Alkaptonuria (AKU) is an ultra-rare genetic disease caused by a deficient activity of the enzyme homogentisate 1,2-dioxygenase (HGD) leading to the accumulation of homogentisic acid (HGA) on ...connective tissues. Even though AKU is a multi-systemic disease, osteoarticular cartilage is the most affected system and the most damaged tissue by the disease. In chondrocytes, HGA causes oxidative stress dysfunctions, which induce a series of not fully characterized cellular responses. In this study, we used a human chondrocytic cell line as an AKU model to evaluate, for the first time, the effect of HGA on autophagy, the main homeostasis system in articular cartilage. Cells responded timely to HGA treatment with an increase in autophagy as a mechanism of protection. In a chronic state, HGA-induced oxidative stress decreased autophagy, and chondrocytes, unable to restore balance, activated the chondroptosis pathway. This decrease in autophagy also correlated with the accumulation of ochronotic pigment, a hallmark of AKU. Our data suggest new perspectives for understanding AKU and a mechanistic model that rationalizes the damaging role of HGA.
•In an in vitro model of Alkaptonuria, HGA cells treatment leads the activation of the autophagic pathway.•In long-term HGA cells treatment, there is a block of autophagic flux and the activation of apoptotic pathway.•HGA induces in cells oxidative stress, mitochondrial damage, nucleolar morphology alteration and ochronotic pigment deposition.
The effect of oral glutathione (GSH) supplementation was studied in obese subjects with and without type 2 diabetes (T2DM) on measures of glucose homeostasis and markers of oxidative stress. Twenty ...subjects (10 patients with T2DM and 10 obese subjects) were recruited for the study, and randomized in a double-blinded placebo-controlled manner to consume either 1000 mg GSH per day or placebo for 3 weeks. Before and after the 3 weeks insulin sensitivity was measured with the hyperinsulinemic-euglycemic clamp and a muscle biopsy was obtained to measure GSH and skeletal muscle mitochondrial hydrogen peroxide (H
2
O
2
) emission rate. Whole body insulin sensitivity increased significantly in the GSH group. Skeletal muscle GSH was numerically increased (∼19%) in the GSH group; no change was seen in GSH to glutathione disulfide ratio. Skeletal muscle mitochondrial H
2
O
2
emission rate did not change in response to the intervention and neither did the urinary excretion of the RNA oxidation product 8-oxo-7,8-dihydroguanosine or the DNA oxidation product 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), although 8-oxodG decreased as a main effect of time. Oral GSH supplementation improves insulin sensitivity in obese subjects with and without T2DM, although it does not alter markers of oxidative stress. The study has been registered in clinicaltrials.gov (NCT02948673).
Novelty:
Reduced glutathione supplementation increases insulin sensitivity in obese subjects with and without T2DM.
H
2
O
2
emission rate from skeletal muscle mitochondria was not affected by GSH supplementation.
Abstract
The fluoroquinolone trovafloxacin (TVX) is associated with a high risk of drug-induced liver injury (DILI). Although part of the liver damage by TVX+TNF relies on neutrophils, we have ...recently demonstrated that liver recruitment of monocytes and neutrophils is delayed by TVX. Here we show that the delayed leukocyte recruitment is caused by a combination of effects which are linked to the capacity of TVX to block the hemichannel pannexin 1. TVX inhibited find-me signal release in apoptotic HepG2 hepatocytes, decelerated freshly isolated human neutrophils toward IL-8 and f-MLF, and decreased the liver expression of ICAM-1. In blood of TVX+TNF-treated mice, we observed an accumulation of activated neutrophils despite an increased MIP-2 release by the liver. Depletion of monocytes and neutrophils caused increased serum concentrations of TNF, IL-6, and MIP-2 in TVX-treated mice as well as in mice treated with the fluoroquinolone levofloxacin, known to have a lower DILI-inducing profile. This supports the idea that early leukocyte recruitment regulates inflammation. In conclusion, disrupted regulation by leukocytes appears to constitute a fundamental step in the onset of TVX-induced liver injury, acting in concert with the capability of TVX to induce hepatocyte cell death. Interference of leukocyte-mediated regulation of inflammation represents a novel mechanism to explain the onset of DILI.
Idiosyncratic drug-induced liver injury (IDILI) is a severe disease that cannot be detected during drug development. It has been shown that hepatotoxicity of some compounds associated with IDILI ...becomes apparent when these are combined in vivo and in vitro with LPS or TNF. Among these compounds trovafloxacin (TVX) induced apoptosis in the liver and increased pro-inflammatory cytokines in mice exposed to LPS/TNF. The hepatocyte survival and the cytokine release after TNF/LPS stimulation relies on a pulsatile activation of NF-κB.
