Mechanisms of nanoparticle-mediated photomechanical cell damage

Laser-assisted killing of gold nanoparticle targeted macrophages was investigated. Using pressure transient detection, flash photography and transmission electron microscopy (TEM) imaging, we studied the mechanism of single cell damage by vapor bubble formation around gold nanospheres induced by nanosecond laser pulses. The influence of the number of irradiating laser pulses and of particle size and concentration on the threshold for acute cell damage was determined. While the single pulse damage threshold is independent of the particle size, the threshold decreases with increasing particle size when using trains of pulses. The dependence of the cell damage threshold on the nanoparticle concentration during incubation reveals that particle accumulation and distribution inside the cell plays a key role in tissue imaging or cell damaging.

Dystrophic cardiomyopathy: role of TRPV2 channels in stretch-induced cell damage

Aims Duchenne muscular dystrophy (DMD), a degenerative pathology of skeletal muscle, also induces cardiac failure and arrhythmias due to a mutation leading to the lack of the protein dystrophin. In cardiac cells, the subsarcolemmal localization of dystrophin is thought to protect the membrane from mechanical stress. The absence of dystrophin results in an elevated stress-induced Ca2+ influx due to the inadequate functioning of several proteins, such as stretch-activated channels (SACs). Our aim was to investigate whether transient receptor potential vanilloid channels type 2 (TRPV2) form subunits of the dysregulated SACs in cardiac dystrophy. Methods and results We defined the role of TRPV2 channels in the abnormal Ca2+ influx of cardiomyocytes isolated from dystrophic mdx mice, an established animal model for DMD. In dystrophic cells, western blotting showed that TRPV2 was two-fold overexpressed. While normally localized intracellularly, in myocytes from mdx mice TRPV2 channels were translocated to the sarcolemma and were prominent along the T-tubules, as indicated by immunocytochemistry. Membrane localization was confirmed by biotinylation assays. Furthermore, in mdx myocytes pharmacological modulators suggested an abnormal activity of TRPV2, which has a unique pharmacological profile among TRP channels. Confocal imaging showed that these compounds protected the cells from stress-induced abnormal Ca2+ signals. The involvement of TRPV2 in these signals was confirmed by specific pore-blocking antibodies and by small-interfering RNA ablation of TRPV2. Conclusion Together, these results establish the involvement of TRPV2 in a stretch-activated calcium influx pathway in dystrophic cardiomyopathy, contributing to the defective cellular Ca2+ handling in this disease.

Loss of sphingosine kinase-1 in carcinoma cells increases formation of reactive oxygen species and sensitivity to doxorubicin-induced DNA damage

Sphingosine kinases (SK) catalyse the formation of sphingosine 1-phosphate, which is a key lipid mediator regulating cell responses such as proliferation, survival and migration. Here we have investigated the effect of targeted inhibition of SK-1 on cell damage and elucidated the mechanisms involved.

Dystrophic cardiomyopathy: amplification of cellular damage by Ca2+ signalling and reactive oxygen species-generating pathways

AIMS: Cardiac myopathies are the second leading cause of death in patients with Duchenne and Becker muscular dystrophy, the two most common and severe forms of a disabling striated muscle disease. Although the genetic defect has been identified as mutations of the dystrophin gene, very little is known about the molecular and cellular events leading to progressive cardiac muscle damage. Dystrophin is a protein linking the cytoskeleton to a complex of transmembrane proteins that interact with the extracellular matrix. The fragility of the cell membrane resulting from the lack of dystrophin is thought to cause an excessive susceptibility to mechanical stress. Here, we examined cellular mechanisms linking the initial membrane damage to the dysfunction of dystrophic heart. METHODS AND RESULTS: Cardiac ventricular myocytes were enzymatically isolated from 5- to 9-month-old dystrophic mdx and wild-type (WT) mice. Cells were exposed to mechanical stress, applied as osmotic shock. Stress-induced cytosolic and mitochondrial Ca(2+) signals, production of reactive oxygen species (ROS), and mitochondrial membrane potential were monitored with confocal microscopy and fluorescent indicators. Pharmacological tools were used to scavenge ROS and to identify their possible sources. Osmotic shock triggered excessive cytosolic Ca(2+) signals, often lasting for several minutes, in 82% of mdx cells. In contrast, only 47% of the WT cardiomyocytes responded with transient and moderate intracellular Ca(2+) signals. On average, the reaction was 6-fold larger in mdx cells. Removal of extracellular Ca(2+) abolished these responses, implicating Ca(2+) influx as a trigger for abnormal Ca(2+) signalling. Our further experiments revealed that osmotic stress in mdx cells produced an increase in ROS production and mitochondrial Ca(2+) overload. The latter was followed by collapse of the mitochondrial membrane potential, an early sign of cell death. CONCLUSION: Overall, our findings reveal that excessive intracellular Ca(2+) signals and ROS generation link the initial sarcolemmal injury to mitochondrial dysfunctions. The latter possibly contribute to the loss of functional cardiac myocytes and heart failure in dystrophy. Understanding the sequence of events of dystrophic cell damage and the deleterious amplification systems involved, including several positive feed-back loops, may allow for a rational development of novel therapeutic strategies.

