Erythropoietin-mediated cardioprotection in hearts subjected to ischemia reperfusion.

Several studies provide evidence that erythropoietin (EPO) could play an important role in the recovery of the heart subjected to ischemia-reperfusion. In this regard, it has been suggested that EPO could be involved in protein kinase B (Akt) activation as a cell survival protein. The aim of the present study was to investigate the effects of EPO on the Akt/glycogen synthase kinase 3 beta (GSK-3ß) pathway in the presence or absence of wortmannin (W, Akt inhibitor) and its relationship with mitochondrial morphology and function preservation in ischemic-reperfused rat hearts. EPO improved the functional recovery of the heart subjected to ischemia-reperfusion, reduced the release of CK and the infarct size, and promoted preservation of the mitochondrial structure. Moreover, it reduced tissue lactate content and preserved glycogen in order to prevent ischemia. The results showed greater Akt activation, accompanied by preservation of swelling and mitochondrial calcium retention capacity, as well as an increase in ATP synthesis capacity. These results were accompanied by an inhibition of GSK-3ß, suggesting regulation of Akt on the opening of the mitochondrial permeability transition pore. All these beneficial effects exerted by acute treatment with EPO were prevented by W. The present study provided novel evidence that EPO not only enhances intrinsic activation of Akt during myocardial ischemia-reperfusion but also promotes GSK-3ß inhibition, contributing to mitochondrial structure and function preservation.

Mechanisms of immune modulation in the tumor microenvironment and implications for targeted therapy.

The efficacy of cancer therapies is limited to a great extent by immunosuppressive mechanisms within the tumor microenvironment (TME). Numerous immune escape mechanisms have been identified. These include not only processes associated with tumor, immune or stromal cells, but also humoral, metabolic, genetic and epigenetic factors within the TME. The identification of immune escape mechanisms has enabled the development of small molecules, nanomedicines, immune checkpoint inhibitors, adoptive cell and epigenetic therapies that can reprogram the TME and shift the host immune response towards promoting an antitumor effect. These approaches have translated into series of breakthroughs in cancer therapies, some of which have already been implemented in clinical practice. In the present article the authors provide an overview of some of the most important mechanisms of immunosuppression within the TME and the implications for targeted therapies against different cancers.

Intracellular DNA sensing by neutrophils and amplification of the innate immune response.

As the first responders, neutrophils lead the innate immune response to infectious pathogens and inflammation inducing agents. The well-established pathogen neutralizing strategies employed by neutrophils are phagocytosis, the action of microbicide granules, the production of ROS, and the secretion of neutrophil extracellular traps (NETs). Only recently, the ability of neutrophils to sense and respond to pathogen-associated molecular patterns is being appreciated. This review brings together the current information about the intracellular recognition of DNA by neutrophils and proposes models of signal amplification in immune response. Finally, the clinical relevance of DNA sensing by neutrophils in infectious and non-infectious diseases including malignancy are also discussed.

Role of autophagy in simulated ischemic-reperfused left atrial myocardium.

BACKGROUND AND AIM: Autophagy has recently emerged as a potential and promising therapeutic approach to maintain cardiac cellular homeostasis. The aim of the present study was to investigate the role of autophagy in the ischemic-reperfused atrial myocardium. METHODS: Isolated rat left atria subjected to simulated ischemia-reperfusion were used. The bathing medium contained either 10 mM d-glucose or 10 mM d-glucose and 1.2 mM palmitate. 3-methyladenine (3-MA) was used as pharmacological autophagy inhibitor. RESULTS: LC3-II/LC3-I ratio, an indicator of autophagosome formation, was significantly enhanced during reperfusion, this increase being slowed by the exposure to high palmitate concentration and prevented by 3-MA. Beclin-1 was significantly increased during reperfusion period in both metabolic conditions, and pharmacological inhibition of AMPK partially prevented LC3-II/LC3-I ratio increase. Autophagy inhibition significantly increased mitochondrial damage and impaired mitochondrial ATP synthesis rate at reperfusion. Tissue ATP content recovery and contractile reserve were also reduced during this period, these effects being more pronounced either in 3-MA treated atria and ischemic-reperfused atria incubated with palmitate. Moreover, severe tachyarrhythmias were observed in the presence of 3-MA, in both metabolic conditions. This phenomenon was partially prevented by mitochondrial inner membrane ion channels blocker, PK11195. CONCLUSION: Present study provides new insights into the role of autophagy in ischemic-reperfused atrial myocardium. The observation of greater deterioration in mitochondrial structure and function when this process was inhibited, suggests an association between autophagy and the structural and functional preservation of mitochondria. Exogenous metabolic substrates, to which the myocardium is exposed during ischemia-reperfusion, might not affect this process.

