Carnosine Counteracts the Molecular Alterations Aß Oligomers-Induced in Human Retinal Pigment Epithelial Cells.

Age-related macular degeneration (AMD) has been described as a progressive eye disease characterized by irreversible impairment of central vision, and unfortunately, an effective treatment is still not available. It is well-known that amyloid-beta (Aß) peptide is one of the major culprits in causing neurodegeneration in Alzheimer's disease (AD). The extracellular accumulation of this peptide has also been found in drusen which lies under the retinal pigment epithelium (RPE) and represents one of the early signs of AMD pathology. Aß aggregates, especially in the form of oligomers, are able to induce pro-oxidant (oxidative stress) and pro-inflammatory phenomena in RPE cells. ARPE-19 is a spontaneously arising human RPE cell line validated for drug discovery processes in AMD. In the present study, we employed ARPE-19 treated with Aß oligomers, representing an in vitro model of AMD. We used a combination of methods, including ATPlite, quantitative real-time PCR, immunocytochemistry, as well as a fluorescent probe for reactive oxygen species to investigate the molecular alterations induced by Aß oligomers. In particular, we found that Aß exposure decreased the cell viability of ARPE-19 cells which was paralleled by increased inflammation (increased expression of pro-inflammatory mediators) and oxidative stress (increased expression of NADPH oxidase and ROS production) along with the destruction of ZO-1 tight junction protein. Once the damage was clarified, we investigated the therapeutic potential of carnosine, an endogenous dipeptide that is known to be reduced in AMD patients. Our findings demonstrate that carnosine was able to counteract most of the molecular alterations induced by the challenge of ARPE-19 with Aß oligomers. These new findings obtained with ARPE-19 cells challenged with Aß1-42 oligomers, along with the well-demonstrated multimodal mechanism of action of carnosine both in vitro and in vivo, able to prevent and/or counteract the dysfunctions elicited by Aß oligomers, substantiate the neuroprotective potential of this dipeptide in the context of AMD pathology.

Adverse Events and Tolerability of Combined Durvalumab and Tremelimumab versus Durvalumab Alone in Solid Cancers: A Systematic Review and Meta-Analysis.

Background: Recently, the combination of durvalumab and tremelimumab, two immune checkpoint inhibitors, for the treatment of different types of cancers has been considered; however, its overall effects, including its safety, are still unclear and need to be further investigated. Objectives: The aim of the present systematic review and meta-analysis was to investigate the safety and tolerability of this combination of drugs. Methods: A systematic review of the literature, based on the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement, was conducted by employing online electronic databases and the American Society of Clinical Oncology (ASCO) Meeting Library. The selection of eligible publications was made following a staged screening and selection process. The software RevMan 5.4 was used to run the quantitative analysis and forest plots, while the Cochrane tool was employed for risk of bias assessment. Results: From the retrieved 157 results, 9 randomized controlled trials involving 3060 patients were included. By comparing the combination of durvalumab and tremelimumab vs. durvalumab monotherapy, it was observed that: adverse events (AEs) ≥ Grade 3 incidence was 32.6% (536/1646) vs. 23.8% (336/1414) (Z = 2.80; p = 0.005; risk ratio (RR) = 1.44), reduced appetite incidence was 10.8% (154/1427) vs. 8.3% (108/1305) (Z = 2.26; p = 0.02; RR = 1.31), diarrhea was reported in 15.6% (229/1473) vs. 8.1% (110/1352) (Z = 5.90; p < 0.00001; RR = 1.91), rash incidence was equal to 11.1% (160/1441) vs. 6.5% (86/1320) (Z = 4.35; p <0.0001; RR = 1.75), pruritis was 13.6% (201/1473) vs. 7.7% (104/1352) (Z = 5.35; p < 0.00001; RR = 1.83), fever was 10.5% (42/399) vs. 6.6% (22/330) (Z = 2.27; p = 0.02; RR = 1.77), discontinuation rate was 18% (91/504) vs. 3% (36/434) (Z = 4.78; p < 0.00001; RR = 2.41), and death rate was 2.6% (13/504) vs. 0.7% (3/434) (Z = 1.90; p = 0.06; RR = 2.77). Conclusions: It was observed that the combined (durvalumab and tremelimumab) vs. monotherapy (durvalumab) is associated with a higher risk of treatment discontinuation, mortality, fever, diarrhea, rash, pruritis, and reduced appetite. This information is relevant and should be disclosed, especially to patients that are currently enrolled in clinical trials considering this combined therapy.

