Surface-dependent biodistribution and proinflammatory properties of semiconductor quantum dots

Methods based on nanotechnologies play a growing role in biomedical research. Quantum dots (QDs) are a group of engineered fluorescent nanoparticles suited for advanced imaging applications. The substitution of the particle’s surface with defined molecular structures could enable the adoption as targeted contrast agents or therapeutic devices for a variety of clinical approaches. However, important aspects such as the basic surfacedependent behavior of non-targeted QDs in the organism and arising health effects upon systemic administration remain incompletely understood. Acute inflammatory effects for instance are often initiated on the microcirculatory level and are probably relevant for cardiovascular pathologies observed in epidemiologic and experimental studies of certain nanoparticles. Most in vitro studies show that the surface structures of QDs and other nanoparticles seem to be predominantly accountable for different cytotoxic effects and a variable potential to liberate proinflammatory cytokines. Currently, no systematic in vivo studies have addressed surface-dependent interactions of QDs on the level of the microcirculation and assessed the resulting impact on biokinetics as well as on proinflammatory parameters. Thus, this thesis aimed to i) analyze the incidence of QDsurface- dependent acute microvascular interactions and their influence on key biokinetic parameters and ii) investigate acute immunomodulatory effects on the multistep process of leukocyte recruitment in vivo. For this, three types of commercially available QDs with different surface modifications: carboxyl-QDs, amine- and polyethylene glycol-QDs (amine-QDs) and polyethylene glycol-QDs (PEG-QDs) were used. The physicochemical characterization was done by dynamic light scattering (DLS) analysis and microscale thermophoresis. In a first set of experiments, circulating half-lives, tissue distribution in different organs, and hepatic as well as renal clearance were measured. Ex vivo analysis of QD tissue distribution was performed on selected tissue samples via transmission electron microscopy (TEM) and two-photon microscopy. By combining reflected-light oblique transillumination (RLOT) and fluorescence in vivo microscopy of the murine M. cremaster, interactions of QDs with components of the microcirculation as well as leukocyte migration parameters were visualized and quantified. The extreme short circulating half-life of anionic carboxyl-QDs was related to pronounced clearance by the mononuclear phagocyte system. Beyond this, further investigations showed, for the first time, that the continuous capillary endothelium of skeletal and heart muscle tissue has the capacity to directly extract carboxyl-QDs from the circulation by means of caveolaemediated endocytosis. Carboxyl-QDs were also taken up by perivascular macrophages in the surgically exposed but not in the native M. cremaster and led to a significant increase of adherent and (subsequently) transmigrated leukocytes in this model. Further experiments provided evidence for a probable involvement of mast cells in the intercellular adhesion molecule-1 (ICAM-1)- and endothelial (E)-selectinmediated modulation of leukocyte recruitment. This process is most likely initiated by the endocytosis of carboxyl-QDs through activated perivascular macrophages. The primary activation of tissue-resident perivascular macrophages seems to be the consequence of tissue damage related to the surgical preparation of the cremaster muscle. This is supposedly a prerequisite for the endocytosis of carboxyl-QDs whereupon endothelial and mast cells seem to be secondarily activated in a paracrine fashion that then leads to an increase in leukocyte recruitment

Surface-dependent biodistribution and proinflammatory properties of semiconductor quantum dots