Role of AMPK in the protective effects exerted by triiodothyronine in ischemic-reperfused myocardium.

Recent studies have provided evidence that triiodothyronine (T3) might play an effective role in the recovery of ischemic myocardium, through the preservation of mitochondrial function and the improvement of energy substrate metabolism. To this respect, it has been suggested that T3 could activate AMP-activated protein kinase (AMPK), the cellular 'fuel-gauge' enzyme, although its role has yet to be elucidated. The aim of the present study was to investigate the effects produced by acute treatment with T3 (60 nM) and the pharmacological inhibition of AMPK by compound C on isolated rat left atria subjected to 75 min simulated ischemia-75 min reperfusion. Results showed that T3 increased AMPK activation during simulated ischemia-reperfusion, while compound C prevented it. At the end of simulated reperfusion, acute T3 treatment increased contractile function recovery and cellular viability conservation. Mitochondrial ultrastructure was better preserved in the presence of T3 as well as mitochondrial ATP production rate and tissue ATP content. Calcium retention capacity, a parameter widely used as an indicator of the resistance of mitochondrial permeability transition pore (MPTP) to opening, and GSK-3ß phosphorylation, a master switch enzyme that limits MPTP opening, were increased by T3 administration. All these beneficial effects exerted by T3 acute treatment were prevented when compound C was co-administrated. The present study provided original evidence that T3 enhances intrinsic activation of AMPK during myocardial ischemia-reperfusion, being this enzyme involved, at least in part, in the protective effects exerted by T3, contributing to mitochondrial structure and function preservation, post-ischemic contractile recovery and conservation of cellular viability.

Differential effects of AMP-activated protein kinase in isolated rat atria subjected to simulated ischemia-reperfusion depending on the energetic substrates available.

AMP-activated protein kinase (AMPK) is a serine-threonine kinase that functions primarily as a metabolic sensor to coordinate anabolic and catabolic processes in the cell, via phosphorylation of multiple proteins involved in metabolic pathways, aimed to re-establish energy homeostasis at a cell-autonomous level. Myocardial ischemia and reperfusion represents a metabolic stress situation for myocytes. Whether AMPK plays a critical role in the metabolic and functional responses involved in these conditions remains uncertain. In this study, in order to gain a deeper insight into the role of endogenous AMPK activation during myocardial ischemia and reperfusion, we explored the effects of the pharmacological inhibition of AMPK on contractile function rat, contractile reserve, tissue lactate production, tissue ATP content, and cellular viability. For this aim, isolated atria subjected to simulated 75 min ischemia-75 min reperfusion (Is-Rs) in the presence or absence of the pharmacological inhibitor of AMPK (compound C) were used. Since in most clinical situations of ischemia-reperfusion the heart is exposed to high levels of fatty acids, the influence of palmitate present in the incubation medium was also investigated. The present results suggest that AMPK activity significantly increases during Is, remaining activated during Rs. The results support that intrinsic activation of AMPK has functional protective effects in the reperfused atria when glucose is the only available energetic substrate whereas it is deleterious when palmitate is also available. Cellular viability was not affected by either of these conditions.

Effects of wortmannin on cardioprotection exerted by ischemic preconditioning in rat hearts subjected to ischemia-reperfusion

