

Tacrine restores cholinergic nicotinic receptors and glucose metabolism in alzheimer patients as visualized by positron emission tomography

Three patients with Alzheimer's disease, a 68-year-old woman with mild dementia and 2 men (aged 64 and 72 years) with moderate dementia were treated orally with the cholinesterase inhibitor tacrine (tetrahydroaminoacridine), 80 mg daily, for several months. The patients were investigated using positron emission tomography (PET) prior to, and after 3 weeks and 3 months of treatment. The PET studies involved a multi-tracer system consisting of [¹⁸F]-fluoro-deoxy-glucose (¹⁸F-FDG) (tracer for glucose metabolism); ¹¹C-butanol (cerebral blood flow) and (S)(−)- and (R)(+)-[N-¹¹C-methyl]-nicotine (nicotinic receptors; cholinergic neural activity). Tacrine treatment increased the uptake of ¹¹C-nicotine to the brain. Significant reduced difference in uptake between the two enantiomers (S)(−)- and (R)(+)¹¹C-nicotine was observed in the frontal and temporal cortices after tacrine treatment in all three patients. The kinetic analysis indicated increased binding of (S)(−)¹¹C-nicotine in brain compatible with a restoration of nicotinic cholinergic receptors. The most pronounced effect was observed after 3 weeks and 3 months treatment in the patient with mild dementia. An increase in cerebral glucose utilization was found in the 68-year-old patient with mild dementia but also slightly in the 64-year-old man with moderate dementia when treated with tacrine for 3 months. Tacrine administration did not affect cerebral blood flow. The PET data obtained after 3 weeks of tacrine treatment was paralleled by improvement in neuropsychological performance. This study shows in vivo by PET neurochemical effects induced in brain by treatment with tacrine to Alzheimer patients. Intervention with tacrine in the early course of the disease might be necessary for clinical improvement.     

In safe hands: a qualitative study on older adults’ experiences of a tailored primary health care unit

Objective: Today’s health care system faces challenges in meeting the needs of older people with multimorbidity. To better cope with these needs, tailored primary health care with geriatric competence and person-centred care has been suggested. The aim of this study was to explore older patients’ experiences of a tailored primary health care unit.Design: This was a qualitative study using semi-structured individual interviews and qualitative content analysis.Setting and patients: Nineteen patients were recruited from a tailored PHC unit for people aged 75 years or older in a region in central Sweden.Methods: The interview data were analysed using inductive category development.Results: In the analysis, the theme In safe hands when in need of primary health care emerged. The interviewees expressed a desire to participate in their own care. Easy access, enough consultation time and a calm environment, along with the PHC professionals’ welcoming and attentive approach enhanced their feeling of being in safe hands. PHC professionals were perceived as having geriatric knowledge and taking responsibility for the care of older patients. Although the interviewees experienced that they received attention for their health conditions, a need for a more preventive approach to care emerged.Conclusion: Older patients highly appreciated their tailored PHC unit and they emphasised that it was an improvement compared to the ordinary PHC centre. This study provides insights into older patients’ experiences, which may be helpful in the ongoing process of improving care for older patients in PHC.

KEY POINTS

• Older patients attending a tailored Primary health care (PHC) unit felt acknowledged, unlike in the ordinary PHC centre, which facilitated their participation in their care.
• The calm environment, specialist geriatric competence and ample patient contact time enabled them to feel secure and taken care of.
• Older patients expressed a need for an incorporation of social services and health promotion visits at the tailored PHC unit.

     

Salicornia dolichostachya organosolv fractionation: towards establishing a halophyte biorefinery

Halophytes are a potential source of lignocellulosic material for biorefinery, as they can be grown in areas unsuitable for the cultivation of crops aimed at food production. To enable the viable use of halophytes in biorefineries, the present study investigated how different organosolv process parameters affected the fractionation of green pressed fibers of Salicornia dolichostachya. We produced pretreated solids characterized by up to 51.3% ± 1.7% cellulose, a significant increase from 25.6% ± 1.3% in untreated fibers. A delignification yield of as high as 60.7%, and hemicellulose removal of as high as 86.1% were also achieved in the current study. The obtained cellulose could be completely converted to glucose via enzymatic hydrolysis within 24 h. The lignin fractions obtained were of high purity, with sugar contamination of only 1.22% w/w and ashes below 1% w/w in most samples. Finally, up to 29.1% ± 0.4% hemicellulose was recovered as a separate product, whose proportion of oligomers to total sugars was 69.9% ± 3.0%. To the best of our knowledge, this is the first report in which Salicornia fibers are shown to be a suitable feedstock for organosolv biomass fractionation. These results expand the portfolio of biomass sources for biorefinery applications.

An organosolv method was developed for the fractionation of fibers of a halophyte plant in a biorefinery approach. Salicornia dolichostachya was used as raw material allowing the production of cellulose, hemicellulose, and lignin fractions.     