Updates on Molecular and Biochemical Development and Progression of Prostate Cancer.

Prostate cancer (PCa) represents the most commonly non-cutaneous diagnosed cancer in men worldwide and occupies a very wide area of preclinical and clinical research. Targeted therapy for any cancer depends on the understanding of the molecular bases and natural behaviour of the diseases. Despite the well-known effect of androgen deprivation on PCa, many patients develop resistance either for antiandrogen therapy or other new treatment modalities such as checkpoint inhibitors and chemotherapy. Comprehensive understanding of the development of PCa as well as of the mechanisms underlying its progression is mandatory to maximise the benefit of the current approved medications or to guide the future research for targeted therapy of PCa. The aim of this review was to provide updates on the most recent mechanisms regarding the development and the progression of PCa. According to the current understanding, future treatment strategies should include more predictive genetic and biomarker analysis to assign different patients to the expected most appropriate and effective treatment.

Lung Surfactant Decreases Biochemical Alterations and Oxidative Stress Induced by a Sub-Toxic Concentration of Carbon Nanoparticles in Alveolar Epithelial and Microglial Cells.

Carbon-based nanomaterials are nowadays attracting lots of attention, in particular in the biomedical field, where they find a wide spectrum of applications, including, just to name a few, the drug delivery to specific tumor cells and the improvement of non-invasive imaging methods. Nanoparticles inhaled during breathing accumulate in the lung alveoli, where they interact and are covered with lung surfactants. We recently demonstrated that an apparently non-toxic concentration of engineered carbon nanodiamonds (ECNs) is able to induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells. Therefore, the complete understanding of their "real" biosafety, along with their possible combination with other molecules mimicking the in vivo milieu, possibly allowing the modulation of their side effects becomes of utmost importance. Based on the above, the focus of the present work was to investigate whether the cellular alterations induced by an apparently non-toxic concentration of ECNs could be counteracted by their incorporation into a synthetic lung surfactant (DPPC:POPG in 7:3 molar ratio). By using two different cell lines (alveolar (A549) and microglial (BV-2)), we were able to show that the presence of lung surfactant decreased the production of ECNs-induced nitric oxide, total reactive oxygen species, and malondialdehyde, as well as counteracted reduced glutathione depletion (A549 cells only), ameliorated cell energy status (ATP and total pool of nicotinic coenzymes), and improved mitochondrial phosphorylating capacity. Overall, our results on alveolar basal epithelial and microglial cell lines clearly depict the benefits coming from the incorporation of carbon nanoparticles into a lung surfactant (mimicking its in vivo lipid composition), creating the basis for the investigation of this combination in vivo.

Carnosine Protects Macrophages against the Toxicity of Aß1-42 Oligomers by Decreasing Oxidative Stress.

Carnosine (ß-alanyl-L-histidine) is a naturally occurring endogenous peptide widely distributed in excitable tissues such as the brain. This dipeptide has well-known antioxidant, anti-inflammatory, and anti-aggregation activities, and it may be useful for treatment of neurodegenerative disorders such as Alzheimer's disease (AD). In this disease, peripheral infiltrating macrophages play a substantial role in the clearance of amyloid beta (Aß) peptides from the brain. Correspondingly, in patients suffering from AD, defects in the capacity of peripheral macrophages to engulf Aß have been reported. The effects of carnosine on macrophages and oxidative stress associated with AD are consequently of substantial interest for drug discovery in this field. In the present work, a model of stress induced by Aß1-42 oligomers was investigated using a combination of methods including trypan blue exclusion, microchip electrophoresis with laser-induced fluorescence, flow cytometry, fluorescence microscopy, and high-throughput quantitative real-time PCR. These assays were used to assess the ability of carnosine to protect macrophage cells, modulate oxidative stress, and profile the expression of genes related to inflammation and pro- and antioxidant systems. We found that pre-treatment of RAW 264.7 macrophages with carnosine counteracted cell death and apoptosis induced by Aß1-42 oligomers by decreasing oxidative stress as measured by levels of intracellular nitric oxide (NO)/reactive oxygen species (ROS) and production of peroxynitrite. This protective activity of carnosine was not mediated by modulation of the canonical inflammatory pathway but instead can be explained by the well-known antioxidant and free-radical scavenging activities of carnosine, enhanced macrophage phagocytic activity, and the rescue of fractalkine receptor CX3CR1. These new findings obtained with macrophages challenged with Aß1-42 oligomers, along with the well-known multimodal mechanism of action of carnosine in vitro and in vivo, substantiate the therapeutic potential of this dipeptide in the context of AD pathology.