Intracisternal application of endotoxin enhances the susceptibility to subsequent hypoxic-ischemic brain damage in neonatal rats.

Perinatal brain damage is associated not only with hypoxic-ischemic insults but also with intrauterine inflammation. A combination of antenatal inflammation and asphyxia increases the risk of cerebral palsy >70 times. The aim of the present study was to determine the effect of intracisternal (i.c.) administration of endotoxin [lipopolysaccharides (LPS)] on subsequent hypoxic-ischemic brain damage in neonatal rats. Seven-day-old Wistar rats were subjected to i.c. application of NaCl or LPS (5 microg/pup). One hour later, the left common carotid artery was exposed through a midline neck incision and ligated with 6-0 surgical silk. After another hour of recovery, the pups were subjected to a hypoxic gas mixture (8% oxygen/92% nitrogen) for 60 min. The animals were randomized to four experimental groups: 1) sham control group, left common carotid artery exposed but not ligated (n = 5); 2) LPS group, subjected to i.c. application of LPS (n = 7); 3) hypoxic-ischemic study group, i.c. injection of NaCl and exposure to hypoxia after ligation of the left carotid artery (n = 17); or 4) hypoxic-ischemic/LPS study group, i.c. injection of LPS and exposure to hypoxia after ligation of the left carotid artery (n = 19). Seven days later, neonatal brains were assessed for neuronal cell damage. In a second set of experiments, rat pups received an i.c. injection of LPS (5 microg/pup) and were evaluated for tumor necrosis factor-alpha expression by immunohistochemistry. Neuronal cell damage could not be observed in the sham control or in the LPS group. In the hypoxic-ischemic/LPS group, neuronal injury in the cerebral cortex was significantly higher than in animals that were subjected to hypoxia/ischemia after i.c. application of NaCl. Injecting LPS intracisternally caused a marked expression of tumor necrosis factor-alpha in the leptomeninges. Applying LPS intracisternally sensitizes the immature rat brain to a subsequent hypoxic-ischemic insult.

Cardiovascular complications of conventional and targeted adjuvant breast cancer therapy

Adjuvant therapy has improved the survival of women with early breast cancer (BC). Meta-analyses suggest that anthracycline-based regimens reduced the annual BC death rate by 40% in women below the age of 50 and 20% in older women. Novel agents designed to modulate abnormal growth factor signaling in and around the BC cell further increase patients' chances of survival. However, both conventional chemotherapeutic agents as well as some of the novel signaling inhibitors can induce important cardiovascular side-effects, potentially attenuating the progress made in recent years. The mechanism of cancer drug-induced cardiovascular complications varies greatly with some compounds inducing irreversible myocardial cell damage, while others lead to temporary cell dysfunction. The challenge of the future will be to prospectively discriminate between irreversible damage which can lead to progressive cardiovascular disease and reversible cardiovascular dysfunctions without further prognostic implications. Since adjuvant therapy for BC is potentially curative, emphasis must be placed on finding treatments combining maximum efficacy with the minimum of long-term side-effects in order to achieve survival with preserved quality of life.

Antioxidant supplementation and endurance training: Win or loss?

