Indicator Of Cell Injury Research Articles

The Impact of a Short-Term Ketogenic Low-Carbohydrate High-Fat Diet on Biomarkers of Intestinal Epithelial Integrity and Gastrointestinal Symptoms.

Endurance exercise can disturb intestinal epithelial integrity, leading to increased systemic indicators of cell injury, hyperpermeability, and pathogenic translocation. However, the interaction between exercise, diet, and gastrointestinal disturbance still warrants exploration. This study examined whether a 6-day dietary intervention influenced perturbations to intestinal epithelial disruption in response to a 25-km race walk. Twenty-eight male race walkers adhered to a high carbohydrate (CHO)/energy diet (65% CHO, energy availability = 40kcal·kg FFM-1·day-1) for 6 days prior to a Baseline 25-km race walk. Athletes were then split into three subgroups: high CHO/energy diet (n = 10); low-CHO, high-fat diet (LCHF: n = 8; <50g/day CHO, energy availability = 40kcal·kg FFM-1·day-1); and low energy availability (n = 10; 65% CHO, energy availability = 15kcal·kg FFM-1·day-1) for a further 6-day dietary intervention period prior to a second 25-km race walk (Adaptation). During both trials, venous blood was collected pre-, post-, and 1hr postexercise and analyzed for markers of intestinal epithelial disruption. Intestinal fatty acid-binding protein concentration was significantly higher (twofold increase) in response to exercise during Adaptation compared to Baseline in the LCHF group (p = .001). Similar findings were observed for soluble CD14 (p < .001) and lipopolysaccharide-binding protein (p = .003), where postexercise concentrations were higher (53% and 36%, respectively) during Adaptation than Baseline in LCHF. No differences in high CHO/energy diet or low energy availability were apparent for any blood markers assessed (p > .05). A short-term LCHF diet increased intestinal epithelial cell injury in response to a 25-km race walk. No effect of low energy availability on gastrointestinal injury or symptoms was observed.

Effect and mechanism of 1, 25(OH)2D3 on myocardial inflammation induced by Coxsackie virus B3 in mice

Objective: To explore the effect and mechanism of 1, 25(OH)2D3 on myocardial inflammation induced by Coxsackie virus B3 (CVB3) in mice. Methods: Wild type (WT) and 1α-hydroxylase knockout [1(OH)ase-/-] male mice were divided into four groups: WT group, WT+CVB3 group, 1(OH)ase-/-+CVB3 group and 1(OH)ase-/-+CVB3+VD3 group, with 8 mice in each group. The indicators for evaluating myocardial cell injury were examined by different methods. The mRNA levels of pro-inflammatory cytokines [interlenkin (IL)-1β, IL-6, interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α)] were determined by quantitative real-time PCR. Hematoxylin-eosin (HE) staining was used to observe the myocardial histopathological changes. The apoptosis of myocardial cells was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and flow cytometry. Fluo-4/AM fluorescence probe was used to detect intracellular calcium ion content. Meanwhile, the expression levels of Ca2+/Calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) protein as well as endoplasmic reticulum stress-related proteins like glucose-related protein 78 (GRP78) and C/EBP homologous protein (CHOP) in the myocardial tissues were detected by Western blot. Results: Compared with WT group, the mRNA levels of pro-inflammatory factors increased in the cardiomyocytes of mice in WT+CVB3 group, including IL-1β (14.88±3.32 vs 1.03±0.02, P=0.009), IL-6 (7.00±1.09 vs 1.81±0.18, P=0.005), IFN-γ (4.70±1.11 vs 1.34±0.34, P=0.006) and TNF-α (17.20±3.22 vs 1.02±0.12, P<0.001). Similarly, the infiltration of inflammatory cells, and the apoptosis rate of cardiomyocytes elevated (16.66%±1.09% vs 7.85%±1.12%, P=0.012). The level of calcium ions in myocardial cytoplasm was significantly higher in WT+CVB3 group than that in the WT group (2.98±1.05 vs 0.96±0.10, P=0.006). Likewise, the expression levels of pCaMKⅡ(1.97±0.34 vs 1.00±0, P<0.001), GRP78 (1.78±0.19 vs 1.00±0, P=0.005) and CHOP (1.62±0.09 vs 1.00±0, P=0.002) in WT+CVB3 group up-regulated. The above myocardial cell injury markers were more significant in the 1(OH)ase-/-+CVB3 group. In the 1(OH)ase-/-+CVB3+VD3 group, 1, 25(OH)2D3 supplementation significantly improved myocardial cell injury indicators. Meanwhile, the specific inhibitors of CaMKⅡ can also reduce the myocardial injury and apoptosis rate of CVB3-infected mice. Conclusion: 1, 25(OH)2D3 deficiency can aggravate myocardial inflammation through over activation of CaMKⅡ.

