Hematoma Resolution In Vivo Is Directed by Activating Transcription Factor 1.

RATIONALE: The efficient resolution of tissue hemorrhage is an important homeostatic function. In human macrophages in vitro, heme activates an AMPK (AMP-activated protein kinase)/ATF1 (activating transcription factor-1) pathway that directs Mhem macrophages through coregulation of HO-1 (heme oxygenase-1; HMOX1) and lipid homeostasis genes. OBJECTIVE: We asked whether this pathway had an in vivo role in mice. METHODS AND RESULTS: Perifemoral hematomas were used as a model of hematoma resolution. In mouse bone marrow-derived macrophages, heme induced HO-1, lipid regulatory genes including LXR (lipid X receptor), the growth factor IGF1 (insulin-like growth factor-1), and the splenic red pulp macrophage gene Spic. This response was lost in bone marrow-derived macrophages from mice deficient in AMPK (Prkab1-/-) or ATF1 (Atf1-/-). In vivo, femoral hematomas resolved completely between days 8 and 9 in littermate control mice (n=12), but were still present at day 9 in mice deficient in either AMPK (Prkab1-/-) or ATF1 (Atf1-/-; n=6 each). Residual hematomas were accompanied by increased macrophage infiltration, inflammatory activation and oxidative stress. We also found that fluorescent lipids and a fluorescent iron-analog were trafficked to lipid-laden and iron-laden macrophages respectively. Moreover erythrocyte iron and lipid abnormally colocalized in the same macrophages in Atf1-/- mice. Therefore, iron-lipid separation was Atf1-dependent. CONCLUSIONS: Taken together, these data demonstrate that both AMPK and ATF1 are required for normal hematoma resolution. Graphic Abstract: An online graphic abstract is available for this article.

A Coumarin-Porphyrin FRET Break-Apart Probe for Heme Oxygenase-1.

Heme oxygenase-1 (HO-1) is a vital enzyme in humans that primarily regulates free heme concentrations. The overexpression of HO-1 is commonly associated with cardiovascular and neurodegenerative diseases including atherosclerosis and ischemic stroke. Currently, there are no known chemical probes to detect HO-1 activity, limiting its potential as an early diagnostic/prognostic marker in these serious diseases. Reported here are the design, synthesis, and photophysical and biological characterization of a coumarin-porphyrin FRET break-apart probe to detect HO-1 activity, Fe-L1. We designed Fe-L1 to "break-apart" upon HO-1-catalyzed porphyrin degradation, perturbing the efficient FRET mechanism from a coumarin donor to a porphyrin acceptor fluorophore. Analysis of HO-1 activity using Escherichia coli lysates overexpressing hHO-1 found that a 6-fold increase in emission intensity at 383 nm was observed following incubation with NADPH. The identities of the degradation products following catabolism were confirmed by MALDI-MS and LC-MS, showing that porphyrin catabolism was regioselective at the α-position. Finally, through the analysis of Fe-L2, we have shown that close structural analogues of heme are required to maintain HO-1 activity. It is anticipated that this work will act as a foundation to design and develop new probes for HO-1 activity in the future, moving toward applications of live fluorescent imaging.

Changes in gastrointestinal cell integrity after marathon running and exercise-associated collapse.

PURPOSE: Endurance exercise and hyperthermia are associated with compromised intestinal permeability and endotoxaemia. The presence of intestinal fatty acid-binding protein (I-FABP) in the systemic circulation suggests intestinal wall damage, but this marker has not previously been used to investigate intestinal integrity after marathon running. METHODS: Twenty-four runners were recruited as controls prior to completing a standard marathon and had sequential I-FABP measurements before and on completion of the marathon, then at four and 24 h later. Eight runners incapacitated with exercise-associated collapse (EAC) with hyperthermia had I-FABP measured at the time of collapse and 1 hour later. RESULTS: I-FABP was increased immediately on completing the marathon (T0; 2593 ± 1373 ng·l-1) compared with baseline (1129 ± 493 ng·l-1; p < 0.01) in the controls, but there was no significant difference between baseline and the levels at four hours (1419 ± 1124 ng·l-1; p = 0.7), or at 24 h (1086 ± 302 ng·l-1; p = 0.5). At T0, EAC cases had a significantly higher I-FABP concentration (15,389 ± 8547 ng.l-1) compared with controls at T0 (p < 0.01), and remained higher at 1 hour after collapse (13,951 ± 10,476 ng.l-1) than the pre-race control baseline (p < 0.05). CONCLUSION: I-FABP is a recently described biomarker whose presence in the circulation is associated with intestinal wall damage. I-FABP levels increase after marathon running and increase further if the endurance exercise is associated with EAC and hyperthermia. After EAC, I-FABP remains high in the circulation for an extended period, suggesting ongoing intestinal wall stress.

