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Background Cardiovascular diseases are the most prominent global health threats. For optimal treatment, myocardial biopsies are an indispensable requirement. Current analysis includes histological and biochemical assays with particular limits. Here we present a first approach utilizing light sheet fluorescence microscopy (LSFM) to visualize the 3D vasculatory architecture in the murine heart after experimental myocardial infarction (MI) and in human cardiac biopsies with simultaneously imaging of various immunological infiltrates for disease investigation. Methods and results For murine hearts, cardiac vasculature was stained using CD31-AF790 antibody, where we found distinct spots of negative staining, which correlated in size and localisation with other markers of cardiac damage such as triphenyl tetratzolium chloride (TTC) staining, ejection fraction (EF) reduction and plasma cardiac troponin I levels. Using a Ly6G-AF647 antibody, we were able to visualize neutrophil infiltrates 24 h after MI, which localized at the border of the I/R-injury zone. 5 d after MI we imaged the healing vasculatory network using the CD31 marker as well as infiltrated macrophages using F4/80 staining. Human cardiac vasculature was stained using CD31-AF790 antibody in a whole-mount staining approach. In short, human samples were fixed in 4% PFA and permeabelized using saponin-based detergents. Antibody staining was conducted at 30 °C in the dark over several days. Immunological infiltrates were visualized using human CD66b, CD16, CD64, CD3 and CD19 antibodies in various, simultaneous approaches. After staining, cardiac samples were dehydrated and cleared using ethyl cinnamate (ECi) for light sheet microscopy. The spatial resolution of cardiac blood vessels was determined by CD31 staining, revealing a highly organized and structured vascular tree. Simultaneous staining for immunological infiltrates showed association with areas of enhanced CD31 staining, displaying activated endothelium at sites of inflammation. Ly6G events in a murine heart Conclusion Light sheet-guided analysis of cardiac samples shows promising results in terms of synchronous vascular and immunological investigation. We highlighted its applicability in identifying specific immune cells based on immune-phenotyping. This novel technique may enable future clinical heart disease investigation with a powerful tool for visualization of 3D cardiac structure and identification of multiple immune cell infiltrates.

Cardioprotection by salvage of the infarct-affected myocardium is an unmet yet highly desired therapeutic goal. To develop new dedicated therapies, experimental myocardial ischemia/reperfusion (I/R) injury would require methods to simultaneously characterize extent and localization of the damage and the ensuing inflammatory responses in whole hearts over time. Here we present a three-dimensional (3D), simultaneous quantitative investigation of key I/R injury-components by combining bleaching-augmented solvent-based non-toxic clearing (BALANCE) using ethyl cinnamate (ECi) with light sheet fluorescence microscopy. This allows structural analyses of fluorescence-labeled I/R hearts with exceptional detail. We discover and 3D-quantify distinguishable acute and late vascular I/R damage zones. These contain highly localized and spatially structured neutrophil infiltrates that are modulated upon cardiac healing. Our model demonstrates that these characteristic I/R injury patterns can detect the extent of damage even days after the ischemic index event hence allowing the investigation of long-term recovery and remodeling processes., Detailed characterization of cardiac damage following ischemia/reperfusion injury and detection of occurring inflammatory responses is important for the development of new therapeutic concepts. Here the authors present a method for the three-dimensional investigation of acute and chronic cardiac injury responses using light sheet fluorescence microscopy.

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory protein and contributes to several different inflammatory and ischemic/hypoxic diseases. MIF was shown to be cardioprotective in experimental myocardial ischemia/reperfusion injury and its expression is regulated by the transcription factor hypoxia-inducible factor (HIF)-1α. We here report on MIF expression in the failing human heart and assess myocardial MIF in different types of cardiomyopathy. Myocardial tissue samples from n = 30 patients were analyzed by quantitative Real-Time PCR. MIF and HIF-1α mRNA expression was analyzed in myocardial samples from patients with ischemic (ICM) and non-ischemic cardiomyopathy (NICM) and from patients after heart transplantation (HTX). MIF expression was elevated in myocardial samples from patients with ICM compared to NICM. Transplanted hearts showed lower MIF levels compared to hearts from patients with ICM. Expression of HIF-1α was analyzed and was shown to be significantly increased in ICM patients compared to patients with NICM. MIF and HIF-1α mRNA is expressed in the human heart. MIF and HIF-1α expression depends on the underlying type of cardiomyopathy. Patients with ICM show increased myocardial MIF and HIF-1α expression. OA gold - CA Pohl