Ozone exposure promotes pyroptosis in rat lungs via the TLR2/4-NF-κB-NLRP3 signaling pathway

ObjectiveOzone has become a major air pollutant in recent years, which leading to a variety of lung diseases. This study aimed to explore the mechanisms of pyroptosis and related signaling pathways in ozone-induced lung injury. MethodsWe exposed 120 Wistar rats to ozone, 20 in each group (half male and half female). Ozone exposure concentrations were 0, 0.12, 0.5, 1.0, 2.0 and 4.0 ppm for 6 h. At the same time, we isolated and cultured type I alveolar epithelial cells, then intervened with high mobility group box 1 protein (HMGB1), hyaluronic acid (HA) and Toll-like receptors 2/4 (TLR2/4) inhibitor. In animal experiments, histopathological experiments, TUNEL, ELISA and biochemical indicators were performed. RT-qPCR and western blot experiments assay were used to detect the expression changes of key factors in relevant signal pathways in vivo and in vitro. ResultsAfter acute ozone exposure, the levels of lung cell injury indicators in bronchoalveolar lavage fluid (BALF), as well as the levels of inflammatory factors in BALF, blood, and lung tissue were significantly increased. Male rats were more sensitive to ozone exposure. Low-concentration ozone exposure caused mild interstitial inflammation in rat lung tissue. Severe inflammation and pulmonary edema appeared with increases in concentration. ELISA results in BALF showed that HMGB1 and HA expressions increased gradually with the increase of ozone exposure concentration. RT-qPCR and Western blot showed that when ozone concentrations increased above 0.5 ppm, the expression of nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3), cleaved caspase-1, and N-gasdermin D (N-GSDMD) in the lung tissue increased significantly, suggesting that ozone exposure induces pyroptosis. At the same time, it was found that ozone exposure activated the nuclear factor kappa B (NF-κB) signal pathway, and increased the mRNA expressions of Toll-like receptors TLR2/4. The results of cell experiments showed that after the addition of HMGB1 and HA, the expression of NF-κB and pyroptosis related indexes increased in type I alveolar epithelial cells, while the corresponding expression decreased after the addition of TLR2/4 inhibitors. ConclusionOzone exposure causes lung injury in a dose- and gender-dependent manner, and is more severe in males. When injured, the levels of HMGB1 and HA in BALF increased, which interact with TLR 2/4 to activate the downstream NF-κB signaling pathway. Further activating the NLRP3 inflammasome complex and regulating the ozone-induced pyroptosis.

Silver nanoparticle pollutants activate oxidative stress responses and rosmarinic acid accumulation in sage.

In this study, physiological and molecular responses of sage (Salvia officinalis) to silver nanoparticles (SNPs) were studied. It is supposed that sage oxidative responses can be activated to overcome the negative effects of SNPs. Results showed the penetration of SNPs via leaf epidermis into the parenchyma cells after foliar application. A significant decrease of photosynthetic pigments and increase of cell injury indicators, the activity of enzymatic antioxidants and also the content of non-enzymatic antioxidants were observed after exposure of sage plants to 50 and 1000 mg l-1 SNPs compared to control plants. Phenolic compounds generally increased, but not in linear response to the dose level. The most abundant phenolic acid, rosmarinic acid (RA), increased more than eightfold at 100 mg l-1 SNPs. Furthermore, the content of RA, salvianolic acid A and B was positively correlated with the activity of phenylalanine ammonia-lyase and RA synthase, but not with tyrosine aminotransferase. It could be concluded that the content of phenolic compounds increased in response to lower SNPs concentrations (50 and 100 mg l-1 ). However, the oxidative stress responses continued above these concentrations.