Anticoagulant action of melagatran: a comparison between neonates and adults using calibrated automated thrombography (CAT)

In the present study, we comparatively evaluated the anticoagulant efficacy of the new direct thrombin inhibitor melagatran in cord vs. adult plasma. In contrast to heparin, melagatran does not require antithrombin as a cofactor. Thus, anticoagulant treatment with melagatran is of special interest in neonatal patients, whose plasma is relatively deficient in antithrombin. We evaluated the anticoagulant action of increasing amounts of melagatran (0.1–2.0 μmol/l) in both cord and adult plasma by means of calibrated automated thrombography (CAT) with respect to the lag time until the onset of thrombin formation, time to thrombin peak maximum (TTP), endogenous thrombin potential (ETP), and thrombin peak height. Melagatran exhibited approximately the same ability to prolong lag times or TTPs in both cord and adult plasma. Similar concentrations (IC₅₀) of melagatran were required to double the lag times (0.44±0.04 μmol/l vs. 0.52±0.05 μmol/l) or to double the TTPs (0.91±0.08 μmol/l vs. 1.06±0.09 μmol/l) in cord vs. adult plasma. Melagatran exhibited a higher ability to suppress ETPs or thrombin peak heights in cord vs. adult plasma. Markedly lower concentrations (IC₅₀) of melagatran were required to suppress ETPs (0.27±0.03 μmol/l vs. 0.70±0.06 μmol/l) or thrombin peak heights by 50% (0.29±0.03 μmol/l vs. 0.53±0.04 μmol/l) in cord vs. adult plasma. We conclude that our results suggest a higher ability of melagatran to suppress thrombin formation in cord vs. adult plasma. Thus, lower amounts of melagatran might be required in neonates undergoing antithrombotic therapy.     

Smad5 is dispensable for adult murine hematopoiesis

Smad5 is known to transduce intracellular signals from bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-beta (TGF-beta) superfamily and are involved in the regulation of hematopoiesis. Recent findings suggest that BMP4 stimulates proliferation of human primitive hematopoietic progenitors in vitro, while early progenitors from mice deficient in Smad5 display increased self-renewal capacity in murine embryonic hematopoiesis. Here, we evaluate the role of Smad5 in the regulation of hematopoietic stem cell (HSC) fate decisions in adult mice by using an inducible MxCre-mediated conditional knockout model. Surprisingly, analysis of induced animals revealed unperturbed cell numbers and lineage distribution in peripheral blood (PB), bone marrow (BM), and the spleen. Furthermore, phenotypic characterization of the stem cell compartment revealed normal numbers of primitive lin(-)Sca-1(+)c-Kit(+) (LSK) cells in Smad5(-)(/)(-) BM. When transplanted in a competitive fashion into lethally irradiated primary and secondary recipients, Smad5-deficient BM cells competed normally with wild-type (wt) cells, were able to provide long-term reconstitution for the hosts, and displayed normal lineage distribution. Taken together, Smad5-deficient HSCs from adult mice show unaltered differentiation, proliferation, and repopulating capacity. Therefore, in contrast to its role in embryonic hematopoiesis, Smad5 is dispensable for hematopoiesis in the adult mouse.     

Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase/aminopeptidase and development of an epoxide hydrolase selective inhibitor