The efficacy of antibiotics in reducing morbidity and mortality from heatstroke - A systematic review.

Severe hyperthermia, for example, classical heatstroke or exertional heatstroke from heatwaves or exercise respectively, or from drug ingestion or other non-infective pyrogens, is associated with a high mortality and morbidity, which may be chronic or permanent. Abolition of lipopolysaccharide, from gram-negative intestinal bacteria translocating into the systemic circulation via an intestinal wall rendered permeable from the hyperthermia, reduces the adverse effects, suggesting that antibiotics against the intestinal bacteria may have a similar effect. A systematic review searching Embase, MEDLINE and PubMed from the earliest date available until 2019 was conducted, according to PRISMA guidelines. Two papers were found which fit the criteria. In one, non-absorbable oral antibiotics were administered prior to the onset of heat stress, which reduced the cardiovascular dysfunction and rise in endotoxaemia, but animals succumbed at a lower temperature. In the second, non-absorbable oral antibiotics, in combination with a laxative and enema, given prior to the onset of heat stress, improved mortality; antibiotics administered after the heat stress did not, but the antibiotics used may have limited action against intestinal bacteria. Only two papers were found; both suggest an improvement in organ dysfunction or mortality after an episode of heat stress. No papers were found that investigate the sole use of antibiotics effective against intestinal bacteria given after the onset of heat stress, although biological plausibility suggest they warrant further research.

APTRA-Based Luminescent Lanthanide Complexes Displaying Enhanced Selectivity for Mg2.

A series of three europium(III) complexes has been created in which an APTRA moiety has been integrated into the sensitising chromophore (APTRA=o-aminophenol-N,N,N-triacetate). The constitutionally isomeric complexes EuL1 and EuL2 feature the APTRA unit linked to a metal-bound pyridine ring through an alkynyl unit, differing according to the disposition of the APTRA substituents relative to the C≡C unit (para-N and para-O). In EuL3 , the APTRA ring is directly bonded to the Eu-coordinated pyridine (para-O). The metal binding affinities for magnesium, calcium and zinc ions have been measured by using emission and excitation spectroscopy. The pyridylalkynylaryl systems, EuL1 and EuL2 , offer superior affinity and selectivity for Mg2+ . The Mg2+ affinities are surprisingly very different from prior studies on structurally related systems that incorporate organic fluorophores as reporters, as opposed to the macrocyclic Eu complex moiety. A much-reduced affinity for calcium and zinc-possibly arising from the lower donor ability of the aryl N or O atoms arising from extended conjugation-means that magnesium ion concentrations can be measured directly in serum for the first time, by using such an approach. An apparent dissociation constant for magnesium binding of Kd =2.4 mm was calculated in the serum background.

Tuning Mg(II) Selectivity: Comparative Analysis of the Photophysical Properties of Four Fluorescent Probes with an Alkynyl-Naphthalene Fluorophore.

Four alkynyl-1-naphthyl fluorophores have been synthesised with tri- or pentadentate ligating groups suited to the binding of magnesium. Their photophysical and binding properties for magnesium, calcium and zinc ions have been assessed using absorption, emission and excitation spectroscopy. Each compound has a pKa value between 6.2 and 5.1 and exhibits no significant pH response around pH 7.2. Enhanced selectivity for Mg2+ over Ca2+ was observed with a pentadentate phosphinate substituted system, compared to its carboxylate analogue, due to a 10-fold lowering of affinity for the Ca2+ ion. In each case, the overall dissociation constants for Mg2+ fall in the low mm range.

The neurological and cognitive consequences of hyperthermia.

An elevated temperature has many aetiologies, both infective and non-infective, and while the fever of sepsis probably confers benefit, there is increasing evidence that the central nervous system is particularly vulnerable to damage from hyperthermia. A single episode of hyperthermia may cause short-term neurological and cognitive dysfunction, which may be prolonged or become permanent. The cerebellum is particularly intolerant to the effects of heat. Hyperthermia in the presence of acute brain injury worsens outcome. The thermotoxicity involved occurs via cellular, local, and systemic mechanisms. This article reviews both the cognitive and neurological consequences and examines the mechanisms of cerebral damage caused by high temperature.

The pathophysiological basis and consequences of fever.

There are numerous causes of a raised core temperature. A fever occurring in sepsis may be associated with a survival benefit. However, this is not the case for non-infective triggers. Where heat generation exceeds heat loss and the core temperature rises above that set by the hypothalamus, a combination of cellular, local, organ-specific, and systemic effects occurs and puts the individual at risk of both short-term and long-term dysfunction which, if severe or sustained, may lead to death. This narrative review is part of a series that will outline the pathophysiology of pyrogenic and non-pyrogenic fever, concentrating primarily on the pathophysiology of non-septic causes.

