Involvement of pro-inflammatory mediators and cell cycle disruption in neuronal cells induced by gliotoxin and ochratoxin A after individual and combined exposure.

Mycotoxins such as gliotoxin (GTX) and ochratoxin A (OTA) are secondary metabolites of Aspergillus and Penicillum found in food and feed. Both mycotoxins have shown to exert a detrimental effect on neuronal activity. The following study was carried out to elucidate the mechanisms by which GTX and OTA exert their toxicity. Non-differentiated SH-SY5Y neuronal-like cells were treated with GTX, OTA and their combinations to assess their cytotoxic effect using the MTT assay during 24, 48 and 72 h of exposure. Based on the results of the cytotoxic assays, cell cycle proliferation and immunological mediators were measured by determining the production of IL-6 and TNF-α using flow cytometry and ELISA, respectively. The IC50 values obtained were 1.24 and 1.35 µM when SH-SY5Y cells were treated with GTX at 48 h and 72 h, respectively. IC50 values of 8.25, 5.49 and 4.5 µM were obtained for OTA treatment at 24 h, 48 h and 72 h, respectively. The SubG0 phase increased in both treatments at 24 and 48 h. On the other hand, IL-6 and TNF-α production was increased in all mycotoxin treatments studied and was more pronounced for [GTX + OTA] after 48 h exposure. The additive and synergistic effect observed by the isobologram analysis between GTX and OTA resulted to a higher cytotoxicity which can be explained by the increased production of IL-6 and TNF-α inflammatory mediators that play an important role in the toxicity mechanism of these mycotoxins.

The Toxic Mechanism of Gliotoxins and Biosynthetic Strategies for Toxicity Prevention.

Gliotoxin is a kind of epipolythiodioxopiperazine derived from different fungi that is characterized by a disulfide bridge. Gliotoxins can be biosynthesized by a gli gene cluster and regulated by a positive GliZ regulator. Gliotoxins show cytotoxic effects via the suppression the function of macrophage immune function, inflammation, antiangiogenesis, DNA damage by ROS production, peroxide damage by the inhibition of various enzymes, and apoptosis through different signal pathways. In the other hand, gliotoxins can also be beneficial with different doses. Low doses of gliotoxin can be used as an antioxidant, in the diagnosis and treatment of HIV, and as an anti-tumor agent in the future. Gliotoxins have also been used in the control of plant pathogens, including Pythium ultimum and Sclerotinia sclerotiorum. Thus, it is important to elucidate the toxic mechanism of gliotoxins. The toxic mechanism of gliotoxins and biosynthetic strategies to reduce the toxicity of gliotoxins and their producing strains are summarized in this review.

In vitro study on aspects of molecular mechanisms underlying invasive aspergillosis caused by gliotoxin and fumagillin, alone and in combination.

Gliotoxin (GT) and fumagillin (FUM) are mycotoxins most abundantly produced by Aspergillus fumigatus during the early stages of infection to cause invasive aspergillosis (IA). Therefore, we hypothesized that GT and FUM could be the possible source of virulence factors, which we put to test adopting in vitro monoculture and the novel integrated multiple organ co-culture (IdMOC) of A549 and L132 cell. We found that (i) GT is more cytotoxic to lung epithelial cells than FUM, and (ii) GT and FUM act synergistically to inflict pathology to the lung epithelial cell. Reactive oxygen species (ROS) is the master regulator of the cytotoxicity of GT, FUM and GT + FUM. ROS may be produced as a sequel to mitochondrial damage and, thus, mitochondria are both the source of ROS and the target to ROS. GT-, FUM- and GT + FUM-induced DNA damage is mediated either by ROS-dependent mechanism or directly by the fungal toxins. In addition, GT, FUM and GT + FUM may induce protein accumulation. Further, it is speculated that GT and FUM inflict epithelial damage by neutrophil-mediated inflammation. With respect to multiple organ cytotoxicity, GT was found to be cytotoxic at IC50 concentration in the following order: renal epithelial cells < type II epithelial cells < hepatocytes < normal lung epithelial cells. Taken together, GT and FUM alone and in combination contribute to exacerbate the damage of lung epithelial cells and, thus, are involved in the progression of IA.