Ischemic preconditioning (IPC) is one of the most powerful interventions to reduce ischemia-reperfusion injury. The aim of the present study was to investigate the involvement of the phosphatidylinositol-3-kinases (PI3Ks) family in cardioprotection exerted by IPC and the relationship between preservation of mitochondrial morphology and ATP synthesis capacity. In this regard, macroautophagy (autophagy) is considered a dynamic process involved in the replacement of aged or defective organelles under physiological conditions. IPC consisted of four 5-min cycles of ischemia-reperfusion followed by sustained ischemia. Wortmannin (W), a PI3K family inhibitor, was added to the perfusion medium to study the involvement of autophagy in the beneficial effects of IPC. In the present study, LC3-II/I expression was significantly increased in the IPC group when compared with the control group. The hearts subjected to IPC showed greater degradation of p62 than control groups, establishing the existence of an autophagic flow. Electron microscopy showed that IPC preserves the structural integrity of mitochondria after ischemia and at the end of reperfusion. Moreover, hearts subjected to IPC exhibited increased mitochondrial ATP synthesis. The beneficial effects of IPC were abolished by W in all trials of this study, abolishing the differences between the IPC and control groups. These results suggest that IPC could partly reduce injury by ischemia-reperfusion (I/R) by decreasing mitochondrial damage and promoting autophagy. Since W is a nonspecific inhibitor of the PI3Ks family, further research is required to confirm participation of PI3K in the response to IPC (AU)

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Effects of wortmannin on cardioprotection exerted by ischemic preconditioning in rat hearts subjected to ischemia-reperfusion.

Ischemic preconditioning (IPC) is one of the most powerful interventions to reduce ischemia-reperfusion injury. The aim of the present study was to investigate the involvement of the phosphatidylinositol-3-kinases (PI3Ks) family in cardioprotection exerted by IPC and the relationship between preservation of mitochondrial morphology and ATP synthesis capacity. In this regard, macroautophagy (autophagy) is considered a dynamic process involved in the replacement of aged or defective organelles under physiological conditions. IPC consisted of four 5-min cycles of ischemia-reperfusion followed by sustained ischemia. Wortmannin (W), a PI3K family inhibitor, was added to the perfusion medium to study the involvement of autophagy in the beneficial effects of IPC. In the present study, LC3-II/I expression was significantly increased in the IPC group when compared with the control group. The hearts subjected to IPC showed greater degradation of p62 than control groups, establishing the existence of an autophagic flow. Electron microscopy showed that IPC preserves the structural integrity of mitochondria after ischemia and at the end of reperfusion. Moreover, hearts subjected to IPC exhibited increased mitochondrial ATP synthesis. The beneficial effects of IPC were abolished by W in all trials of this study, abolishing the differences between the IPC and control groups. These results suggest that IPC could partly reduce injury by ischemia-reperfusion (I/R) by decreasing mitochondrial damage and promoting autophagy. Since W is a nonspecific inhibitor of the PI3Ks family, further research is required to confirm participation of PI3K in the response to IPC.

Effects of 3-methyladenine on isolated left atria subjected to simulated ischaemia-reperfusion.

Although autophagy is a prominent feature of myocardial ischaemia and reperfusion, its functional significance is unclear and controversial. In order to gain a deeper insight into the role of autophagy in myocardial ischaemia-reperfusion, we explored the effects of the pharmacological inhibitor of autophagy 3-methyladenine (3-MA). Isolated rat atria subjected to simulated 75-min ischaemia/75-min reperfusion (Is-Rs) in the presence or absence of 3-MA were used. The LC3-II/LC3-I ratio, an indicator of autophagosome formation, did not increase after ischaemia either in the presence or absence of 3-MA, but there was significant enhancement during reperfusion, which was prevented by the presence of 3-MA. The autophagy inhibitor also increased p62 protein, one of the specific substrates degraded through the autophagy-lysosomal pathway. Electron micrographs showed double membrane autophagosome-like structures during reperfusion, which were absent in atria subjected to Is-Rs in the presence of 3-MA. These findings suggest that this agent inhibited the autophagic flux under the present experimental conditions. Inhibition of autophagy during Is-Rs was accompanied by a high incidence of tachyarrhythmias during reperfusion, and a decrease in the maximal inotropic response to ß-adrenergic and to calcium stimulation at the end of Is-Rs. Deterioration of mitochondrial morphology and function, without affecting cell viability, was observed in atria subjected to Is-Rs in the presence of 3-MA. The present results suggest an association between the inhibition of autophagy and functional alterations of the cells that have undergone sublethal stress, and have been able to recover in this experimental model of ischaemia-reperfusion.

Elevated expression of cancer/testis antigen FSIP1 in ER-positive breast tumors.