We set out to evaluate the dynamic activation of NF-κB in response to TVX + TNF or LPS models, both in mouse and human cells. Remarkably, TVX prolonged the first translocation of NF-κB induced by TNF both in vivo and in vitro. The prolonged p65 translocation caused by TVX was associated with an increased phosphorylation of IKK and MAPKs and accumulation of inhibitors of NF-κB such as IκBα and A20 in HepG2. Coherently, TVX suppressed further TNF-induced NF-κB translocations in HepG2 leading to decreased transcription of ICAM-1 and inhibitors of apoptosis. TVX prolonged LPS-induced NF-κB translocation in RAW264.7 macrophages increasing the secretion of TNF. In summary, this study presents new, relevant insights into the mechanism of TVX-induced liver injury underlining the resemblance between mouse and human models. In this study we convincingly show that regularly used toxicity models provide a coherent view of relevant pathways for IDILI. We propose that assessment of the kinetics of activation of NF-κB and MAPKs is an appropriate tool for the identification of hepatotoxic compounds during drug development.
•TVX disturbed TNF/LPS-induced NF-kB nuclear translocation.•TVX altered expression of NF-kB-dependent genes (e.g. IKBα, A20, TNF, ICAM-1)•TVX altered phosphorylation of IKK and MAPKs.•TVX effects illustrate a clear cell-stress related response linked to hepatotoxicity
To evaluate the effectiveness of hydrogen peroxide (HP) use as a disinfectant in the hospital water network for the control of
spp. colonization.
Following the detection of high levels of
...contamination in a 136-bed general hospital water network, an HP treatment of the hot water supply (25 mg/L) was adopted. During a period of 34 months, the effectiveness of HP on
colonization was assessed.
was isolated in accordance with ISO-11731 and identification was carried out by sequencing of the
gene.
Before HP treatment,
sg 2-15 was isolated in all sites with a mean count of 9950 ± 8279 cfu/L. After one-month of HP treatment, we observed the disappearance of
2-15, however other
species previously not seen were found;
1 was isolated in one out of four sampling sites (2000 cfu/L) and other non-
species were present in all sites (mean load 3000 ± 2887 cfu/L). Starting from September 2013, HP treatment was modified by adding food-grade polyphosphates, and in the following months, we observed a progressive reduction of the mean load of all species (
< 0.05), resulting in substantial disappearance of
colonization.
Hydrogen peroxide demonstrated good efficacy in controlling
. Although in the initial phases of treatment it appeared unable to eliminate all
species, by maintaining HP levels at 25 mg/L and adding food-grade polyphosphates, a progressive and complete control of colonization was obtained.
Background
Promising therapies for food allergy are emerging, mostly based on animal experimentation. However, different mouse strains are used, which may make it hard to compare experiments. The ...current study investigated whether the immunological differences between C3H/HeOuJ (C3H) and BALB/c mice lead to differences in efficacy of peanut‐specific immunotherapy.
Methods
After sensitization using peanut extract (PE), C3H and BALB/c mice received oral immunotherapy (OIT) by intragastric dosing for three weeks. Hereafter, mice were exposed to PE via the intradermal, intragastric and intraperitoneal route, to determine allergic outcomes. Furthermore, PE‐specific antibody and cytokine production were determined and the number of various immune cells at different time points during the study were measured.
Results
OIT protected C3H mice against anaphylaxis, whereas no anaphylaxis was seen in BALB/c mice. In contrast, OIT induced an increase in MMCP‐1 levels in BALB/c mice but not in C3H mice. No effect of OIT on the acute allergic skin response was observed in either strain. Specific antibody responses showed similar patterns in both strains for IgA and IgG1. IgE levels were a tenfold higher in BALB/c mice and after the intragastric challenge (day 70) OIT‐treated BALB/c mice showed induced IgE levels. Moreover, in C3H mice IgG2a levels were higher and increased in response to OIT and challenges. After the final challenge, but not at other timepoints MLN‐derived lymphocytes from OIT‐treated BALB/c mice produced less IL‐13 and IL‐5 compared to control‐treated mice, whereas no differences were seen in case of C3H mice.
Conclusions
Taken together, these results show that the C3H strain is more suitable to study clinical outcomes of OIT, whereas the BALB/c strain is more optimal to study T cell responses.
Scope
A major downside of oral immunotherapy (OIT) for food allergy is the risk of severe side effects. Non‐digestible short‐ and long‐chain fructo‐oligosaccharides (scFOS/lcFOS) reduce allergy ...development in murine models. Therefore, it is hypothesized that scFOS/lcFOS can also support the efficacy of OIT in a peanut allergy model.