Gliotoxin Aggravates Experimental Autoimmune Encephalomyelitis by Triggering Neuroinflammation.

Gliotoxin (GTX) is the major and the most potent mycotoxin that is secreted by Aspergillus fumigatus, which is capable of injuring and killing microglial cells, astrocytes, and oligodendrocytes. During the last years, studies with patients and experimental models of multiple sclerosis (MS), which is an autoimmune disease of the central nervous system (CNS), suggested that fungal infections are among the possible initiators or aggravators of this pathology. The deleterious effect can occur through a direct interaction of the fungus with the CNS or by the toxin release from a non-neurological site. In the present work, we investigated the effect of GTX on experimental autoimmune encephalomyelitis (EAE) development. Female C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein and then intraperitoneally injected with three doses of GTX (1 mg/kg b.w., each) on days 4, 7, and 10. GTX aggravated clinical symptoms of the disease in a dose-dependent way and this outcome was concomitant with an increased neuroinflammation. CNS analyses revealed that GTX locally increased the relative expression of inflammatory genes and the cytokine production. Our results indicate that GTX administered in a non-neuronal site was able to increase neuroinflammation in EAE. Other mycotoxins could also be deleterious to many neurological diseases by similar mechanisms.

Myelin repair stimulated by CNS-selective thyroid hormone action.

Oligodendrocyte processes wrap axons to form neuroprotective myelin sheaths, and damage to myelin in disorders, such as multiple sclerosis (MS), leads to neurodegeneration and disability. There are currently no approved treatments for MS that stimulate myelin repair. During development, thyroid hormone (TH) promotes myelination through enhancing oligodendrocyte differentiation; however, TH itself is unsuitable as a remyelination therapy due to adverse systemic effects. This problem is overcome with selective TH agonists, sobetirome and a CNS-selective prodrug of sobetirome called Sob-AM2. We show here that TH and sobetirome stimulated remyelination in standard gliotoxin models of demyelination. We then utilized a genetic mouse model of demyelination and remyelination, in which we employed motor function tests, histology, and MRI to demonstrate that chronic treatment with sobetirome or Sob-AM2 leads to significant improvement in both clinical signs and remyelination. In contrast, chronic treatment with TH in this model inhibited the endogenous myelin repair and exacerbated disease. These results support the clinical investigation of selective CNS-penetrating TH agonists, but not TH, for myelin repair.

Gliotoxin destructs the pulmonary epithelium barrier function by reducing cofilin oligomer formation to promote the dissolution of actin stress fibers.

The destruction of pulmonary epithelium is a major feature of lung diseases caused by the fungal pathogen Aspergillus fumigatus (A. fumigatus). Gliotoxin, a major mycotoxin of A. fumigatus, is widely postulated to be associated with the tissue invasion. However, the mechanism is unclear. In this study, we first discovered that cofilin, a regulator of actin dynamics in the pulmonary epithelial cells, existed mainly in the form of oligomer, which kept it unable to depolymerize actin filaments. Gliotoxin could reduce the formation of cofilin oligomer and promote the release of active cofilin monomer by regulating cofilin phosphorylation balance. Then, the active cofilin induced the dissolution of actin stress fibers to result in the disruption of pulmonary epithelium barrier function. Collectively, our study revealed a novel mechanism of gliotoxin destructing lung epithelium barrier function and for the first time indicated the role of cofilin oligomer in this process.

Gliotoxin penetrates and impairs the integrity of the human blood-brain barrier in vitro.