Methods based on nanotechnologies play a growing role in biomedical research. Quantum dots (QDs) are a group of engineered fluorescent nanoparticles suited for advanced imaging applications. The substitution of the particle’s surface with defined molecular structures could enable the adoption as targeted contrast agents or therapeutic devices for a variety of clinical approaches. However, important aspects such as the basic surfacedependent behavior of non-targeted QDs in the organism and arising health effects upon systemic administration remain incompletely understood. Acute inflammatory effects for instance are often initiated on the microcirculatory level and are probably relevant for cardiovascular pathologies observed in epidemiologic and experimental studies of certain nanoparticles. Most in vitro studies show that the surface structures of QDs and other nanoparticles seem to be predominantly accountable for different cytotoxic effects and a variable potential to liberate proinflammatory cytokines. Currently, no systematic in vivo studies have addressed surface-dependent interactions of QDs on the level of the microcirculation and assessed the resulting impact on biokinetics as well as on proinflammatory parameters. Thus, this thesis aimed to i) analyze the incidence of QDsurface- dependent acute microvascular interactions and their influence on key biokinetic parameters and ii) investigate acute immunomodulatory effects on the multistep process of leukocyte recruitment in vivo. For this, three types of commercially available QDs with different surface modifications: carboxyl-QDs, amine- and polyethylene glycol-QDs (amine-QDs) and polyethylene glycol-QDs (PEG-QDs) were used. The physicochemical characterization was done by dynamic light scattering (DLS) analysis and microscale thermophoresis. In a first set of experiments, circulating half-lives, tissue distribution in different organs, and hepatic as well as renal clearance were measured. Ex vivo analysis of QD tissue distribution was performed on selected tissue samples via transmission electron microscopy (TEM) and two-photon microscopy. By combining reflected-light oblique transillumination (RLOT) and fluorescence in vivo microscopy of the murine M. cremaster, interactions of QDs with components of the microcirculation as well as leukocyte migration parameters were visualized and quantified. The extreme short circulating half-life of anionic carboxyl-QDs was related to pronounced clearance by the mononuclear phagocyte system. Beyond this, further investigations showed, for the first time, that the continuous capillary endothelium of skeletal and heart muscle tissue has the capacity to directly extract carboxyl-QDs from the circulation by means of caveolaemediated endocytosis. Carboxyl-QDs were also taken up by perivascular macrophages in the surgically exposed but not in the native M. cremaster and led to a significant increase of adherent and (subsequently) transmigrated leukocytes in this model. Further experiments provided evidence for a probable involvement of mast cells in the intercellular adhesion molecule-1 (ICAM-1)- and endothelial (E)-selectinmediated modulation of leukocyte recruitment. This process is most likely initiated by the endocytosis of carboxyl-QDs through activated perivascular macrophages. The primary activation of tissue-resident perivascular macrophages seems to be the consequence of tissue damage related to the surgical preparation of the cremaster muscle. This is supposedly a prerequisite for the endocytosis of carboxyl-QDs whereupon endothelial and mast cells seem to be secondarily activated in a paracrine fashion that then leads to an increase in leukocyte recruitment

Optimized dispersion of nanoparticles for biological in vitro and in vivo studies

Background: The aim of this study was to establish and validate a practical method to disperse nanoparticles in physiological solutions for biological in vitro and in vivo studies. Results: TiO(2) (rutile) dispersions were prepared in distilled water, PBS, or RPMI 1640 cell culture medium. Different ultrasound energies, various dispersion stabilizers (human, bovine, and mouse serum albumin, Tween 80, and mouse serum), various concentrations of stabilizers, and different sequences of preparation steps were applied. The size distribution of dispersed nanoparticles was analyzed by dynamic light scattering and zeta potential was measured using phase analysis light scattering. Nanoparticle size was also verified by transmission electron microscopy. A specific ultrasound energy of 4.2 x 10(5) kJ/m(3) was sufficient to disaggregate TiO(2) (rutile) nanoparticles, whereas higher energy input did not further improve size reduction. The optimal sequence was first to sonicate the nanoparticles in water, then to add dispersion stabilizers, and finally to add buffered salt solution to the dispersion. The formation of coarse TiO(2) (rutile) agglomerates in PBS or RPMI was prevented by addition of 1.5 mg/ml of human, bovine or mouse serum albumin, or mouse serum. The required concentration of albumin to stabilize the nanoparticle dispersion depended on the concentration of the nanoparticles in the dispersion. TiO(2) (rutile) particle dispersions at a concentration lower than 0.2 mg/ml could be stabilized by the addition of 1.5 mg/ml albumin. TiO(2) (rutile) particle dispersions prepared by this method were stable for up to at least 1 week. This method was suitable for preparing dispersions without coarse agglomerates (average diameter < 290 nm) from nanosized TiO(2) (rutile), ZnO, Ag, SiO(x), SWNT, MWNT, and diesel SRM2975 particulate matter. Conclusion: The optimized dispersion method presented here appears to be effective and practicable for preparing dispersions of nanoparticles in physiological solutions without creating coarse agglomerates

Plasminogen Activator Inhibitor-1 Promotes Neutrophil Infiltration and Tissue Injury on Ischemia–Reperfusion