Leukotriene (LT) A₄ hydrolase/aminopeptidase (LTA4H) is a bifunctional zinc metalloenzyme that catalyzes the committed step in the formation of the proinflammatory mediator LTB₄. Recently, the chemotactic tripeptide Pro-Gly-Pro was identified as an endogenous aminopeptidase substrate for LTA₄ hydrolase. Here, we determined the crystal structure of LTA₄ hydrolase in complex with a Pro-Gly-Pro analog at 1.72 Å. From the structure, which includes the catalytic water, and mass spectrometric analysis of enzymatic hydrolysis products of Pro-Gly-Pro, it could be inferred that LTA₄ hydrolase cleaves at the N terminus of the palindromic tripeptide. Furthermore, we designed a small molecule, 4-(4-benzylphenyl)thiazol-2-amine, denoted ARM1, that inhibits LTB₄ synthesis in human neutrophils (IC₅₀ of ∼0.5 μM) and conversion of LTA₄ into LTB₄ by purified LTA4H with a K_i of 2.3 μM. In contrast, 50- to 100-fold higher concentrations of ARM1 did not significantly affect hydrolysis of Pro-Gly-Pro. A 1.62-Å crystal structure of LTA₄ hydrolase in a dual complex with ARM1 and the Pro-Gly-Pro analog revealed that ARM1 binds in the hydrophobic pocket that accommodates the ω-end of LTA₄, distant from the aminopeptidase active site, thus providing a molecular basis for its inhibitory profile. Hence, ARM1 selectively blocks conversion of LTA₄ into LTB₄, although sparing the enzyme’s anti-inflammatory aminopeptidase activity (i.e., degradation and inactivation of Pro-Gly-Pro). ARM1 represents a new class of LTA₄ hydrolase inhibitor that holds promise for improved anti-inflammatory properties.Leukotriene (LT) A₄ hydrolase/aminopeptidase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that catalyzes the formation of the potent chemotactic agent LTB₄, a key lipid mediator in the innate immune response (1, 2). Previous work has shown that LTA₄ hydrolase (LTA4H) is an aminopeptidase with high affinity for N-terminal arginines of various synthetic tripeptides (3, 4). The two enzyme activities of LTA4H are exerted via distinct but overlapping active sites and depend on the catalytic zinc, bound within the signature HEXXH-(X)₁₈-E, typical of M1 metallopeptidases (5–7). In LTA4H, His295, His299, and Glu318 are the zinc-binding ligands, whereas Glu296 is the general base catalyst for peptide hydrolysis (8, 9).LTA4H’s crystal structure has been determined (10). The enzyme folds into an N-terminal domain, a catalytic domain, and a C-terminal domain, each with ∼200 amino acids. The interface of the domains forms a cavity, where the active site is located (Fig. 1). The cavity narrows at the zinc-binding site, forming a tunnel into the catalytic domain. The opening and wider parts of the cavity are highly polar; the tunnel is more hydrophobic. The cavity is mostly defined by the catalytic and C-terminal domains; part of the tunnel is defined by the N-terminal domain. Bound substrate is in contact with all three domains.Open in a separate windowFig. 1.Position and extension of the active center in LTA4H. Cartoon representation of the structure of LTA4H with a tunnel for LTA₄ (red mesh) and peptide substrates (blue mesh). The catalytic zinc (yellow sphere) is located in a wide section of the active site from which a narrow, L-shaped, hydrophobic tunnel protrudes ∼15 Å deeper into the protein. LTA₄ is believed to bind with its ω-end at the end of the hydrophobic tunnel. The volume of the active center was calculated in CAVER (31).Recently, it was discovered that LTA4H cleaves and inactivates the chemotactic tripeptide Pro-Gly-Pro, thus identifying a previously unrecognized endogenous, physiologically significant aminopeptidase substrate (11). Inasmuch as Pro-Gly-Pro attracts neutrophils and promotes inflammation, these data also suggest that LTA4H plays dual and opposite roles during an inflammatory response (i.e., production of chemotactic LTB₄, as well as inactivation of chemotactic Pro-Gly-Pro). Previous efforts to develop inhibitors of LTA4H have used the aminopeptidase activity for screening purposes, and these molecules therefore block both catalytic activities of LTA4H (12).Here, we used crystallography, MS, and a stable peptide analog to determine the binding mode of Pro-Gly-Pro at the active site of LTA4H, as well as the mechanism of peptide cleavage. Based on the structure, we also designed a lead compound that selectively blocks the conversion of LTA₄ into LTB₄, although sparing the hydrolysis of Pro-Gly-Pro.     

Activities of Fosfomycin and Rifampin on Planktonic and Adherent Enterococcus faecalis Strains in an Experimental Foreign-Body Infection Model

Enterococcal implant-associated infections are difficult to treat because antibiotics generally lack activity against enterococcal biofilms. We investigated fosfomycin, rifampin, and their combinations against planktonic and adherent Enterococcus faecalis (ATCC 19433) in vitro and in a foreign-body infection model. The MIC/MBC_log values were 32/>512 μg/ml for fosfomycin, 4/>64 μg/ml for rifampin, 1/2 μg/ml for ampicillin, 2/>256 μg/ml for linezolid, 16/32 μg/ml for gentamicin, 1/>64 μg/ml for vancomycin, and 1/5 μg/ml for daptomycin. In time-kill studies, fosfomycin was bactericidal at 8× and 16× MIC, but regrowth of resistant strains occurred after 24 h. With the exception of gentamicin, no complete inhibition of growth-related heat production was observed with other antimicrobials on early (3 h) or mature (24 h) biofilms. In the animal model, fosfomycin alone or in combination with daptomycin reduced planktonic counts by ≈4 log₁₀ CFU/ml below the levels before treatment. Fosfomycin cleared planktonic bacteria from 74% of cage fluids (i.e., no growth in aspirated fluid) and eradicated biofilm bacteria from 43% of cages (i.e., no growth from removed cages). In combination with gentamicin, fosfomycin cleared 77% and cured 58% of cages; in combination with vancomycin, fosfomycin cleared 33% and cured 18% of cages; in combination with daptomycin, fosfomycin cleared 75% and cured 17% of cages. Rifampin showed no activity on planktonic or adherent E. faecalis, whereas in combination with daptomycin it cured 17% and with fosfomycin it cured 25% of cages. Emergence of fosfomycin resistance was not observed in vivo. In conclusion, fosfomycin showed activity against planktonic and adherent E. faecalis. Its role against enterococcal biofilms should be further investigated, especially in combination with rifampin and/or daptomycin treatment.