Typically, free radicals are thought of as perpetrators of cell damage, ageing, even cancer, whereas antioxidants are seen as the defence against these threats. Accordingly, antioxidants are among the most common sports supplements used by amateur and professional athletes. However, the sensibility of this practice has recently been challenged in the scientific literature. This article briefly summarizes both positive and negative physiological effects of free radicals and antioxidants, culminating with emphasis on the signalling roles played by free radicals during training adaptations and the ability of superfluous antioxidants to weaken these desired signals, as revealed in several recent publications. The aim of this article is not to explicitly condemn antioxidant supplementation by athletes, but to underscore complexity of the situation and to champion efforts to achieve a deeper understanding of circumstances (e.g. dosage, timing, and setting) that might deem antioxidant supplementation as either largely beneficial or largely detrimental for endurance athletes in training.

Destruction of lymphoid organ architecture and hepatitis caused by CD4+ T cells

Immune responses have the important function of host defense and protection against pathogens. However, the immune response also causes inflammation and host tissue injury, termed immunopathology. For example, hepatitis B and C virus infection in humans cause immunopathological sequel with destruction of liver cells by the host's own immune response. Similarly, after infection with lymphocytic choriomeningitis virus (LCMV) in mice, the adaptive immune response causes liver cell damage, choriomeningitis and destruction of lymphoid organ architecture. The immunopathological sequel during LCMV infection has been attributed to cytotoxic CD8(+) T cells. However, we now show that during LCMV infection CD4(+) T cells selectively induced the destruction of splenic marginal zone and caused liver cell damage with elevated serum alanin-transferase (ALT) levels. The destruction of the splenic marginal zone by CD4(+) T cells included the reduction of marginal zone B cells, marginal zone macrophages and marginal zone metallophilic macrophages. Functionally, this resulted in an impaired production of neutralizing antibodies against LCMV. Furthermore, CD4(+) T cells reduced B cells with an IgM(high)IgD(low) phenotype (transitional stage 1 and 2, marginal zone B cells), whereas other B cell subtypes such as follicular type 1 and 2 and germinal center/memory B cells were not affected. Adoptive transfer of CD4(+) T cells lacking different important effector cytokines and cytolytic pathways such as IFNγ, TNFα, perforin and Fas-FasL interaction did reveal that these cytolytic pathways are redundant in the induction of immunopathological sequel in spleen. In conclusion, our results define an important role of CD4(+) T cells in the induction of immunopathology in liver and spleen. This includes the CD4(+) T cell mediated destruction of the splenic marginal zone with consecutively impaired protective neutralizing antibody responses.

Vapor bubble generation around gold nano-particles and its application to damaging of cells

We investigated vapor bubbles generated upon irradiation of gold nanoparticles with nanosecond laser pulses. Bubble formation was studied both with optical and acoustic means on supported single gold nanoparticles and single nanoparticles in suspension. Formation thresholds determined at different wavelengths indicate a bubble formation efficiency increasing with the irradiation wavelength. Vapor bubble generation in Bac-1 cells containing accumulations of the same particles was also investigated at different wavelengths. Similarly, they showed an increasing cell damage efficiency for longer wavelengths. Vapor bubbles generated by single laser pulses were about half the cell size when inducing acute damage.

Adverse cardiac events during catecholamine vasopressor therapy: a prospective observational study