Objective Ischemia-reperfusion (I/R) injury significantly contributes to organ dysfunction and failure after myocardial infarction, stroke, and transplantation. In addition to its established role in the fibrinolytic system, plasminogen activator inhibitor-1 has recently been implicated in the pathogenesis of I/R injury. The underlying mechanisms remain largely obscure. Approach and Results Using different in vivo microscopy techniques as well as ex vivo analyses and in vitro assays, we identified that plasminogen activator inhibitor-1 rapidly accumulates on microvascular endothelial cells on I/R enabling this protease inhibitor to exhibit previously unrecognized functional properties by inducing an increase in the affinity of 2 integrins in intravascularly rolling neutrophils. These events are mediated through low-density lipoprotein receptor-related protein-1 and mitogen-activated protein kinase-dependent signaling pathways that initiate intravascular adherence of these immune cells to the microvascular endothelium. Subsequent to this process, extravasating neutrophils disrupt endothelial junctions and promote the postischemic microvascular leakage. Conversely, deficiency of plasminogen activator inhibitor-1 effectively reversed leukocyte infiltration, microvascular dysfunction, and tissue injury on experimental I/R without exhibiting side effects on microvascular hemostasis. Conclusions Our experimental data provide novel insights into the nonfibrinolytic properties of the fibrinolytic system and emphasize plasminogen activator inhibitor-1 as a promising target for the prevention and treatment of I/R injury

Paradoxical antidepressant effects of alcohol are related to acid sphingomyelinase and its control of sphingolipid homeostasis

Alcohol is a widely consumed drug that can lead to addiction and severe brain damage. However, alcohol is also used as self-medication for psychiatric problems, such as depression, frequently resulting in depression-alcoholism comorbidity. Here, we identify the first molecular mechanism for alcohol use with the goal to self-medicate and ameliorate the behavioral symptoms of a genetically induced innate depression. An induced over-expression of acid sphingomyelinase (ASM), as was observed in depressed patients, enhanced the consumption of alcohol in a mouse model of depression. ASM hyperactivity facilitates the establishment of the conditioned behavioral effects of alcohol, and thus drug memories. Opposite effects on drinking and alcohol reward learning were observed in animals with reduced ASM function. Importantly, free-choice alcohol drinking—but not forced alcohol exposure—reduces depression-like behavior selectively in depressed animals through the normalization of brain ASM activity. No such effects were observed in normal mice. ASM hyperactivity caused sphingolipid and subsequent monoamine transmitter hypo-activity in the brain. Free-choice alcohol drinking restores nucleus accumbens sphingolipid- and monoamine homeostasis selectively in depressed mice. A gene expression analysis suggested strong control of ASM on the expression of genes related to the regulation of pH, ion transmembrane transport, behavioral fear response, neuroprotection and neuropeptide signaling pathways. These findings suggest that the paradoxical antidepressant effects of alcohol in depressed organisms are mediated by ASM and its control of sphingolipid homeostasis. Both emerge as a new treatment target specifically for depression-induced alcoholism. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00401-016-1658-6) contains supplementary material, which is available to authorized users

Neutral sphingomyelinase mediates the co-morbidity trias of alcohol abuse, major depression and bone defects

Mental disorders are highly comorbid and occur together with physical diseases, which are often considered to arise from separate pathogenic pathways. We observed in alcohol-dependent patients increased serum activity of neutral sphingomyelinase. A genetic association analysis in 456,693 volunteers found associations of haplotypes of SMPD3 coding for NSM-2 (NSM) with alcohol consumption, but also with affective state, and bone mineralisation. Functional analysis in mice showed that NSM controls alcohol consumption, affective behaviour, and their interaction by regulating hippocampal volume, cortical connectivity, and monoaminergic responses. Furthermore, NSM controlled bone–brain communication by enhancing osteocalcin signalling, which can independently supress alcohol consumption and reduce depressive behaviour. Altogether, we identified a single gene source for multiple pathways originating in the brain and bone, which interlink disorders of a mental–physical co-morbidity trias of alcohol abuse—depression/anxiety—bone disorder. Targeting NSM and osteocalcin signalling may, thus, provide a new systems approach in the treatment of a mental–physical co-morbidity trias

Delayed hemolysis, elevated liver enzymes, low platelet count syndrome in succession of switches of preventive anticoagulant treatment in a 41-year-old patient with a history of recurrent assisted implantation failures: a case report