Skeletal muscle tissue has a remarkable high regenerative capacity. The underlying cellular events are governed by complex signaling processes, and the proliferation of skeletal myoblasts is a key initial event. The role of nitric oxide (NO) in cell cycle regulation is well-appreciated. Nitrite, an NO oxidation product, is a stable source for NO-like bioactivity particularly in cases when oxygen shortage compromises NO-synthases activity. Although numerous studies suggest that nitrite effects are largely related to NO-dependent signaling, emerging evidence also implicates that nitrite itself can activate protein pathways albeit under physiological, normoxic conditions. This includes a recently demonstrated cyclic guanosine monophosphate-(cGMP)-independent enhancement of endothelial cell proliferation. Whether nitrite itself has the potential to affect myoblast proliferation and metabolism with or without activation of the canonical NO/cGMP pathway to subsequently support muscle cell regeneration is not known. Here we show that nitrite increases proliferation and metabolic activity of murine cultured myoblasts dose-dependently. This effect is not abolished by the NO scavenger 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimida-zoline-1-oxyl-3 oxide and does not affect intracellular cGMP levels, implicating a cGMP-independent mechanism. Nitrite circumvents the rapamycin induced attenuation of myoblast proliferation and enhances mTOR activity. Our results provide evidence for a novel potential physiological and therapeutic approach of nitrite in skeletal muscle regeneration processes under normoxia independent of NO and cGMP.

Increasing age involves a number of detrimental changes in the cardiovascular system and particularly on the large arteries. It deteriorates vascular integrity and leads to increased vascular stiffness entailing hypertension with increased cardiovascular morbidity and mortality. The consequences of continuous oxidative stress and damages to biomolecules include altered gene expression, genomic instability, mutations, loss of cell division and cellular responses to increased stress. Many studies have been performed in aged C57BL/6 mice; however, analyses of the age-related changes that occur at a gene expression level and transcriptional profile in vascular tissue have not been elucidated in depth. To determine the changes of the vascular transcriptome, we conducted gene expression microarray experiments on aortas of adult and old mice, in which age-related vascular dysfunction was confirmed by increased stiffness and associated systolic hypertension. Our results highlight differentially expressed genes overrepresented in Gene Ontology categories. Molecular interaction and reaction pathways involved in vascular functions and disease, within the transforming growth factor-beta (TGF-β) pathway, the renin-angiotensin system and the detoxification systems are displayed. Our results provide insight to an altered gene expression profile related to age, thus offering useful clues to counteract or prevent vascular aging and its detrimental consequences.

The analytical validation of a possible biomarker is the first step in the long translational process from basic science to clinical routine. Although the chemokine-like cytokine macrophage migration inhibitory factor (MIF) has been investigated intensively in experimental approaches to various disease conditions, its transition into clinical research is just at the very beginning. Because of its presence in preformed storage pools, MIF is the first cytokine to be released under various stimulation conditions. In the first proof-of-concept studies, MIF levels correlated with the severity and outcome of various disease states. In a recent small study with acute coronary syndrome patients, elevation of MIF was described as a new factor for risk assessment. When these studies are compared, not only MIF levels in diseased patients differ, but also MIF levels in healthy control groups are inconsistent. Blood MIF concentrations in control groups vary between 0.56 and 95.6 ng/ml, corresponding to a 170-fold difference. MIF concentrations in blood were analyzed by ELISA. Other than the influence of this approach due to method-based variations, the impact of preanalytical processing on MIF concentrations is unclear and has not been systematically studied yet. Before large randomized studies are performed to determine the impact of circulating MIF on prognosis and outcome and before MIF is characterized as a diagnostic marker, an accurate protocol for the determination of reproducible MIF levels needs to be validated. In this study, the measurement of MIF in the blood of healthy volunteers was investigated focusing on the potential influence of critical preanalytical factors such as anticoagulants, storage conditions, freeze/thaw stability, hemolysis, and dilution. We show how to avoid pitfalls in the measurement of MIF and that MIF concentrations are highly susceptible to preanalytical factors. MIF serum concentrations are higher than plasma concentrations and show broader ranges. MIF concentrations are higher in samples processed with latency than in those processed directly and strongly correlate with hemoglobin in plasma. Neither storage temperature nor storage length or dilution or repeated freezing and thawing influenced MIF concentrations in plasma. Preanalytical validation of MIF is essential. In summary, we suggest using plasma and not serum samples when determining circulating MIF and avoiding hemolysis by processing samples immediately after blood drawing.