Mitochondria-targeting biocompatible fluorescent BODIPY probes.

An increase in the mitochondrial membrane potential (MMP) is a characteristic feature of cancer and cardiovascular disease. Therefore, it remains of crucial importance to develop new and improved fluorescent probes that are sensitive to the MMP, to report on mitochondrial health and function. Reported here are the design, synthesis, photophysical properties and biological characterisation of a series of BODIPY dyes, BODIPY-Mito-n, for mitochondria-targeted fluorescence imaging applications. Six BODIPY-Mito-n analogues were synthesised under mild conditions, and displayed excellent fluorescence quantum yields of between 0.59 and 0.72 in aqueous environments at physiological pH (pH = 7.4). The incorporation of poly(ethylene glycol) (PEG) chains to the triarylphosphonium cation moiety significantly improved the biocompatibility of the probes (BODIPY-Mito-6, IC50 > 50 µM). All BODIPY-Mito-n compounds demonstrated a high MMP-sensitive localisation in the mitochondria, with Pearson's correlation coefficients (PCC) of between 0.76 and 0.96. Compounds BODIPY-Mito-2 and BODIPY-Mito-6 revealed the highest sensitivity to the MMP, with a decrease in the emission intensity of 62% and 75%, respectively following MMP depolarisation. It is anticipated that the highest MMP sensitivity and enhanced biocompatibility of BODIPY-Mito-6 could lead to the development of new probes for mitochondrial imaging in the future.

Critical illness aspects of heatstroke: A hot topic.

Heatstroke represents the most severe end of the heat illness spectrum, and is increasingly seen in those undergoing exercise or exertion ('exertional heatstroke') and those exposed to high ambient temperatures, for example in heatwaves ('classical heatstroke'). Both forms may be associated with significant thermal injury, leading to organ dysfunction and the need for admission to an intensive care unit. The process may be exacerbated by translocation of bacteria or endotoxin through an intestinal wall rendered more permeable by the hyperthermia. This narrative review highlights the importance of early diagnosis, rapid cooling and effective management of complications. It discusses the incidence, clinical features and treatment of heatstroke, and discusses the possible role of intestinal permeability and advances in follow-up and recovery of this condition. Optimum treatment involves an integrated input from prehospital, emergency department and critical care teams, along with follow-up by rehabilitation teams and, if appropriate, sports or clinical physiologists.

Familial hepatic rupture in vascular Ehlers-Danlos syndrome in pregnancy with atypical thromboses.

This case highlights the importance of genetic testing over fibroblast testing and presents the first published thromboelastometry data in vascular Ehlers-Danlos syndrome.

Effect of a fever in viral infections - the 'Goldilocks' phenomenon?

Acute infections, including those due to Coronaviridae and other viruses, often stimulate a febrile response. A mild fever appears to improve outcome; it appears to diminish viral replication by several mechanisms, including virion entry into host cells and genome transcription, and improving host defence mechanisms against the pathogen. However, a fever may also damage host cellular and tissue function and increase metabolic demands. At temperatures at the lower end of the febrile range, the benefit of the fever appears to outweigh the detrimental effects. However, at higher temperatures, the outcome worsens, suggesting that the disadvantages of fever on the host predominate. A non-infective fever is associated with a worse outcome at lower temperatures, suggesting that hyperthermia carries less benefit in the absence of infection. This review discusses the risks and benefits of a fever on the host response, focusing on the effects of a fever on viral replication and host response, and the detrimental effect on the host.

Enzyme-activated probes in optical imaging: a focus on atherosclerosis.

Enzyme-activated probes enable complex biological processes to be studied in real-time. A wide range of enzymes are modulated in diseases, including cancer, inflammatory diseases and cardiovascular disease, and have the potential to act as vital diagnostic and prognostic biomarkers to monitor and report on disease progression. In this perspective article, we discuss suitable design characteristics of enzyme-activated fluorescent probes for ex vivo and in vivo optical imaging applications. With a particular focus on atherosclerosis imaging, we highlight recent approaches to report on the activity of cathepsins (K and B), matrix metalloproteinases (MMP-2 and MMP-9), thrombin, heme oxygenase-1 (HO-1) and myeloperoxidase (MPO).

A fluorescent probe for the discrimination of oxidation states of palladium.

Palladium-based catalysts are widely used in pharmaceutical industries, which can sometimes cause palladium contamination in pharmaceutical drug manufacture. It is important to separately quantify the different oxidation states of palladium (Pd0 and Pd2+) in pharmaceuticals as they react with scavengers differently. Although palladium sensors have been under intense investigation, oxidation state differentiators are very rare. Here, we report a simple porphyrin-coumarin conjugate, PPIX-L2, that can selectively discriminate between the oxidation states of palladium. The reaction of PPIX-L2 with Pd0 showed a 24-fold fluorescence increase of the coumarin emission, meanwhile, the presence of Pd2+ led to a 98% quenching of the porphyrin emission. Fluorescent responses of PPIX-L2 towards Pd0 and Pd2+ are specific, and its sensitivity towards both palladium species is significantly increased with a detection limit of 75 nM and 382 nM for Pd0 and Pd2+ respectively.