Abstract Background For the past decades the mean age of primiparae in Western societies is constantly increasing. At the same time, there is a growing demand for assisted reproductive technologies such as in vitro fertilization and intracytoplasmic sperm injection. Subsequently, a higher prevalence of pregnancy-associated diseases such as gestational hypertension and preeclampsia is observed. To improve pregnancy rates after in vitro fertilization/intracytoplasmic sperm injection and to reduce the risk of pregnancy-associated diseases with a cardiovascular pathophysiology, two anticoagulants are the focus of current research: low molecular weight heparin and acetylsalicylic acid (aspirin). Case presentation A 41-year-old white woman, gravida 3, para 0, received low molecular weight heparin to reduce the risk of abortion after five unsuccessful intracytoplasmic sperm injections and two miscarriages. She autonomously discontinued the medication with low molecular weight heparin at 12 weeks and 2 days of gestation and took aspirin instead until 24 weeks and 2 days of gestation as preeclampsia prophylaxis. However, the pregnancy ended with an urgent cesarean section at 27 weeks and 4 days of gestation due to a fast progressing hemolysis, elevated liver enzyme levels, and low blood platelet count syndrome, a potentially life-threatening variant of preeclampsia. Conclusion Based on the current demographic trend toward late-in-life pregnancy it is mandatory to establish clear guidelines concerning preventive treatment options of preeclampsia for patients with risk factors. The establishment of a special first-trimester screening for these women should be discussed. Moreover, it is necessary to raise the awareness among physicians of these contemporary issues to guarantee the best possible medical care

Serine proteases mediate leukocyte recruitment and hepatic microvascular injury in the acute phase following extended hepatectomy

ObjectivePost-hepatectomy liver failure (PHLF) is the main limitation of extended liver resection. The molecular mechanism and the role of leukocytes in the development of PHLF remain to be unveiled. We aimed to address the impact of serine proteases (SPs) on the acute phase after liver resection by intravitally analyzing leukocyte recruitment and changes in hemodynamics and microcirculation of the liver. MethodsC57BL/6 mice undergoing 60% partial hepatectomy were treated with aprotinin (broad-spectrum SP inhibitor), tranexamic acid (plasmin inhibitor), or vehicle. Sham-operated animals served as controls. In vivo fluorescence microscopy was used to quantify leukocyte-endothelial interactions immediately after, as well as 120 min after partial hepatectomy in postsinusoidal venules, along with measurement of sinusoidal perfusion rate and postsinusoidal shear rate. Recruitment of leukocytes, neutrophils, T cells, and parameters of liver injury were assessed in tissue/blood samples. ResultsLeukocyte recruitment, sinusoidal perfusion failure rate, and shear rate were significantly increased in mice after 60% partial hepatectomy compared to sham-operated animals. The inhibition of SPs or plasmin significantly attenuated leukocyte recruitment and improved the perfusion rate in the remnant liver. ICAM-1 expression and neutrophil recruitment significantly increased after 60% partial hepatectomy and were strongly reduced by plasmin inhibition. ConclusionsEndothelial activation and leukocyte recruitment in the liver in response to the increment of sinusoidal shear rate were hallmarks in the acute phase after liver resection. SPs mediated leukocyte recruitment and contributed to the impairment of sinusoidal perfusion in an ICAM-1-dependent manner in the acute phase after liver resection

Chronic Serotonergic Overstimulation Mimicking Panic Attacks in a Patient with Parkinson’s Disease Receiving Additional Antidepressant Treatment with Moclobemide

Background. The pharmacological treatment options of Parkinson’s disease (PD) have considerably evolved during the last decades. However, therapeutic regimes are complicated due to individual differences in disease progression as well as the occurrence of complex nonmotor impairments such as mood and anxiety disorders. Antidepressants in particular are commonly prescribed for the treatment of depressive symptoms and anxiety in PD. Case Presentation. In this case report, we describe a case of a 62-year-old female patient with PD and history of depressive symptoms for which she had been treated with moclobemide concurrent with anti-Parkinson medications pramipexole, rasagiline, and L-DOPA+benserazide retard. An increase in the dosage of moclobemide 12 months prior to admission progressively led to serotonergic overstimulation and psychovegetative exacerbations mimicking the clinical picture of an anxiety spectrum disorder. After moclobemide and rasagiline were discontinued based on the hypothesis of serotonergic overstimulation, the patient’s psychovegetative symptoms subsided. Conclusions. The specific pharmacological regime in this case probably caused drug-drug interactions resulting in a plethora of psychovegetative symptoms. Likely due to the delayed onset of adverse effects, physicians had difficulties in determining the pharmacologically induced serotonin toxicity. This case report emphasizes the complexity of pharmacological treatments and the importance of drug-drug interaction awareness in the treatment of PD patients with complicating nonmotor dysfunctions such as depression