Resuscitation and growth kinetics of sub-lethally injured Listeria monocytogenes strains following fluorescence activated cell sorting (FACS)

This study investigated the effect of acid (pH4.2), osmotic (10% NaCl) and heat (55°C for 30min) stress induced injury on Listeria monocytogenes strains ATCC19115, 69, 159/10 and 243 using differential plating and flow cytometry coupled with membrane integrity indicators, thiazole orange (TO) and propidium iodide (PI) staining. Growth kinetics of injured cells sorted by fluorescence activated cell sorting (FACS) were studied at 4, 25 and 37°C. The percentage of cell injury detectable by both flow cytometry and differential plating varied significantly among strains and stress treatments (p<0.0001). Based on flow cytometry and TO/PI staining, acid stress caused the highest level of injury followed by heat and osmotic stress. Following cell sorting, acid and osmotic stress injured cells were capable of resuscitation and re-growth while heat injured cells (except for strain 69) were incapable of re-growth despite having a high level of membrane intact cells. The lag phase duration (λ) of sorted stress injured cells resuscitated in brain heart infusion (BHI) broth was significantly influenced by strain variations (p<0.0001), stress treatments (p=0.007) and temperature of resuscitation (p≤0.001). Following repair, the maximum specific growth rate (μmax) of resuscitated cells was not different from untreated control cells regardless of strain differences and stress treatments. Only temperature had a significant effect (p<0.0001) on growth rate. Sorted cells were also capable of growth at 4°C, with the time to detectable growth (≥1.40Log10CFUml−1) ranging from 3 to 15days. Overall, re-growth potential of sorted cells showed that while membrane integrity was a good indicator of cell injury and viability loss for acid and osmotic stress, it was not a sufficient indicator of heat stress injury. Once injured cells repair the cellular damage, their growth rate is not different from non-injured cells regardless of form of stress and strain differences. Thus highlighting the potential food safety risks of stress injured L. monocytogenes cells.

Protective effects of diltiazem against vascular endothelial cell injury induced by angiotensin-II and hypoxia

To provide pharmacological data for future clinical studies, this study investigated the protective effects of diltiazem on vascular endothelial cell (VEC) injury induced by angiotensin-II (AngII), hypoxia, and a combination of both treatments. The concentration of intracellular free calcium and the mitochondrial membrane potential in VEC were assessed as indicators of cell injury. An in vivo hypoxic animal model was used to test the protective effect of diltiazem on vascular endothelial tissues. Our study showed that AngII and hypoxia decreased the mitochondrial membrane potential in VEC, which was significantly inhibited by diltiazem. Diltiazem protected against VEC injury induced by the increased concentration of intracellular free calcium, which was associated with AngII and hypoxia. Diltiazem reduced the apoptosis of rat VEC under a sustained hypoxic condition. In addition, it reduced AngII and endothelin I levels in rat vascular endothelial tissues. Our study confirmed that AngII and hypoxia induced VEC injury by regulating the levels of mitochondrial membrane potential and intracellular free calcium. Diltiazem, a calcium channel blocker, protected VEC from AngII- and hypoxia-induced injury.

Screening of Tomatoes Germplasm for Heat Stress Tolerance Under Controlled Conditions