PURPOSE: To determine the incidence of and risk factors for adverse cardiac events during catecholamine vasopressor therapy in surgical intensive care unit patients with cardiovascular failure. METHODS: The occurrence of any of seven predefined adverse cardiac events (prolonged elevated heart rate, tachyarrhythmia, myocardial cell damage, acute cardiac arrest or death, pulmonary hypertension-induced right heart dysfunction, reduction of systemic blood flow) was prospectively recorded during catecholamine vasopressor therapy lasting at least 12 h. RESULTS: Fifty-four of 112 study patients developed a total of 114 adverse cardiac events, an incidence of 48.2 % (95 % CI, 38.8-57.6 %). New-onset tachyarrhythmia (49.1 %), prolonged elevated heart rate (23.7 %), and myocardial cell damage (17.5 %) occurred most frequently. Aside from chronic liver diseases, factors independently associated with the occurrence of adverse cardiac events included need for renal replacement therapy, disease severity (assessed by the Simplified Acute Physiology Score II), number of catecholamine vasopressors (OR, 1.73; 95 % CI, 1.08-2.77; p = 0.02) and duration of catecholamine vasopressor therapy (OR, 1.01; 95 % CI, 1-1.01; p = 0.002). Patients developing adverse cardiac events were on catecholamine vasopressors (p < 0.001) and mechanical ventilation (p < 0.001) for longer and had longer intensive care unit stays (p < 0.001) and greater mortality (25.9 vs. 1.7 %; p < 0.001) than patients who did not. CONCLUSIONS: Adverse cardiac events occurred in 48.2 % of surgical intensive care unit patients with cardiovascular failure and were related to morbidity and mortality. The extent and duration of catecholamine vasopressor therapy were independently associated with and may contribute to the pathogenesis of adverse cardiac events.

A metabolic enzyme as a primary virulence factor of Mycoplasma mycoides subsp. mycoides small colony

During evolution, pathogenic bacteria have developed complex interactions with their hosts. This has frequently involved the acquisition of virulence factors on pathogenicity islands, plasmids, transposons, or prophages, allowing them to colonize, survive, and replicate within the host. In contrast, Mycoplasma species, the smallest self-replicating organisms, have regressively evolved from gram-positive bacteria by reduction of the genome to a minimal size, with the consequence that they have economized their genetic resources. Hence, pathogenic Mycoplasma species lack typical primary virulence factors such as toxins, cytolysins, and invasins. Consequently, little is known how pathogenic Mycoplasma species cause host cell damage, inflammation, and disease. Here we identify a novel primary virulence determinant in Mycoplasma mycoides subsp. mycoides Small Colony (SC), which causes host cell injury. This virulence factor, released in significant amounts in the presence of glycerol in the growth medium, consists of toxic by-products such as H2O2 formed by l-alpha-glycerophosphate oxidase (GlpO), a membrane-located enzyme that is involved in the metabolism of glycerol. When embryonic calf nasal epithelial cells are infected with M. mycoides subsp. mycoides SC in the presence of physiological amounts of glycerol, H2O2 is released inside the cells prior to cell death. This process can be inhibited with monospecific anti-GlpO antibodies.

Macrophage activating syndrome is associated with lobular hepatitis and severe bile duct injury with cholestasis

Macrophage activating syndrome (MAS) is a rare hematological disorder associated with uncontrolled systemic T-cell activation. Persistent fever, fatigue and hepatosplenomegaly are frequent clinical manifestations, whereas hyperferritinemia, elevated serum lactate dehydrogenase levels and cytopenia are key criteria for the diagnosis of MAS. The nature of liver pathology in MAS has been partially elucidated but destructive biliary lesions have been rarely described. This report illustrates four cases of MAS developing marked cholestasis, leading to one case of biliary cirrhosis necessitating liver transplantation. Histologically, liver involvement was characterized in all cases by acute lobular hepatitis, marked hepatocyte apoptosis and small bile duct injury similar to the vanishing bile duct syndrome. Immuno-histological studies showed that the inflammatory changes and bile duct lesions were dominated by the presence of activated macrophages and T-cells, in particular CD8+ lymphocytes, and in part NK-cells. These findings suggest that in MAS, various T-cell triggers such as infection, autoimmune disease and malignancy might result in the release of cytokines, which in turn activate macrophages to trigger a systemic acute phase response and local tissue damage. This communication suggests that a macrophage, T- and NK-cell network is operational in the pathogenesis of the cholangiocyte, hepatocyte and sinus endothelial cell damage in MAS.