AIM: The study aimed to identify and characterize highly specific breast tumor biomarkers. METHODS: A microarray data set comprised of 513 diverse normal and tumor mRNA samples was analyzed to identify breast tumor biomarkers with minimal expression in normal tissues. RESULTS: FSIP1 was identified as a breast tumor biomarker with elevated mRNA expression in breast tumors and minimal expression in most normal tissues except the testis. Quantitative real-time PCR confirmed the elevated expression of FSIP1 mRNA in breast tumors and revealed a significant correlation with ER-positive status. Immunofluorescence staining of breast tumor sections showed that the majority of breast tumors examined in this study (20 out of 22) expressed detectable FSIP1 protein, with significantly higher than average expression in ER-positive versus ER-negative breast tumors. CONCLUSION: The prevalence and uniformity of FSIP1 expression in breast tumors, taken together with the highly restricted expression in normal tissues, suggests that FSIP1 may be an attractive target for breast cancer immunotherapy.

COL10A1 expression is elevated in diverse solid tumor types and is associated with tumor vasculature.

AIM: The identification of molecular markers that are upregulated in multiple tumor types could lead to novel diagnostic and therapeutic strategies. The authors screened a panel of RNAs prepared from diverse tumors and tumor cell lines, and compared them with normal tissues and cultured somatic cell types, in order to identify candidate genes expressed in a broad spectrum of tumor types. MATERIALS & METHODS: Gene expression microarray analysis was carried out on 128 individual tumor samples representing over 20 tumor types, 85 samples representing 31 diverse normal tissue types, 68 tumor cell lines and 97 diverse normal primary cell cultures. Genes were ranked for elevated expression across a large number and variety of tumors relative to normal tissues. RESULTS & CONCLUSION: COL10A1 was identified as a gene with restricted expression in most normal tissues and elevated expression in many diverse tumor types. By contrast, COL10A1 expression was undetectable in the 68 tumor cell lines surveyed in this study. Immunofluorescence studies localized collagen, type X, α-1 (collagen X) staining to tumor vasculature in breast tumors, whereas the vasculature of normal breast tissue was either collagen X-negative or had markedly lower levels. The tumor microenvironment-specific expression of collagen X, together with its localization in the vasculature, may facilitate its use as a novel target for the diagnosis and treatment of diverse solid tumor types.

Restricted feeding improves postischemic recovery of Langendorff-perfused rat hearts.

The goal of the present study was to assess the effects of a restricted feeding schedule (RFS) on postischemic contractile recovery in relation to triacylglycerol (TAG), glycogen, and ATP content. Glucose-6-phosphate dehydrogenase (G6PDH) activity, reduced/oxidized glutathione ratio (GSH/GSSG), and thiobarbituric acid reactive substances (TBARS) levels were also determined. Isolated rat hearts entrained to daily RFS (2 h food access starting at 1200) or fed ad libitum (FED) for 3 weeks were Langendorff-perfused (25 min ischemia, 30 min reperfusion) with Krebs-Ringer bicarbonate solution (10 mmol/L glucose). RFS improved the recovery of contractility and reduced creatine kinase (CK) release upon reperfusion. Further, at the end of reperfusion, RFS hearts exhibited increased G6PDH activity and repletion of tissue glycogen, TAG, and ATP that was not observed in the FED hearts. GSH/GSSG at the end of reperfusion fell to the same value in both nutritional states, and TBARS levels were higher in the RFS hearts. In conclusion, RFS improved postischemic functional recovery, which was accompanied by a reduction in CK release and a striking energy recovery. Although enhanced G6PDH activity was displayed, RFS was unable to reduce lipid peroxidation, supporting a clear dissociation between protection against mechanical dysfunction and CK release on the one hand and oxidative damage on the other.

Involvement of mitochondrial permeability transition, glutathione status, pentose phosphate pathway and oxidative damage in the protective effect of fasting on the ischaemic-reperfused rat heart.