Purpose D-dopachrome tautomerase (MIF-2 or DDT) is a member of the macrophage migration inhibitory factor (MIF) superfamily and a close structural homolog to MIF. Circulating MIF-2 has been described to be elevated in patients suffering from sepsis, severe burn injury and after surgery. We sought to evaluate the prognostic value of MIF-2 in critically ill patients. Methods A total of 72 patients were studied upon admission to the medical intensive care unit (ICU). MIF and MIF-2 levels were assessed and compared to healthy controls. Clinical data, various laboratory parameters and mortality were assessed. Results We found significantly elevated levels of MIF-2 and MIF at admission to the ICU in critically ill patients compared to healthy controls. MIF-2 levels were associated with disease severity as measured by APACHE II scores. MIF-2 levels in ICU patients correlated with biomarkers reflecting organ damage, but were not influenced by acute or chronic kidney disease. Kaplan-Meier analysis revealed distinctly elevated mortality in patients with high plasma MIF-2 levels. Conclusions MIF-2 levels are elevated in critically ill patients and linked to parameters of organ damage, supporting its value as a potential tool for the assessment of prognosis in critical illness.

Background Macrophage migration inhibitory factor (MIF) is known to amplify the immune response in septic animal models. Few clinical data support this pro-inflammatory role in septic patients. Renal replacement therapy (RRT) as adjuvants in the complex therapy of sepsis has been proposed as a possible approach to eliminate elevated circulating cytokines. Since recent data suggest that MIF can be effectively removed from the circulating blood pool in patients with chronic kidney disease, we here aimed to investigate whether RRT in septic shock can lower plasma levels of this pro-inflammatory cytokine in septic shock patients. Methods An observational single-center study on an internist intensive care unit (ICU) was conducted. MIF plasma levels and mortality of n = 25 patients with septic shock were assessed with a previously validated method for reliable MIF values. The effect of continuous renal replacement therapy (CRRT) on daily MIF levels and mortality was assessed by comparing patients with and without need for CRRT due to acute kidney injury (AKI). Results MIF plasma levels in patients undergoing CRRT due to septic AKI were steadily decreased compared to those from patients without CRRT hinting at a MIF removal by hemodialysis. MIF release during ICU stay as assessed by MIFAUC was lower in patients undergoing CRRT, and Kaplan-Meier analysis revealed a distinctly lower mortality in patients undergoing CRRT. Analysis of daily MIF levels showed that patients who did not survive septic shock exhibited steadily higher MIF plasma levels and higher MIFAUC compared to those surviving sepsis. Low MIF levels were closely associated with improved survival. Conclusions This is the first study investigating the effect of efficient MIF removal from the plasma pool of patients with septic shock. Reduction of high circulating MIF by CRRT therapy was accompanied by improved survival. Thus, targeted removal of MIF from the circulating blood pool might be a promising approach to reduce mortality in severe sepsis. Electronic supplementary material The online version of this article (doi:10.1186/s40560-016-0163-2) contains supplementary material, which is available to authorized users.

Introduction Following successful resuscitation from cardiac arrest (CA), neurological impairment and other types of organ dysfunction cause significant morbidity and mortality—a condition termed post-cardiac arrest syndrome. Whole-body ischemia/reperfusion with oxygen debt activates immunologic and coagulation pathways increasing the risk of multiple organ failure and infection. We here examined the role of the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) in post-cardiac arrest syndrome. Methods MIF plasma levels of n =16 patients with return of spontaneous circulation (ROSC) after CA were assessed with a previously validated method and compared to markers of systemic inflammation and cellular damage. ICU patients without former CA and healthy volunteers served as controls. Results MIF levels in patients after ROSC were higher compared to those in healthy volunteers and ICU patients without CA. Kaplan–Meyer analysis revealed a distinctly elevated mortality since day one that further increased towards an elevated 60-days-mortality in patients with high plasma MIF. ROC curve identified plasma MIF as a predictor for mortality in patients after CA. Correlation with inflammatory parameters revealed that high MIF levels did not mirror post CA inflammatory syndrome, but distinctive cellular damage after ROSC as there were strong correlations with markers of cellular damage like LDH and GOT/GPT. Conclusion High MIF levels were associated with elevated 60-days-mortality and high MIF predicted mortality after CA. We found a close relation between circulating MIF levels and cellular damage, but not with an inflammatory syndrome.