Oncological Response and Predictive Biomarkers for the Checkpoint Inhibitors in Castration-Resistant Metastatic Prostate Cancer: A Systematic Review and Meta-Analysis.

Recently, checkpoint inhibitors have been investigated in metastatic prostate cancer, however their overall effect is unclear and needs to be further investigated. OBJECTIVES: The aim of this systematic review is to investigate the oncological response of metastatic castration-resistant prostate cancer patients to immune checkpoint inhibitors. METHODS: Based on the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement, a systematic review of the literature was conducted through online electronic databases and the American Society of Clinical Oncology (ASCO) Meeting Library. Eligible publications were selected after a staged screening and selection process. RevMan 5.4 software was employed to run the quantitative analysis and forest plots. Risk of bias assessment was conducted using the Cochrane tool and Newcastle-Ottawa Scale for the randomized and non-randomized trials, respectively. RESULTS: From the 831 results retrieved, 8 studies including 2768 patients were included. There was no significant effect on overall survival (OS) (overall response (OR) = 0.98; Z = 0.42; p = 0.67). Meanwhile, progression-free survival (PFS) was significantly better with immune checkpoint inhibitors administration (OR = 0.85; Z = 3.9; p < 0.0001). The subgroup analysis for oncological outcomes based on programmed death ligand 1 (PD-L1) positivity status displayed no significant effect, except on prostate-specific antigen response rate (PSA RR) (OR = 3.25; Z = 2.29; p = 0.02). Based on DNA damage repair (DDR), positive patients had a significantly better PFS and a trend towards better OS and overall response rate (ORR); the ORR was 40% in positive patients compared to 20% in the negative patients (OR = 2.46; Z = 1.3; p = 0.19), while PSA RR was 23.5% compared to 14.3% (OR = 1.88; Z = 0.88; p = 0.38). Better PFS was clearly associated with DDR positivity (OR = 0.70; Z = 2.48; p = 0.01) with a trend towards better OS in DDR positive patients (OR = 0.71; Z = 1.38; p = 0.17). Based on tumor mutation burden (TMB), ORR was 46.7% with high TMB versus 8.8% in patients with low TMB (OR = 11.88; Z = 3.0; p = 0.003). CONCLUSIONS: Checkpoint inhibitors provide modest oncological advantages in metastatic castration-resistant prostate cancer. There are currently no good predictive indicators that indicate a greater response in some patients.

A New Human Blood-Retinal Barrier Model Based on Endothelial Cells, Pericytes, and Astrocytes.

Blood-retinal barrier (BRB) dysfunction represents one of the most significant changes occurring during diabetic retinopathy. We set up a high-reproducible human-based in vitro BRB model using retinal pericytes, retinal astrocytes, and retinal endothelial cells in order to replicate the human in vivo environment with the same numerical ratio and layer order. Our findings showed that high glucose exposure elicited BRB breakdown, enhanced permeability, and reduced the levels of junction proteins such as ZO-1 and VE-cadherin. Furthermore, an increased expression of pro-inflammatory mediators (IL-1ß, IL-6) and oxidative stress-related enzymes (iNOS, Nox2) along with an increased production of reactive oxygen species were observed in our triple co-culture paradigm. Finally, we found an activation of immune response-regulating signaling pathways (Nrf2 and HO-1). In conclusion, the present model mimics the closest human in vivo milieu, providing a valuable tool to study the impact of high glucose in the retina and to develop novel molecules with potential effect on diabetic retinopathy.