Forty four tomato (Solanum lycopersicum) lines originating from USA including three wild accessions were evaluated under controlled conditions for heat tolerance using the three physiological parameters electrolyte leakage, chlorophyll fluorescence and stomatal conductance. Tomato plants in their vegetative stage two months after sowing were placed in a growth chamber at 27 degrees for one week. Control measurements were taken prior to the heat shock treatment of 44°C for four hours. As selection of heat tolerant and sensitive genotypes proved difficult with stomatal conductance, a preliminary selection of twelve genotypes was made based on electrolyte leakage and fluorescence,, and measurements were repeated in triplicate.Chlorophyll fluorescence, i.e. the ratio of variable to maximal fluorescence in both dark-adapted (Fv/Fm) and light-adapted leaves (Fv’/Fm’), was measured using an infra-red gas analyser (IRGA, Licor 6400) fitted with a fluorescence chamber. Both Fv/Fm and Fv’/Fm’ decreased in the heat-sensitive genotypes, however Fv’/Fm’ was a better predictor for heat-shock sensitivity.Electrolyte leakage was measured by collecting the leaf discs from control and heat-shocked plants. Electrical conductivity (EC) was measured as an indication of cell injury due to electrolytes leakage. Electrolyte leakage was significantly increased in heat-sensitive genotypes and it was 32.92μmhos/cm in sensitive genotype compare with 22.1μmhos/cm in tolerance genotype.Significant variation in heat tolerance was observed between genotypes. Electrolyte leakage proved to be a more reliable parameter compared to chlorophyll fluorescence in differentiating heat tolerant and heat sensitive genotypes of tomato under controlled conditions. This method can potentially be used to short list larger sets of germplasm under controlled conditions that will reduce cost of testing the bigger sets under field settings.

Cellular injury evidenced by impedance technology and infrared microspectroscopy

Fourier Transform Infrared (FTIR) spectroscopy is finding increasing biological application, for example in the analysis of diseased tissues and cells, cell cycle studies and investigating the mechanisms of action of anticancer drugs. Cancer treatment studies routinely define the types of cell-drug responses as either total cell destruction by the drug (all cells die), moderate damage (cell deterioration where some cells survive) or reversible cell cycle arrest (cytostasis). In this study the loss of viability and related chemical stress experienced by cells treated with the medicinal plant, Plectranthus ciliatus, was investigated using real time cell electronic sensing (RT-CES) technology and FTIR microspectroscopy. The use of plants as medicines is well established and ethnobotany has proven that crude extracts can serve as treatments against various ailments. The aim of this study was to determine whether FTIR microspectroscopy would successfully distinguish between different types of cellular injury induced by a potentially anticancerous plant extract. Cervical adenocarcinoma (HeLa) cells were treated with a crude extract of Pciliatus and cells monitored using RT-CES to characterize the type of cellular responses induced. Cell populations were then investigated using FTIR microspectroscopy and statistically analysed using One-way Analysis of Variance (ANOVA) and Principal Component Analysis (PCA). The plant extract and a cancer drug control (actinomycin D) induced concentration dependent cellular responses ranging from nontoxic, cytostatic or cytotoxic. Thirteen spectral peaks (915cm−1, 933cm−1, 989cm−1, 1192cm−1, 1369cm−1, 1437cm−1, 1450cm−1, 1546cm−1, 1634cm−1, 1679cm−1 1772cm−1, 2874cm−1 and 2962cm−1) associated with cytotoxicity were significantly (p value<0.05, one way ANOVA, Tukey test, Bonferroni) altered, while two of the bands were also indicative of early stress related responses. In PCA, poor separation between nontoxic and cytostatic responses was evident while clear separation was linked to cytotoxicity. RT-CES detected morphological changes as indicators of cell injury and could distinguish between viable, cytostatic and cytotoxic responses. FTIR microspectroscopy confirmed that cytostatic cells were viable and could still recover while also describing early cellular stress related responses on a molecular level.

In vitro cardioprotective effect of Wusen Erlian Granules through the inhibition of Coxsackievirus B3 replication.

Wusen Erlian Granules are a traditional Chinese medicine and we sought to determine the antiviral activity of Wusen Erlian Granules against Coxsackievirus B3 infection. First, cytotoxicity of Wusen Erlian Granules was determined in cultured cardiomyocytes isolated from day-old Wister rat pups. Later, cardiomyocytes were infected with Coxsackievirus B3 and the protective effect of Wusen Erlian Granules against cell injury was compared with that of ribavirin. Cell injury indicators including myoglobin, MB isozyme of Creatine Kinase, and cardiac Troponin were assessed by enzyme-linked immunosorbent assay (ELISA) and antiviral effect was assessed by MTT assay. We found that the 50%-Toxic (TC50) and 50%-Effective (EC50) concentrations of Wusen Erlian Granules were 394.05 and 30.26μg/ml, respectively. Following infection of cardiomyocytes with Coxsackievirus B3, cell injury index of Wusen Erlian Granules, as determined by ELISA, was 125μg/ml which yielded significant protection from virus-induced cell damage. The antiviral activity of Wusen Erlian Granules i.e., therapeutic index in MTT assay was higher (13.02) than that of ribavirin (6.93). It was, therefore, concluded that the Wusen Erlian Granules exerted better antiviral effect than ribavirin using Coxsackievirus B3 in vitro infection model in terms of rat cardiomyocytes protection from virus-induced cell injury.