Methods and Results
After sensitization to peanut extract (PE) using cholera toxin, C3H/HeOuJ mice are fed a 1% scFOS/lcFOS or control diet and receive OIT (1.5 or 15 mg PE). Hereafter, mice are exposed to PE via different routes to determine the safety and efficacy of treatment in clinical outcomes, PE‐specific antibody production, and numbers of various immune cells. scFOS/lcFOS increases short‐chain fatty acid levels in the caecum and reduce the acute allergic skin response and drop in body temperature after PE exposure. Interestingly, 15 mg and 1.5 mg OIT with scFOS/lcFOS induce protection against anaphylaxis, whereas 1.5 mg OIT alone does not. OIT, with or without scFOS/lcFOS, induces PE‐specific immunoglobulin (Ig) IgG and IgA levels and increases CD103+ dendritic cells in the mesenteric lymph nodes.
Conclusions
scFOS/lcFOS and scFOS/lcFOS combined with low dose OIT are able to protect against a peanut‐allergic anaphylactic response.
A major downside of oral immunotherapy (OIT) for food allergy is the risk of side effects. Dietary supplementation with nondigestible short‐ and long‐chain fructo‐oligosaccharides (scFOS/lcFOS) (FF), after peanut allergy development, is shown to reduce the allergic reaction upon peanut exposure. Interestingly, a high dose of OIT and low dose OIT combined with FF also induced protection against an allergic reaction, whereas low dose OIT alone did not. This suggests that combining OIT with FF enables the use of a lower dose of OIT.
Idiosyncratic drug‐induced liver injury (iDILI) has a poorly understood pathogenesis. However, iDILI is often associated with inflammatory stress signals in human patients as well as animal models. ...Tumor necrosis factor (TNF) and neutrophils play a key role in onset of trovafloxacin (TVX)‐induced iDILI, but the exact role of neutrophils and other leukocytes remains to be defined. We therefore set out to study the kinetics of immunological changes during the development of TVX‐induced iDILI in the established murine model of acute liver injury induced by administration of TVX and TNF. Initially, TNF stimulated the appearance of leukocytes, in particular neutrophils, into the liver of TVX‐treated mice, but even more so in control mice treated with the non‐DILI inducing analogue levofloxacin (LVX) or saline as vehicle (Veh). This difference was apparent at 2 hours after TNF administration, but at 4 hours, the relative neutrophil amounts were reduced again in Veh‐ and LVX‐treated mice whereas the amounts in TVX‐treated mice remained at the same increased level as at 2 hours. The influx of monocytes/macrophages, which was unaffected in Veh‐ and LVX‐treated mice was markedly reduced or even absent in TVX‐treated mice. Unlike controls, mice receiving TVX + TNF display severe hepatotoxicity with clear pathology and apoptosis, coagulated hepatic vessels and increased alanine aminotransferase levels and interleukin 6/10 ratios. Findings indicate that TVX delays the acute influx of neutrophils and monocytes/macrophages. Considering their known anti‐inflammatory functions, the disruption of influx of these innate immune cells may hamper the resolution of initial cytotoxic effects of TVX and thus contribute to liver injury development.
The role of innate immune system in the onset of idiosyncratic drug‐induced liver injury is still matter of debate. Recent findings revealed how inflammation represents a key player in idiosyncratic drug‐induced liver injury. We evaluated the effects of trovafloxacin on the inflammation induced by tumor necrosis factor in mouse and found that trovafloxacin delays neutrophil and monocyte influx in mice livers. Considering the known neutrophil and monocyte anti‐inflammatory functions, the observed effect may hinder the regulation of inflammation contributing to the development of liver injury.
The frequency and clinical features of drug-related taste and/or smell impairments are currently unclear.
The aim of this study was to identify major drug classes associated with taste and smell ...alterations reported to the Italian spontaneous adverse drug reaction (ADR) reporting database.
The association between drug and altered taste or smell was investigated by case/non-case methodology. The reporting odds ratio (ROR) was used as a measure of disproportionality. Cases were defined as patients with at least one ADR related to taste or smell impairments. The non-cases included all patients without any ADRs related to taste or smell alterations.
According to the selection criteria, 52 166 reports were included in the analysis. Overall, 182 cases of drug-related taste and/or smell dysfunctions were identified. Statistically significant unadjusted RORs were reported for macrolides (n = 31; 7.1; 95% CI 4.8, 10.5), terbinafine (the only drug reported within the group of antimycotics belonging to the Anatomical Therapeutic Chemical class D01AE) n = 17; 76.4; 95% CI 44.0, 132.6, fluoroquinolones (n = 15; 1.7; 95% CI 1.0, 2.8) and protein kinase inhibitors (n = 10; 4.0; 95% CI 2.1, 7.7). When RORs were adjusted for sex and age category, the disproportion remained statistically significant for all of the previously mentioned drug classes.
Taste and/or smell abnormalities are common, sometimes unexpected and often persistent complaints of patients during pharmacological treatments. Physicians should be aware of the impact of these ADRs on patients' quality of life.
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