Modulation of Pro-Oxidant and Pro-Inflammatory Activities of M1 Macrophages by the Natural Dipeptide Carnosine.

Carnosine is a natural endogenous dipeptide widely distributed in mammalian tissues, existing at particularly high concentrations in the muscles and brain and possesses well-characterized antioxidant and anti-inflammatory activities. In an in vitro model of macrophage activation, induced by lipopolysaccharide + interferon-gamma (LPS + IFN-γ), we here report the ability of carnosine to modulate pro-oxidant and pro-inflammatory activities of macrophages, representing the primary cell type that is activated as a part of the immune response. An ample set of parameters aimed to evaluate cytotoxicity (MTT assay), energy metabolism (HPLC), gene expressions (high-throughput real-time PCR (qRT-PCR)), protein expressions (western blot) and nitric oxide production (qRT-PCR and HPLC), was used to assess the effects of carnosine on activated macrophages challenged with a non cytotoxic LPS (100 ng/mL) + IFN-γ (600 U/mL) concentration. In our experimental model, main carnosine beneficial effects were: (1) the modulation of nitric oxide production and metabolism; (2) the amelioration of the macrophage energy state; (3) the decrease of the expressions of pro-oxidant enzymes (Nox-2, Cox-2) and of the lipid peroxidation product malondialdehyde; (4) the restoration and/or increase of the expressions of antioxidant enzymes (Gpx1, SOD-2 and Cat); (5) the increase of the transforming growth factor-ß1 (TGF-ß1) and the down-regulation of the expressions of interleukins 1ß and 6 (IL-1ß and IL-6) and 6) the increase of the expressions of Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1). According to these results carnosine is worth being tested in the treatment of diseases characterized by elevated levels of oxidative stress and inflammation (atherosclerosis, cancer, depression, metabolic syndrome, and neurodegenerative diseases).

Inflammation as the Common Biological Link Between Depression and Cardiovascular Diseases: Can Carnosine Exert a Protective Role?

Several epidemiological studies have clearly shown the high co-morbidity between depression and Cardiovascular Diseases (CVD). Different studies have been conducted to identify the common pathophysiological events of these diseases such as the overactivation of the hypothalamic- pituitary-adrenal axis and, most importantly, the dysregulation of immune system which causes a chronic pro-inflammatory status. The biological link between depression, inflammation, and CVD can be related to high levels of pro-inflammatory cytokines, such as IL-1ß, TNF-α, and IL-6, released by macrophages which play a central role in the pathophysiology of both depression and CVD. Pro-inflammatory cytokines interfere with many of the pathophysiological mechanisms relevant to depression by upregulating the rate-limiting enzymes in the metabolic pathway of tryptophan and altering serotonin metabolism. These cytokines also increase the risk to develop CVD, because activation of macrophages under this pro-inflammatory status is closely associated with endothelial dysfunction and oxidative stress, a preamble to atherosclerosis and atherothrombosis. Carnosine (ß-alanyl-L-histidine) is an endogenous dipeptide which exerts a strong antiinflammatory activity on macrophages by suppressing reactive species and pro-inflammatory cytokines production and altering pro-inflammatory/anti-inflammatory macrophage polarization. This dipeptide exhibits antioxidant properties scavenging reactive species and preventing oxidative stress-induced pathologies such as CVD. In the present review we will discuss the role of oxidative stress and chronic inflammation as common pathophysiological events both in depression and CVD and the preclinical and clinical evidence on the protective effect of carnosine in both diseases as well as the therapeutic potential of this dipeptide in depressed patients with a high co-morbidity of cardiovascular diseases.

Optimization of a microchip electrophoresis method with electrochemical detection for the determination of nitrite in macrophage cells as an indicator of nitric oxide production.