A novel low pHo‐induced Cl‐ conductance in nodose ganglion neurons is neuroprotective during simulated ischemia: implication for vagal afferent activity in heart failure (667.9)

We found that vagal afferent neurons isolated from nodose ganglia exhibit a large and prolonged low pHo‐conditioned Cl‐ current (25±5 pA/pF, n=60), when exposed to 3 brief pulse of low pHo (6.0) solution delivered at 20sec intervals. The current was similar to the one carried by the “Swell‐Activated Cl‐ Channel” (SAC) during hypoosmolarity. The activation pathways of this Cl‐ conductance are through NADPH oxidase (NOX)‐dependent production of ROS (dihydroethidine fluorescence). Since a similar Cl‐ conductance was found to be protective during ischemic preconditioning in cardiomyocytes (Draz et al 1999) we tested the hypothesis that the activation of this current may also be protective in cultured neurons exposed to “simulated ischemia” (low glucose, hypoxia and pH5.0). We measured the LDH release as an indication of cell injury. Without low pHo preconditioning the supernatant LDH concentration averaged 82±4 μU/ml and 441±62 μU/ml after exposure to 1 and 3 hours of simulated ischemia. In contrast, supernatant LDH was significantly reduced to 58±10 μU/ml and 227±25 μU/ml at 1 and 3 hours when neurons were preconditioned by activating the Cl‐ current before simulated ischemia. The protective effect of preconditioning was partially reversed by SAC channel inhibitor tamoxifen (10μM) (393±99 μU/ml at 3hours).Conclusions and Clinical Implications: A low pHo‐induced activation of a Cl‐ conductance in nodose neurons is NOX mediated and provides a putative autocrine protective mechanism for preservation of cardiac vagal afferents during ischemia or heart failure.Grant Funding Source: Supported by NIH HL 14388

T Cells and Blood Vessels

Remarkable advances in our understanding of innate and adaptive immunity have shed light on why inflammation is centrally involved in the pathogenesis of many diseases traditionally not viewed as primarily inflammatory in nature. One key area of progress has been the discovery and study of innate immune receptors, referred to as pattern recognition receptors (PRRs), such as toll-like receptors and nod-like receptors. PRRs recognize molecules commonly produced by microbial pathogens, broadly defined as pathogen associated molecular patterns (PAMPs), but PRRs also recognize self molecules that are indicators of cell injury or death. These self molecules, called damage-associated molecular patterns (DAMPs), include reactive oxygen species, intracellular crystalline deposits, normal molecules in abnormal locations (ATP, heat shock proteins, nuclear proteins), and many others.1,2 DAMPs may be induced or change location because of cell injury or death by any number of causes (genetic, traumatic, toxic, metabolic), and their recognition by innate immune system receptors will initiate responses that include local and systemic inflammation. This is why diseases with just about any underlying cause may be expected to have an inflammatory component. Importantly, the innate immune system has evolved to amplify and modify responses to best combat diverse infections, but these amplified and specialized responses, if inappropriately targeted against self, may themselves become intrinsic to the progression of disease processes. One way this can occur is by innate immune stimulation of adaptive immune responses mediated by T lymphocytes. In fact, normal protective T cell-mediated immunity requires a kick-start by the innate immune system, a condition that ensures that we do not readily mount powerful and potentially harmful adaptive immune responses when there is no real damage or danger present. Article see p 2529 One of major ways in which innate immunity promotes T cell-mediated immunity is through the activation of antigen-presenting …

Antiphospholipid (aPL) antibodies promote the release of plasma membrane-derived vesicles which inhibit the phagocytosis of apoptotic cells - role in Systemic Lupus Erythematosus (SLE) (135.9)