1. Fasting, which increases the catabolism of fatty acids, gives functional protection to the ischaemic-reperfused heart. To obtain further knowledge of this cardioprotective effect, changes in mitochondrial permeability transition (MPT) were measured by the entrapment of 2-deoxy-[(3)H]-glucose (2-DG). We also assessed whether MPT is associated with changes in glutathione status, the activity of glucose-6-phosphate-dehydrogenase (G6PDH) and tissue oxidative damage, estimated by the measurement of Thiobarbituric acid-reactive substances (TBARS). 2. Spontaneously beating hearts of fed and 24 h fasted rats were Langendorff perfused with Krebs'-Ringer bicarbonate solution (10 mmol/L glucose) and exposed to 25 min global ischaemia, followed by 30 min reperfusion. 3. Ischaemia-reperfusion resulted in a fourfold increase in mitochondrial entrapment of 2-DG in the fed group. This response was 29% lower in the fasted group, but there were no concomitant changes in total retention of 2-DG in the heart. Fasting increased the activity of G6PDH by a factor of 1.4 and caused a 2.8-fold increase in the ratio of reduced glutathione to oxidized glutathione (GSH:GSSG) at the end of the pre-ischaemic period. Ischaemia-reperfusion did not affect G6PDH activity, but reduced the GSH:GSSG ratio in both the fed and fasted groups by 50%. Therefore, the GSH:GSSG ratio remained higher in the fasted group. Fasting also decreased cellular levels of TBARS by approximately 25%. Lipolysis of endogenous triacylglycerol was increased during the pre-ischaemic period in the fasted group. 4. These data suggest that the enhancement of fatty acid catabolism that occurs in fasting activates mechanisms that tend to reduce oxidative damage and limit MPT.

Protection of ischaemic-reperfused rat heart by dimethylamiloride is associated with inhibition of mitochondrial permeability transition.

1. The aim of the present study was to assess whether protection afforded by the Na(+)/H(+) exchanger blocker dimethylamiloride (DMA) is associated with inhibition of mitochondrial permeability transition (MPT). The effects of DMA were compared with those of cyclosporine (Cs) A, an inhibitor of MPT. 2. Rat hearts were Langendorff perfused with Krebs'-bicarbonate medium containing 10 mmol/L glucose and were subjected to 25 min no-flow global ischaemia and 30 min reperfusion in the presence or absence of 10 micromol/L DMA or 0.2 micromol/L CsA. Cell viability was measured using tetrazolium stain. The MPT was determined by loading hearts with 2-deoxy-[(3)H]-glucose (2DG), which enters mitochondria only during MPT. Total heart 2DG content as an estimation of the extent of tissue damage was also measured. To assess whether DMA has any direct effect on glycolysis, a cell-free heart extract containing all the glycolytic enzymes was used. 3. Dimethylamiloride improved functional recovery (rate-pressure product) from 24 +/- 7 to 68 +/- 11% (P < 0.01) at reperfusion end, attenuated the increase in left ventricular end-diastolic pressure (from 29 +/- 7 to 6 +/- 3% 10 min after reperfusion onset; P < 0.01), improved cell viability (from 21.2 +/- 6.6 to 69.6 +/- 7.1% at reperfusion end; P < 0.05) and lessened lactate accumulation at the end of ischaemia (119 +/- 15 vs 163 +/- 14 micromol/g dry weight; P < 0.05). Dimethylamiloride limited MPT: 2DG mitochondrial entrapment, being 33.1 +/- 14.2 and 96.3 +/- 14.0 at reperfusion end in the treated and control hearts, respectively (P < 0.05), and concomitantly raised total 2DG content (51.3 +/- 4.4 vs 86.8 +/- 1.7 x 10(3) d.p.m./g wet weight in control and treated groups, respectively; P < 0.05). Cyclosporine A improved functional recovery and attenuated the amplitude of ventricular diastolic pressure in ischaemic-reperfused hearts. It also reduced mitochondrial entrapment (67.3 +/- 7.7%; P < 0.05 vs control) and increased total cell 2DG content (162.3 +/- 1.3 x 10(3) d.p.m./g wet weight; P < 0.01 vs control) at the end of reperfusion. Dimethylamiloride did not affect glucose consumption and lactate production in the cell-free heart extract. 4. In conclusion, DMA protects against the noxious effects of ischaemia-reperfusion and inhibits MPT, coinciding with present and previous findings concerning the effects of CsA. Dimethylamiloride also diminished lactate accumulation, although it did not exhibit any direct effect on glycolysis. These data suggest that blockade of Na(+)/H(+) exchange by DMA attenuates the extent of MPT in ischaemic-reperfused rat heart.

Fine specificity of natural killer T cells against GD3 ganglioside and identification of GM3 as an inhibitory natural killer T-cell ligand.