Nitric oxide (NO) is involved in many biological functions, including blood pressure regulation, the immune response, and neurotransmission. However, excess production of NO can lead to the generation of reactive nitrogen species and nitrosative stress and has been linked to several neurodegenerative diseases and cardiovascular disorders. Because NO is short-lived and generally difficult to detect, its primary stable degradation product, nitrite, is frequently monitored in its place. In this paper, an improved method using microchip electrophoresis with electrochemical detection (ME-EC) was developed for the separation and detection of nitrite in cell lysates. A separation of nitrite from several electroactive cell constituents and interferences was optimized, and the effect of sample and buffer conductivity on peak efficiency was explored. It was found that the addition of 10 mM NaCl to the run buffer caused stacking of the nitrite peak and improved limits of detection. A platinum black working electrode was also evaluated for the detection of nitrite and other electroactive cellular species after electrophoretic separation. The use of a modified platinum working electrode resulted in 2.5-, 1.7-, and 7.2-fold signal enhancement for nitrite, ascorbic acid, and hydrogen peroxide, respectively, and increased the sensitivity of the method for nitrite twofold. The optimized ME-EC method was used to compare nitrite production by native and lipopolysaccharide-stimulated RAW 264.7 macrophage cells.

Carnosine Decreases PMA-Induced Oxidative Stress and Inflammation in Murine Macrophages.

Carnosine is an endogenous dipeptide composed of ß-alanine and L-histidine. This naturally occurring molecule is present at high concentrations in several mammalian excitable tissues such as muscles and brain, while it can be found at low concentrations in a few invertebrates. Carnosine has been shown to be involved in different cellular defense mechanisms including the inhibition of protein cross-linking, reactive oxygen and nitrogen species detoxification as well as the counteraction of inflammation. As a part of the immune response, macrophages are the primary cell type that is activated. These cells play a crucial role in many diseases associated with oxidative stress and inflammation, including atherosclerosis, diabetes, and neurodegenerative diseases. In the present study, carnosine was first tested for its ability to counteract oxidative stress. In our experimental model, represented by RAW 264.7 macrophages challenged with phorbol 12-myristate 13-acetate (PMA) and superoxide dismutase (SOD) inhibitors, carnosine was able to decrease the intracellular concentration of superoxide anions (O2-•) as well as the expression of Nox1 and Nox2 enzyme genes. This carnosine antioxidant activity was accompanied by the attenuation of the PMA-induced Akt phosphorylation, the down-regulation of TNF-α and IL-6 mRNAs, and the up-regulation of the expression of the anti-inflammatory mediators IL-4, IL-10, and TGF-ß1. Additionally, when carnosine was used at the highest dose (20 mM), there was a generalized amelioration of the macrophage energy state, evaluated through the increase both in the total nucleoside triphosphate concentrations and the sum of the pool of intracellular nicotinic coenzymes. Finally, carnosine was able to decrease the oxidized (NADP+)/reduced (NADPH) ratio of nicotinamide adenine dinucleotide phosphate in a concentration dependent manner, indicating a strong inhibitory effect of this molecule towards the main source of reactive oxygen species in macrophages. Our data suggest a multimodal mechanism of action of carnosine underlying its beneficial effects on macrophage cells under oxidative stress and inflammation conditions.

Carnosine Prevents Aß-Induced Oxidative Stress and Inflammation in Microglial Cells: A Key Role of TGF-ß1.

Carnosine (ß-alanyl-L-histidine), a dipeptide, is an endogenous antioxidant widely distributed in excitable tissues like muscles and the brain. Carnosine is involved in cellular defense mechanisms against oxidative stress, including the inhibition of amyloid-beta (Aß) aggregation and the scavenging of reactive species. Microglia play a central role in the pathogenesis of Alzheimer's disease, promoting neuroinflammation through the secretion of inflammatory mediators and free radicals. However, the effects of carnosine on microglial cells and neuroinflammation are not well understood. In the present work, carnosine was tested for its ability to protect BV-2 microglial cells against oligomeric Aß1-42-induced oxidative stress and inflammation. Carnosine prevented cell death in BV-2 cells challenged with Aß oligomers through multiple mechanisms. Specifically, carnosine lowered the oxidative stress by decreasing NO and O2-• intracellular levels as well as the expression of iNOS and Nox enzymes. Carnosine also decreased the secretion of pro-inflammatory cytokines such as IL-1ß, simultaneously rescuing IL-10 levels and increasing the expression and the release of TGF-ß1. Carnosine also prevented Aß-induced neurodegeneration in mixed neuronal cultures challenged with Aß oligomers, and these neuroprotective effects were completely abolished by SB431542, a selective inhibitor of the type-1 TGF-ß receptor. Our data suggest a multimodal mechanism of action of carnosine underlying its protective effects on microglial cells against Aß toxicity with a key role of TGF-ß1 in mediating these protective effects.