Abstract Plasma Membrane-derived Vesicles (PMVs) are membrane-coated vesicles of diameter 0.1 to 1 µm, carrying various proteins inherent in their parental cells. PMVs are released when cells undergo activation/apoptosis via blebbing and shedding. Exposition of phosphotidylserine (PS) on the outer membrane leaflet could explain a role for PMVs in phagocytosis and thrombosis. Increased PMV release, although controlled, is a sign of cellular dysfunction and indicator of cell injury or stress. Patients with inflammatory disease such as Systemic Lupus Erythematosus (SLE) show increased PMV levels in plasma, accompanied by persistent apoptosis and defective clearance which correlates with disease activity. Although still unclear, impaired clearance of apoptotic cells (ACs) may represent a mechanism for the development or enhancement of SLE. Both PMVs and ACs express PS on the outer leaflet of the lipid bilayer as a marker for phagocytosis. We found that PMVs dose dependently inhibit the phagocytosis of ACs by competing for the PS receptor on macrophages. Phosphotidylserine is directly involved as annexin V-labelled PMVs reversed the PMV-mediated inhibition of phagocytosis. We further confirmed elevated PMV levels in SLE patient plasma and showed that the aPL antibodies from the plasma of such patients induced the release of PMVs. These findings show that PMVs can modulate phagocytosis of ACs and suggest an alternative mechanism for PMV enhancement of inflammatory responses.

Pomegranate extract mouth rinsing effects on saliva measures relevant to gingivitis risk

Pomegranate components have properties that could promote oral health, including reducing the risk of gingivitis. The present study examined young adults (n = 32, split evenly among both genders), for the effects of 4 weeks of thrice daily mouth rinsing with the pomegranate (Punica granatum L.) extract PomElla dissolved in water. This treatment changed salivary measures relevant to oral health including gingivitis. The changes were: reduced total protein (which can correlate with plaque forming bacteria readings), reduced activities of aspartate aminotransferase (an indicator of cell injury), reduced alpha-glucosidase activity (a sucrose degrading enzyme), increased activities of the antioxidant enzyme ceruloplasmin (which could give better protection against oral oxidant stress) and increased radical scavenging capacity (though this increase was significant only by nonparametric statistical analysis). A placebo of cornstarch in water did not affect these measures. These data raise the possibility of using pomegranate extracts in oral health products such as toothpaste and mouthwashes.

Recovery of heat-injured Listeria innocua

Listeria innocua was subjected to thermal inactivation and the extent of heat-injured cells was quantified. Cultures were heated in liquid medium for different times, using temperatures in the range of 52.5 to 65.0 °C, and plated on Tryptic Soy Agar with 0.6% yeast extract (TSAYE) used as non-selective medium and on TSAYE plus 5% NaCl (TSAYE + NaCl) and Palcam agar with selective supplement (Palcam agar) as selective media. The difference observed in counts in non-selective and in selective media gave an indication of cell injury during the heat treatment. D- and z- values were calculated for all conditions considered. For each temperature, D-values obtained using non-selective recovery procedures were higher than the ones obtained using the two selective media. When comparing the selective media, it can be concluded that Palcam agar allowed recovery and growth of thermally injured cells and so it was less inhibitor than TSAYE + NaCl. Another important result was the influence of temperature on the degree of cellular injury. As temperature increases, the degree of heat-injured cells also increases, and consequently concern has to be taken with the temperature and the counting medium used in food processing studies. The results of this work clearly demonstrated that selective media used for Listeria monocytogenes enumeration/detection might not be suitable for the recovery of heat-injured cells, which can dangerously underestimate the presence of this foodborne pathogen.

Quantitative kinetics of damage and recovery of cytoskeletal structure by means of image analysis

The cytoskeleton is a network of proteins which structurally and dynamically organise the cytoplasm of living cells. Microtubules are among its constituents. Morphological alterations of microtubules are related to functional impairment. Therefore cytoskeletal morphology is a valuable indicator of cell injury and functionality. This paper focuses on the comparison between normal and altered cytoskeletal microtubules by means of image analysis and classification with the aim of replacing visual assessment. Morphology has been quantified by the extraction of some descriptors yielded by spatial differentiation, fractal analysis and Fourier analysis followed by non-linear filtering. The principal component analysis of these descriptors has led to image recognition and has been applied to hepatocytes and fibroblasts exposed to some xenobiotics. In the case of hepatocytes, images have been ranked according to the severity of cytoskeletal damage, a dose–response relation has been derived from the regression of the first principal component and the percentage of structural recovery after exposure has been estimated.