Suppression of transforming growth factor beta signalling aborts caerulein induced pancreatitis and eliminates restricted stimulation at high caerulein concentrations

BACKGROUND: Transforming growth factors betas (TGF-betas) are implicated in pancreatic tissue repair but their role in acute pancreatitis is not known. To determine whether endogenous TGF-betas modulate the course of caerulein induced acute pancreatitis, caerulein was administered to wild-type (FVB-/-) and transgenic mice that are heterozygous (FVB+/-) for expression of a dominant negative type II TGF-beta receptor. METHODS: After 7 hourly supramaximal injections of caerulein, the pancreas was evaluated histologically and serum was assayed for amylase and lipase levels. Next, the effects of caerulein on amylase secretion were determined in mouse pancreatic acini, and cholecystokinin (CCK) receptor expression was assessed. RESULTS: The normal mouse pancreas was devoid of inflammatory cells whereas the pancreas from transgenic mice contained lymphocytic infiltrates. Caerulein injection in wild-type mice resulted in 6- and 36-fold increases in serum amylase and lipase levels, respectively, increased serum trypsinogen activation peptide (TAP) levels, gross oedema and a marked inflammatory response in the pancreas that consisted mainly of neutrophils and macrophages. By contrast, FVB+/- mice exhibited minimal alterations in response to caerulein with attenuated neutrophil-macrophage infiltrates. Moreover, acini from FVB+/- mice did not exhibit restricted stimulation at high caerulein concentrations, even though CCK receptor mRNA levels were not decreased. CONCLUSION: Our findings indicate that a functional TGF-beta signalling pathway may be required for caerulein to induce acute pancreatitis and for the CCK receptor to induce acinar cell damage at high ligand concentrations. Our results also support the concept that restricted stimulation at high caerulein concentrations contributes to the ability of caerulein to induce acute pancreatitis.

Substrate delivery and ionic balance disturbance after severe human head injury

The most important early pathomechanism in traumatic brain injury (TBI) is alteration of the resting membrane potential. This may be mediated via voltage, or agonist-dependent ion channels (e.g. glutamate-dependent channels). This may result in a consequent increase in metabolism with increased oxygen consumption, in order to try to restore ionic balance via the ATP-dependent pumps. We hypothesize that glutamate is an important agonist in this process and may induce an increase in lactate, potassium and brain tissue CO2, and hence a decrease in brain pH. Further we propose that an increase in lactate is thus not an indicator of anaerobic metabolic conditions as has been thought for many years. We therefore analyzed a total of 85 patients with TBI, Glasgow Coma Scale (GCS) < 8 using microdialysis, brain tissue oxygen, CO2 and pH monitoring. Cerebral blood flow studies (CBF) were performed to test the relationship between regional cerebral blood flow (rCBF) and the metabolic determinants. Glutamate was significantly correlated with lactate (p < 0.0001), potassium (p < 0.0001), brain tissue pH (p = 0.0005), and brain tissue CO2 (p = 0.006). rCBF was inversely correlated with glutamate, lactate and potassium. 44% of high lactate values were observed in brain with tissue oxygen values, above the threshold level for cell damage. These results support the hypothesis of a glutamate driven increase in metabolism, with secondary traumatic depolarization and possibly hyperglycolysis. Further, we demonstrate evidence for lactate production in aerobic conditions in humans after TBI. Finally, when reduced regional cerebral blood flow (rCBF) is observed, high dialysate glutamate, lactate and potassium values are usually seen, suggesting ischemia worsens these TBI-induced changes.

Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense

Although eosinophils are considered useful in defense mechanisms against parasites, their exact function in innate immunity remains unclear. The aim of this study is to better understand the role of eosinophils within the gastrointestinal immune system. We show here that lipopolysaccharide from Gram-negative bacteria activates interleukin-5 (IL-5)- or interferon-gamma-primed eosinophils to release mitochondrial DNA in a reactive oxygen species-dependent manner, but independent of eosinophil death. Notably, the process of DNA release occurs rapidly in a catapult-like manner--in less than one second. In the extracellular space, the mitochondrial DNA and the granule proteins form extracellular structures able to bind and kill bacteria both in vitro and under inflammatory conditions in vivo. Moreover, after cecal ligation and puncture, Il5-transgenic but not wild-type mice show intestinal eosinophil infiltration and extracellular DNA deposition in association with protection against microbial sepsis. These data suggest a previously undescribed mechanism of eosinophil-mediated innate immune responses that might be crucial for maintaining the intestinal barrier function after inflammation-associated epithelial cell damage, preventing the host from uncontrolled invasion of bacteria.