Sub-Toxic Human Amylin Fragment Concentrations Promote the Survival and Proliferation of SH-SY5Y Cells via the Release of VEGF and HspB5 from Endothelial RBE4 Cells.

Human amylin is a 37-residue peptide hormone (hA1-37) secreted by ß-cells of the pancreas and, along with insulin, is directly associated with type 2 diabetes mellitus (T2DM). Amyloid deposits within the islets of the pancreas represent a hallmark of T2DM. Additionally, amylin aggregates have been found in blood vessels and/or brain of patients with Alzheimer's disease, alone or co-deposited with ß-amyloid. The purpose of this study was to investigate the neuroprotective potential of human amylin in the context of endothelial-neuronal "cross-talk". We initially performed dose-response experiments to examine cellular toxicity (quantified by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] MTT assay) of different hA17⁻29 concentrations in endothelial cells (RBE4). In the culture medium of these cells, we also measured heat shock protein B5 (HspB5) levels by ELISA, finding that even a sub-toxic concentration of hA17⁻29 (3 µM) produced an increase of HspB5. Using a cell medium of untreated and RBE4 challenged for 48 h with a sub-toxic concentration of hA17⁻29, we determined the potential beneficial effect of their addition to the medium of neuroblastoma SH-SY5Y cells. These cells were subsequently incubated for 48 h with a toxic concentration of hA17⁻29 (20 µM). We found a complete inhibition of hA17⁻29 toxicity, potentially related to the presence in the conditioned medium not only of HspB5, but also of vascular endothelial growth factor (VEGF). Pre-treating SH-SY5Y cells with the anti-Flk1 antibody, blocking the VEGF receptor 2 (VEGFR2), significantly decreased the protective effects of the conditioned RBE4 medium. These data, obtained by indirectly measuring VEGF activity, were strongly corroborated by the direct measurement of VEGF levels in conditioned RBE4 media as detected by ELISA. Altogether, these findings highlighted a novel role of sub-toxic concentrations of human amylin in promoting the secretion of proteic factors by endothelial cells (HspB5 and VEGF) that support the survival and proliferation of neuron-like cells.

Non-toxic engineered carbon nanodiamond concentrations induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells.

Engineered nanoparticles are finding a wide spectrum of biomedical applications, including drug delivery and capacity to trigger cytotoxic phenomena, potentially useful against tumor cells. The full understanding of their biosafety and interactions with cell processes is mandatory. Using microglial (BV-2) and alveolar basal epithelial (A549) cells, in this study we determined the effects of engineered carbon nanodiamonds (ECNs) on cell viability, nitric oxide (NO) and reactive oxygen species (ROS) production, as well as on energy metabolism. Particularly, we initially measured decrease in cell viability as a function of increasing ECNs doses, finding similar cytotoxic ECN effects in the two cell lines. Subsequently, using apparently non-cytotoxic ECN concentrations (2 µg/mL causing decrease in cell number < 5%) we determined NO and ROS production, and measured the concentrations of compounds related to energy metabolism, mitochondrial functions, oxido-reductive reactions, and antioxidant defences. We found that in both cell lines non-cytotoxic ECN concentrations increased NO and ROS production with sustained oxidative/nitrosative stress, and caused energy metabolism imbalance (decrease in high energy phosphates and nicotinic coenzymes) and mitochondrial malfunctioning (decrease in ATP/ADP ratio).These results underline the importance to deeply investigate the molecular and biochemical changes occurring upon the interaction of ECNs (and nanoparticles in general) with living cells, even at apparently non-toxic concentration. Since the use of ECNs in biomedical field is attracting increasing attention the complete evaluation of their biosafety, toxicity and/or possible side effects both in vitro and in vivo is mandatory before these highly promising tools might find the correct application.