Method for identifying neuronal cells suffering zinc toxicity by use of a novel fluorescent sensor

During excitotoxic brain damage, injured neurons accumulate an anomalous, pathological burden of weakly bound, rapidly exchangeable Zn 2+ that diffusely fills the soma, nucleus and proximal dendrites. Mounting evidence indicates that this Zn 2+ is a major contributing factor in the subsequent demise of the damaged neurons. Thus, identifying, imaging, and characterizing zinc-filled cells have become essential steps in understanding excitotoxicity. Here we demonstrate that a new fluorescent stain for zinc can rather selectively and quite vividly label zinc-filled neurons in frozen histologic sections. The method is more sensitive and selective than the existing stain TSQ, and simpler than the Timm–Danscher silver staining techniques. A previously unobserved population of apparently injured cells in the dentate gyrus has been discovered with the new reagent. Whereas cells viewed in situ in normal, healthy tissue virtually never display any perikaryal staining by histochemical methods for zinc [Histochemistry, 71 (1981) 1; Int. Rev. Neurobiol. 31 (1989) 145], injured cells stain intensely for zinc in culture [J. Neurosci. 17 (1997) 9554], acute slice preparations [J. Histochem. Cytochem. 47 (1999) 969; J. Neurosci. 22 (2002) 1273] and in tissue harvested in vivo [Science 272 (1996) 1013; Annu. Rev. Neurosci. 21 (1998) 347]. Thus, the presence of rapidly-exchangeable, “stainable” perikaryal zinc may be taken as an indicator of cell injury [J. Nutr. 130 (2000) 1471S; Biometals 14 (2001) 353].

Endothelial cell injury in venule and capillary induced by contrast ultrasonography

The aim of the present study was to test the hypothesis that microvascular endothelial cells (EC) are subject to the bioeffects induced by contrast ultrasound (US) because of their proximity to the circulating microbubbles. We examined EC injury in each microvessel section (arteriole, capillary or venule) in rat mesenteries among the following five groups: three controls (sham operation, microbubble injection alone, US exposure with saline injection), and two contrast-US groups (US exposure at a 1-Hz or 30-Hz frame rate with microbubble injection). Propidium iodide (PI), a fluorescent indicator of cell injury, was employed to visualize impaired EC. PI-positive nuclei were equally few among the three controls. Contrast-US increased PI-positive cells in capillaries (1-Hz frame rate, 2.4 ± 2.2 cells per 0.1-mm vessel length, p = 0.09; 30-Hz frame rate, 4.3 ± 1.8 cells, p < 0.01) and in venules (1-Hz frame rate, 4.1 ± 2.5 cells, p < 0.05; 30-Hz frame rate, 13.8 ± 3.6 cells, p < 0.01) compared with sham operation (0.10 ± 0.22 cells). The finding indicates that diagnostic contrast US potentially causes EC injury, particularly in venules and capillaries.

Context-based therapy: A conceptual framework for transplantation tolerance

Allotransplantation has improved dramatically in the past 2 decades, mostly as a result of more potent immunosuppression and increasingly effective prophylaxis against opportunistic infections. It is likely, however, that the field has entered a period of diminishing returns, such that increased immunosuppression will minimally increase graft survival and markedly augment morbidity. The authors suggest that gains in graft and patient survival will best be achieved by pursuing strategies promoting immune tolerance and selecting therapies that enhance physiologic immune adaptation rather than those that indiscriminately suppress immune function. Principles of tolerance as they relate to allotransplantation are discussed. The authors introduce the concept of context-based therapy. This approach places an emphasis on preserving specificity during immune intervention by augmenting or avoiding interference with antigen receptor function. Graft acceptance is fostered by altering contextual signals influencing the outcome of antigen recognition such as intrinsic indicators of cell injury, costimulatory signals, the activation state of antigen presenting cells, or the local environment of immune engagement. The manipulation of immune thresholds is also introduced as an additional concept influencing tolerance. Supportive data from nonhuman primate and human trials are presented.