Exercise hyperthermia induces greater changes in gastrointestinal permeability than equivalent passive hyperthermia.

Hyperthermia and exertional heat illness increase gastrointestinal (GI) permeability, although whether the latter is only via hyperthermia is unclear. The aim of this pilot study was to determine whether different changes in GI permeability, characterized by an increased plasma lactulose:rhamnose concentration ratio ([L:R]), occurred in exercise hyperthermia in comparison to equivalent passive hyperthermia. Six healthy adult male participants (age 25 ± 5 years, mass 77.0 ± 6.7 kg, height 181 ± 6 cm, peak oxygen uptake [ V·O2peak ] 48 ± 8 ml.kg-1 .min-1 ) underwent exercise under hot conditions (Ex-Heat) and passive heating during hot water immersion (HWI). Heart rate (HR), rectal temperature (TCORE ), rating of perceived exertion (RPE), and whole-body sweat loss (WBSL) were recorded throughout the trials. The L:R ratio, peak HR, change in HR, and change in RPE were higher in Ex-Heat than HWI, despite no differences in trial duration, peak core temperature or WBSL. L:R was strongly correlated (p < 0.05) with HR peak (r = 0.626) and change in HR (r = 0.615) but no other variable. The greater L:R in Ex-Heat, despite equal TCORE responses to HWI, indicates that increased cardiovascular strain occurred during exercise, and exacerbates hyperthermia-induced GI permeability at the same absolute temperature.

Association between collapse and serum creatinine and electrolyte concentrations in marathon runners: a 9-year retrospective study.

OBJECTIVE: Abnormal biochemical measurements have previously been described in runners following marathons. The incidence of plasma sodium levels outside the normal range has been reported as 31%, and the incidence of raised creatinine at 30%. This study describes the changes seen in electrolytes and creatinine in collapsed (2010-2019 events) and noncollapsed (during the 2019 event) runners during a UK marathon. METHODS: Point-of-care sodium, potassium, urea and creatinine estimates were obtained from any collapsed runner treated by the medical team during the Brighton Marathons, as part of their clinical care, and laboratory measurements from control subjects. RESULTS: Results from 224 collapsed runners were available. Serum creatinine was greater than the normal range in 68.9%. About 6% of sodium results were below, and 3% above the normal range, with the lowest 132 mmol/l. Seventeen percent of potassium readings were above the normal range; the maximum result was 8.4 mmol/l, but 97% were below 6.0 mmol/l. In the control group, mean creatinine was significantly raised in both the collapse and control groups, with 55.4% meeting the criteria for acute kidney injury, but had resolved to baseline after 24 h. Sodium concentration but not the potassium was significantly raised after the race compared with baseline, but only 15% were outside the normal range. CONCLUSION: In this study, incidence of a raised creatinine was higher than previously reported. However, the significance of such a rise remains unclear with a similar rise seen in collapsed and noncollapsed runners, and resolution noted within 24 h. Abnormal sodium concentrations were observed infrequently, and severely abnormal results were not seen, potentially reflecting current advice to drink enough fluid to quench thirst.

'Cool runnings': heat stroke in cool conditions.

Heat stroke is rare and usually occurs in warm conditions. It is often fatal. This paper reports six runners from two road-running events who developed heat stroke despite cool conditions and who improved quickly with immediate treatment.

The efficacy of steroids in reducing morbidity and mortality from extreme hyperthermia and heatstroke-A systematic review.

Severe hyperthermia from classical or exertional heatstroke, or from drug ingestion or other noninfective pyrogens, is associated with a high mortality and morbidity. A systemic pro-inflammatory response occurs during heatstroke, characterized by elevated cytokines with endotoxemia from elevated lipopolysaccharide (LPS) levels. Corticosteroids reduce LPS and cytokine levels, suggesting that they may improve outcome. A systematic review searching Embase, MEDLINE, and PubMed from the earliest date available until September 2019 was conducted, according to the PRISMA guidelines, with five papers identified. In four studies, systemic steroids administered before or at the onset of heat stress improved mortality or reduced organ dysfunction. Survival time was greatest when steroid administration preceded heat stress. In one study, a nonsignificant increase in mortality was seen. A dose response was observed, with higher doses extending survival time. Animal studies suggest that steroids improve mortality and/or organ dysfunction after an episode of heat stress or extreme hyperthermia.