Monitoring carnosine uptake by RAW 264.7 macrophage cells using microchip electrophoresis with fluorescence detection.

Carnosine, a dipeptide found in a variety of tissues, is believed to possess antioxidant properties. It serves as a scavenger of reactive nitrogen and oxygen species (RNOS), which are important stress mediators of pro-inflammatory conditions and can lead to macrophage activation. In this study, intracellular concentrations of carnosine in murine RAW 264.7 macrophage cells were determined using microchip electrophoresis with laser-induced fluorescence detection following derivatization with naphthalene-2,3-dicarboxaldehyde and cyanide. The method was linear from 25 nM to 5 µM with a limit of detection in cell lysate samples of 65 nM. Using the method of standard additions, the basal intracellular content of carnosine in macrophage cells was determined to be 0.079 ± 0.02 nmol/106 cells. The uptake of carnosine by these cells was then investigated under both physiological and pro-inflammatory conditions. There was a 2.8-fold increase in carnosine uptake for macrophages exposed to lipopolysaccharide and interferon-γ prior to incubation, compared to the controls. This suggests that macrophages may use carnosine uptake as a defense mechanism under pro-inflammatory conditions. Future studies will investigate the role of the carnosine transporter in carnosine uptake and its possible correlation with cell morphological changes observed after stimulation.

Microchip electrophoresis with laser-induced fluorescence detection for the determination of the ratio of nitric oxide to superoxide production in macrophages during inflammation.

It is well known that excessive production of reactive oxygen and nitrogen species is linked to the development of oxidative stress-driven disorders. In particular, nitric oxide (NO) and superoxide (O2•-) play critical roles in many physiological and pathological processes. This article reports the use of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and MitoSOX Red in conjunction with microchip electrophoresis and laser-induced fluorescence detection for the simultaneous detection of NO and O2•- in RAW 264.7 macrophage cell lysates following different stimulation procedures. Cell stimulations were performed in the presence and absence of cytosolic (diethyldithiocarbamate) and mitochondrial (2-methoxyestradiol) superoxide dismutase (SOD) inhibitors. The NO/O2•- ratios in macrophage cell lysates under physiological and proinflammatory conditions were determined. The NO/O2•- ratios were 0.60 ± 0.07 for unstimulated cells pretreated with SOD inhibitors, 1.08 ± 0.06 for unstimulated cells in the absence of SOD inhibitors, and 3.14 ± 0.13 for stimulated cells. The effect of carnosine (antioxidant) or Ca2+ (intracellular messenger) on the NO/O2•- ratio was also investigated. Graphical Abstract Simultaneous detection of nitric oxide and superoxide in macrophage cell lysates.

Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages.

Excess nitric oxide (NO) production occurs in several pathological states, including neurodegeneration, ischemia, and inflammation, and is generally accompanied by increased oxidative/nitrosative stress. Carnosine [ß-alanine-histidine (ß-Ala-His)] has been reported to decrease oxidative/nitrosative stress-associated cell damage by reducing the amount of NO produced. In this study, we evaluated the effect of carnosine on NO production by murine RAW 264.7 macrophages stimulated with lipopolysaccharides + interferon-γ. Intracellular NO and intracellular and extracellular nitrite were measured by microchip electrophoresis with laser-induced fluorescence and by the Griess assay, respectively. Results showed that carnosine causes an apparent suppression of total NO production by stimulated macrophages accompanied by an unexpected simultaneous drastic increase in its intracellular low toxicity endproduct, nitrite, with no inhibition of inducible nitric oxide synthase (iNOS). ESI-MS and NMR spectroscopy in a cell-free system showed the formation of multiple adducts (at different ratios) of carnosine-NO and carnosine-nitrite, involving both constituent amino acids (ß-Ala and His) of carnosine, thus providing a possible mechanism for the changes in free NO and nitrite in the presence of carnosine. In stimulated macrophages, the addition of carnosine was also characterized by changes in the expression of macrophage activation markers and a decrease in the release of IL-6, suggesting that carnosine might alter M1/M2 macrophage ratio. These results provide evidence for previously unknown properties of carnosine that modulate the NO/nitrite ratio of stimulated macrophages. This modulation is also accompanied by changes in the release of pro-inflammatory molecules, and does not involve the inhibition of iNOS activity.