Distinct glycolysis inhibitors determine retinal cell sensitivity to glutamate-mediated injury.

In this study, we analyzed how distinct glycolysis inhibitors influenced the redox status of retinal cells, used as a neuronal model. Three different approaches were used to inhibit glycolysis: the cells were submitted to iodoacetic acid (IAA), an inhibitor of glyceraldehyde 3-phosphate dehydrogenase, to 2-deoxy-glucose (DG) in glucose-free medium, which was used as a substitute of glucose, or in the absence of glucose. The redox status of the cells was evaluated by determining the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). By the analysis of dose-response curves of MTT reduction, IAA showed values of IC50 = 7.02 x 10(-5) M, whereas DG showed values of IC50 = 7.42 x 10(-4) M. Upon 30 min-incubation, glucose deprivation, per se, did not significantly affect MTT reduction. We also evaluated the reduction of MTT as an indicator of cell injury by exposing the cells to 100 microM glutamate during the decrement of glycolysis function. In the presence of glutamate, for 2 h, there was a decrease in MTT reduction, which was potentiated in the presence of DG (10-20% decrease), in the presence of IAA (about 30% decrease) or in glucose-free medium (about 30% decrease). Major changes observed by the MTT assay, upon exposure to glutamate, indicative of changes in the redox status of retinal cells, were concomitant with variations in intracellular ATP. Under glucose deprivation, endogenous ATP decreased significantly from 38.9+/-4.4 to 13.3+/-0.7 nmol/mg protein after exposure to 100 microM glutamate. The results support a different vulnerability of retinal cells after being exposed to distinct forms of glycolysis inhibition.

Altered Oxygen Toxicity with Variable Overexpression of Heme Oxygenase-1 in Hamster Fibroblasts ♦ 352

Heme oxygenase (HO) is the rate limiting enzyme in bilirubin production. The HO-1 isoform is inducible by a variety of oxidant stresses including hyperoxia(1). In its reaction, heme, a prooxidant is degraded, bilirubin, an antioxidant is formed(2). Thus, the overall effect of HO may be to protect against oxidative stress. Previous results from rat fibroblasts transfected with an expression vector containing the rat HO-1 cDNA have led us to speculate that there may be a beneficial threshold of HO-1 overexpression. To further investigate this hypothesis, we have established a variable overexpression model of HO-1 using the Tet-Off™ Gene Expression System (Clontech) which is based on the tetracycline resistance system found in E. coli(3). In this system, gene expression is controlled by the interaction of a tet-responsive transcriptional activator protein and a tet-responsive element where gene expression is tightly regulated by varying concentrations of either tetracycline or doxycycline (Dox) in a dose dependent manner. After insertion of the full length rat HO-1 cDNA into the pBI-L response plasmid, two consecutive stable transfections were performed and >60 stable colonies were screened in order to find a cell line allowing for regulated variable HO-1 overexpression. This cell line was grown in 75cm flasks to >80% confluence. To verify HO-1 overexpression, total HO activity was measured by gas chromatography and HO-1 protein was assessed by Western analysis. By varying the concentration of Dox between 0 and 10ng/ml, we were able to regulate HO-1 expression between 1.5 and 20 fold. Once confluent, cell lines expressing a range of HO-1 protein were exposed to hyperoxia (95% O2/5% CO2) for 72 h and non-specific indicators of cell injury were measured by Trypan blue exclusion and LDH release. Lipid peroxidation, protein oxidation, and total glutathione were also evaluated as specific markers of oxygen toxicity. Lower HO-1 overexpression was associated with increased protection against oxygen damage; however, higher expression was associated with increased markers of cell injury. In this model, we conclude that there is a beneficial threshold of HO-1 overexpression beyond which detrimental effects become apparent. We suggest that measures to enhance protection against oxygen toxicity by HO-1 overexpression be undertaken with a thorough understanding of the potential dual nature of HO-1.