Cerebral fungal infections represent an important public health concern, where a key element of pathophysiology is the ability of the fungi to cross the blood-brain barrier (BBB). Yet the mechanism used by micro-organisms to cross such a barrier and invade the brain parenchyma remains unclear. This study investigated the effects of gliotoxin (GTX), a mycotoxin secreted by Aspergillus fumigatus, on the BBB using brain microvascular endothelial cells (BMECs) derived from induced pluripotent stem cells (iPSCs). We observed that both acute (2 h) and prolonged (24 h) exposure to GTX at the level of 1 µM or higher compromised BMECs monolayer integrity. Notably, acute exposure was sufficient to disrupt the barrier function in iPSC-derived BMECs, resulting in decreased transendothelial electrical resistance (TEER) and increased fluorescein permeability. Further, our data suggest that such disruption occurred without affecting tight junction complexes, via alteration of cell-matrix interactions, alterations in F-actin distribution, through a protein kinase C-independent signaling. In addition to its effect on the barrier function, we have observed a low permeability of GTX across the BBB. This fact can be partially explained by possible interactions of GTX with membrane proteins. Taken together, this study suggests that GTX may contribute in cerebral invasion processes of Aspergillus fumigatus by altering the blood-brain barrier integrity without disrupting tight junction complexes.

Impact of prenatal immune challenge on the demyelination injury during adulthood.

AIM: Brain inflammation is associated with several brain diseases such as multiple sclerosis (MS), a disease characterized by demyelination. Whether prenatal immune challenge affects demyelination-induced inflammation in the white matter during adulthood is unclear. In the present study, we used a well-established experimental model of focal demyelination to assess whether prenatal immune challenge affects demyelination-induced inflammation. METHODS: Pregnant rats were injected with either lipopolysaccharide (100 µg/kg, ip) or pyrogen-free saline. A 2 µL solution of the gliotoxin ethidium bromide (0.04%) was stereotaxically infused into the corpus callosum of adult male offspring. The extent of demyelination lesion was assessed using Luxol fast blue (LFB) staining. Oligodendrocyte precursor cells, mature oligodendrocytes, markers of cellular gliosis, and inflammation were monitored in the vicinity of the demyelination lesion area. RESULTS: Prenatal lipopolysaccharide reduced the size of the demyelination lesion during adulthood. This reduced lesion was associated with enhanced density of mature oligodendrocytes and reduced density of microglial cells in the vicinity of the demyelination lesion. Such reduction in microglial cell density was accompanied by a reduced activation of the nuclear factor κB signaling pathway. CONCLUSION: These data strongly suggest that prenatal immune challenge dampens the extent of demyelination during adulthood likely by reprogramming the local brain inflammatory response to demyelinating insults.

Impact of Mycotoxins Secreted by Aspergillus Molds on the Inflammatory Response of Human Corneal Epithelial Cells.

Exposure to molds and mycotoxins not only contributes to the onset of respiratory disease, it also affects the ocular surface. Very few published studies concern the evaluation of the effect of mycotoxin exposure on ocular cells. The present study investigates the effects of aflatoxin B1 (AFB1) and gliotoxin, two mycotoxins secreted by Aspergillus molds, on the biological activity of the human corneal epithelial (HCE) cells. After 24, 48, and 72 h of exposure, cellular viability and inflammatory response were assessed. Both endpoint cell viability colorimetric assays and continuous cell impedance measurements, providing noninvasive real-time assessment of the effect on cells, were performed. Cytokine gene expression and interleukin-8 release were quantified. Gliotoxin appeared more cytotoxic than AFB1 but, at the same time, led to a lower increase of the inflammatory response reflecting its immunosuppressive properties. Real-time cell impedance measurement showed a distinct profile of cytotoxicity for both mycotoxins. HCE cells appeared to be a well-suited in vitro model to study ocular surface reactivity following biological contaminant exposure. Low, but persistent inflammation, caused by environmental factors, such as fungal toxins, leads to irritation and sensitization, and could be responsible for allergic manifestations which, in turn, could lead to mucosal hyper-reactivity.

Propentofylline reduces glial scar development following gliotoxic damage in the rat brainstem.

OBJECTIVE: This study aimed to evaluate the effect of propentofylline administration on astrocytic response following gliotoxic injury. METHOD: Wistar rats were injected with ethidium bromide into the cisterna pontis and treated or not with propentofylline (12.5mg/kg/day, intraperitoneal) during the experimental period. Brainstem sections were collected from 15 to 31 days after gliotoxic injection and processed for GFAP immunohistochemistry. RESULTS AND CONCLUSION: Results demonstrate that propentofylline decreased astrocytic activation until the 21st day, suggesting that this drug may have a role in reducing glial scar development following injury.