Receptor-mediated toxicity of human amylin fragment aggregated by short- and long-term incubations with copper ions.

Human amylin (hA1-37) is a polypeptide hormone secreted in conjunction with insulin from the pancreatic ß-cells involved in the pathogenesis of type 2 diabetes mellitus (T2DM). The shorter fragment hA17-29 than full-length peptide is capable to form amyloids "in vitro". Here, we monitored the time course of hA17-29 ß-amyloid fibril and oligomer formation [without and with copper(II)], cellular toxicity of different amyloid aggregates, and involvement of specific receptors (receptor for advanced glycation end-products, RAGE; low-affinity nerve growth factor receptor, p75-NGFR) in aggregate toxicity. Fibril and oligomer formation of hA17-29 incubated at 37 °C for 0, 48, and 120 h, without or with copper(II), were measured by the thioflavin T fluorescence assay and ELISA, respectively. Toxicity of hA17-29 aggregates and effects of anti-RAGE and anti-p75-NGFR antibodies were evaluated on neuroblastoma SH-SY5Y viability. Fluorescence assay of hA17-29 indicates an initial slow rate of soluble fibril formation (48 h), followed by a slower rate of insoluble aggregate formation (120 h). The highest quantity of oligomers was recorded when hA17-29 was pre-aggregated for 48 h in the presence of copper(II) showing also the maximal cell toxicity (-44% of cell viability, p < 0.01 compared to controls). Anti-RAGE or anti-p75-NGFR antibodies almost abolished cell toxicity of hA17-29 aggregates. These results indicate that copper(II) influences the aggregation process and hA17-29 toxicities are especially attributable to oligomeric aggregates. hA17-29 aggregate toxicity seems to be mediated by RAGE and p75-NGFR receptors which might be potential targets for new drugs in T2DM treatment.

Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence.

Superoxide, a naturally produced reactive oxygen species (ROS) in the human body, is involved in many pathological and physiological signaling processes. However, if superoxide formation is left unregulated, overproduction can lead to oxidative damage to important biomolecules, such as DNA, lipids, and proteins. Superoxide can also lead to the formation of peroxynitrite, an extremely hazardous substance, through its reaction with endogenously produced nitric oxide. Despite its importance, quantitative information regarding superoxide production is difficult to obtain due to its high reactivity and low concentrations in vivo. MitoHE, a fluorescent probe that specifically reacts with superoxide, was used in conjunction with microchip electrophoresis (ME) and laser-induced fluorescence (LIF) detection to investigate changes in superoxide production by RAW 264.7 macrophage cells following stimulation with phorbol 12-myristate 13-acetate (PMA). Stimulation was performed in the presence and absence of the superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC) and 2-metoxyestradiol (2-ME). The addition of these inhibitors resulted in an increase in the amount of superoxide specific product (2-OH-MitoE(+)) from 0.08 ± 0.01 fmol (0.17 ± 0.03 mM) in native cells to 1.26 ± 0.06 fmol (2.5 ± 0.1 mM) after PMA treatment. This corresponds to an approximately 15-fold increase in intracellular concentration per cell. Furthermore, the addition of 3-morpholino-sydnonimine (SIN-1) to the cells during incubation resulted in the production of 0.061 ± 0.006 fmol (0.12 ± 0.01 mM) of 2-OH-MitoE(+) per cell on average. These results demonstrate that indirect superoxide detection coupled with the use of SOD inhibitors and a separation method is a viable method to discriminate the 2-OH-MitoE(+) signal from possible interferences.