GD3, a ganglioside expressed on melanoma, is the only tumour-associated glycolipid described to date that can induce a CD1d-restricted natural killer T (NKT)-cell response. We analysed the fine specificity of GD3-reactive NKT cells and discovered that immunization with GD3 induced two populations of GD3-reactive NKT cells. One population was CD4+ CD8- and was specific for GD3; the other population was CD4- CD8- and cross-reacted with GM3 in a CD1d-restricted manner, but did not cross-react with GM2, GD2, or lactosylceramide. This indicated that the T-cell receptors reacting with GD3 recognize glucose-galactose linked to at least one N-acetyl-neuraminic acid but will not accommodate a terminal N-acetylgalactosamine. Immunization with GM2, GM3, GD2, or lactosylceramide did not induce an NKT-cell response. Coimmunization of GM3-loaded antigen-presenting cells (APCs) with GD3-loaded APCs suppressed the NKT-cell response to GD3 in a CD1d-restricted manner. This suppressive effect was specific for GM3 and was a local effect lasting 2-4 days. In vitro, GM3-loaded APCs also suppressed the interleukin-4 response, but not the interferon-gamma response, of NKT cells to alpha-galactosylceramide. However, there was no effect on the T helper type 2 responses of conventional T cells. We found that this suppression was not mediated by soluble factors. We hypothesize that GM3 induces changes to the APC that lead to suppression of T helper type 2-like NKT-cell responses.

Role of endogenous nitric oxide in classic preconditioning in rat hearts.

Ischemic preconditioning (IPC) protects the heart against subsequent sustained ischemia reperfusion (RP). Despite many triggers and signaling pathways, which seem to be involved in IPC, the IPC-mechanisms remain a controversial issue. One of them is endogenous production of nitric oxide (NO). To assess the role of NO in IPC and its relation with glycogen and glycolysis, the effects of inhibiting NO synthase with L-NAME (50 microM) were examined in IPC rat hearts perfused with medium containing 10 mM glucose. Left ventricular developed pressure-rate product (RPP) and end diastolic pressure (EDP), lactate and glycogen contents, and cell viability were measured. Global ischemia (25 min) was followed by 30 min RP. IPC consisted in one cycle of 3 min ischemia-5 min RP. IPC reduced EDP and improved RP recovery of RPP. L-NAME had no effects on the non-IPC group but abolished these effects of IPC. IPC reduced ischemic decrease of glycogen and the acceleration of glycolysis, and improved cell viability. L-NAME did not affect these effects of IPC. The results suggest that NO is ineffective on the noxious effects of ischemia-RP in non-IPC hearts and on the effects of IPC on cell viability, glycogenolysis and glycolysis whereas it is only involved in functional protection.

Tiña del cuero cabelludo por Microsporum canis en una mujer adulta / Tinea capitis in the elderly: an unusual situation

La tiña del cuero cabelludo es una enfermedad muy rara en adultos. Presentamos una tinea capitis causada por Microsporum canis en una paciente sana postmenopáusica. Se discuten los factores predisponentes de la dermatofitosis en los pacientes adultos y se cuestiona el uso de terbinafina como tratamiento de primera elección en esta entidad, debido a la aparición de resistencias

Tinea capitis is very rare in adults. We report a new case of tinea capitis caused by Microsporum canis in a healthy postmenopausal woman. We argue the predisposing factors of the dermatophytoses in adult patients and discuss about the use of terbinafine like first line of treatment in this entity, because of the apparition of resistances

Right place, right time, right peptide: DO keeps DM focused.

Peptide loading of major histocompatibility class II molecules is catalyzed in late endosomal and lysosomal compartments of cells by the catalytic action of human leukocyte antigen (HLA)-DM (H-2M in mice). In B cells, dendritic cells and thymic epithelial cells, the peptide loading of class II molecules is modified by the expression of the non-classical class II molecule, HLA-DO (H-2O in mice). Collectively, studies to date support that DO/H-2O expression inhibits the presentation of antigens acquired by cells via fluid phase endocytosis. However, in B cells, the expression of H-2O promotes the presentation of antigens internalized by the B-cell receptor. In this review, we summarize the literature pertaining to DO assembly, transport, and function, with an emphasis on the function of DO/H-2O.