Background— Revascularization is an adaptive repair mechanism that restores blood flow to undersupplied ischemic tissue. Nitric oxide plays an important role in this process. Whether dietary nitrate, serially reduced to nitrite by commensal bacteria in the oral cavity and subsequently to nitric oxide and other nitrogen oxides, enhances ischemia-induced remodeling of the vascular network is not known. Methods and Results— Mice were treated with either nitrate (1 g/L sodium nitrate in drinking water) or sodium chloride (control) for 14 days. At day 7, unilateral hind-limb surgery with excision of the left femoral artery was conducted. Blood flow was determined by laser Doppler. Capillary density, myoblast apoptosis, mobilization of CD34 + /Flk-1 + , migration of bone marrow–derived CD31 + /CD45 − , plasma S-nitrosothiols, nitrite, and skeletal tissue cGMP levels were assessed. Enhanced green fluorescence protein transgenic mice were used for bone marrow transplantation. Dietary nitrate increased plasma S-nitrosothiols and nitrite, enhanced revascularization, increased mobilization of CD34 + /Flk-1 + and migration of bone marrow–derived CD31 + /CD45 − cells to the site of ischemia, and attenuated apoptosis of potentially regenerative myoblasts in chronically ischemic tissue. The regenerative effects of nitrate treatment were abolished by eradication of the nitrate-reducing bacteria in the oral cavity through the use of an antiseptic mouthwash. Conclusions— Long-term dietary nitrate supplementation may represent a novel nutrition-based strategy to enhance ischemia-induced revascularization.

The cDNAs encoding an intestinal defensin (def1) and lysozyme (lys1) of the reduviid bug Triatoma brasiliensis have been amplified by PCR using specific oligonucleotide primers and 5'- and 3'-RACE, cloned and sequenced. The 576 bp clone has an open reading frame of 282 bp and encodes a pre-prodefensin with 94 amino acid residues, containing a putative signal and activation peptide cleavage site at Ser19 and Arg51, respectively. The genomic DNA contains a second defensin gene with similar characteristics, 88.3% identity and also one intron of 107 nucleotides. The 538 bp clone has an open reading frame of 417 bp, encoding a pre-lysozyme with 139 amino acid residues. The putative signal peptide is cleaved at alanine 18. Using whole mount in situ hybridization, high expression of both genes has been found, distributed uniformly throughout the entire cardia and the blood-storing stomach and to a much lower extent in the digesting small intestine. Using quantitative real-time PCR, the expression level of def1 was also shown to be very low in small intestine, rectum and salivary glands; in the stomach, expression was 500-2500 times higher than in the cardia and fat body. No expression of lys1 could be detected in the salivary glands and rarely a very low expression in the small intestine, rectum and fat body. Lys1 expression in the stomach was 60-300 times higher than in the cardia. Comparing the levels in unfed fifth instars and up to 15 days after feeding, a strong def1 induction was evident in the fat body at 15 days after feeding and in the stomach a maximum level of def1 and lys1 at 5 days after feeding.

After the previous characterization of one trypsin gene (Try1) of the human body louse Pediculus humanus, genes encoding a second trypsin (Try2) and a chymotrypsin (Chy1) have been cloned using degenerate serine proteinase primers and 5¢- and 3¢-RACE, and sequenced. The deduced 259 and 267 amino acid se- quences of Try2 and Chy1 show an identity of 33%- 40% to trypsinogens and chymotrypsinogens of other insects. Considering previously published partial se- quences, P. humanus possesses at least one Try1 gene, five variants/isoforms of Try2 and six variants/isoforms of Chy1. The genomic DNA of Try2 contains three in- trons and Chy1 contains five introns. Using whole mount in situ hybridization, gene expression of Try1, Try2 and Chy1 has been localized not only in the dis- tensible anterior region of the midgut of lice but some- times also in the area following the distensible region. The Try2 gene was always expressed at much lower levels than Try1 or Chy1. This lower expression, the constitutive expression of Try1 and Chy1 at 1, 2, 6, 12 and 24 h after feeding of adults and the regional differ- ences have been verified in quantitative real-time PCR.