Propentofylline reduces glial scar development following gliotoxic damage in the rat brainstem / Propentofilina reduz o desenvolvimento da cicatriz glial após dano gliotóxico no tronco encefálico de ratos

ABSTRACT Propentofylline is a xanthine derivative that depresses activation of glial cells, whose responses contribute to neural tissue damage during inflammation. Ethidium bromide injection into the central nervous system induces local oligodendroglial and astrocytic loss, resulting in primary demyelination, neuroinflammation and blood-brain barrier disruption. Surviving astrocytes present a vigorous reaction around the injury site with increased immunoreactivity to glial fibrillary acidic protein (GFAP). Objective This study aimed to evaluate the effect of propentofylline administration on astrocytic response following gliotoxic injury. Method Wistar rats were injected with ethidium bromide into the cisterna pontis and treated or not with propentofylline (12.5mg/kg/day, intraperitoneal) during the experimental period. Brainstem sections were collected from 15 to 31 days after gliotoxic injection and processed for GFAP immunohistochemistry. Results and Conclusion Results demonstrate that propentofylline decreased astrocytic activation until the 21st day, suggesting that this drug may have a role in reducing glial scar development following injury.

RESUMO A propentofilina é uma xantina que deprime a ativação das células gliais, cujas respostas contribuem para o dano neural durante inflamação. A injeção de brometo de etídio no sistema nervoso central induz a perda oligodendroglial e astrocitária, resultando em desmielinização, neuroinflamação e ruptura da barreira hematoencefálica. Os astrócitos sobreviventes apresentam vigorosa reação ao redor da lesão com aumento da imunorreatividade à proteína glial fibrilar ácida (GFAP). Objetivo Este estudo objetivou avaliar o efeito da propentofilina sobre a resposta astrocitária após injúria gliotóxica. Método Ratos Wistar foram injetados com brometo de etídio na cisterna basal e tratados ou não com propentofilina (12.5mg/kg/dia, intraperitoneal). Amostras do tronco encefálico foram coletadas dos 15 aos 31 dias pós-injeção do gliotóxico e processadas para estudo ultraestrutural e imuno-histoquímico para GFAP. Resultados e Conclusão Os resultados demonstram que a propentofilina reduziu a ativação astrocitária até o 21o dia, sugerindo que essa droga pode atuar na redução da cicatriz glial após injúria.

Gliotoxin Suppresses Macrophage Immune Function by Subverting Phosphatidylinositol 3,4,5-Trisphosphate Homeostasis.

UNLABELLED: Aspergillus fumigatus, an opportunistic fungal pathogen, spreads in the environment by releasing numerous conidia that are capable of reaching the small alveolar airways of mammalian hosts. In otherwise healthy individuals, macrophages are responsible for rapidly phagocytosing and eliminating these conidia, effectively curbing their germination and consequent invasion of pulmonary tissue. However, under some circumstances, the fungus evades phagocyte-mediated immunity and persists in the respiratory tree. Here, we report thatA. fumigatusescapes macrophage recognition by strategically targeting phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] metabolism through gliotoxin, a potent immunosuppressive mycotoxin. Time-lapse microscopy revealed that, in response to the toxin, macrophages cease to ruffle, undergo abrupt membrane retraction, and fail to phagocytose large targets effectively. Gliotoxin was found to prevent integrin activation and interfere with actin dynamics, both of which are instrumental for phagocytosis; similar effects were noted in immortalized and primary phagocytes. Detailed studies of the underlying molecular mechanisms of toxicity revealed that inhibition of phagocytosis is attributable to impaired accumulation of PtdIns(3,4,5)P3and the associated dysregulation of downstream effectors, including Rac and/or Cdc42. Strikingly, in response to the diacylglycerol mimetic phorbol 12-myristate 13-acetate, gliotoxin-treated macrophages reactivate beta integrins, reestablish actin dynamics, and regain phagocytic capacity, despite the overt absence of plasmalemmal PtdIns(3,4,5)P3 Together, our findings identify phosphoinositide metabolism as a critical upstream target of gliotoxin and also indicate that increased diacylglycerol levels can bypass the requirement for PtdIns(3,4,5)P3signaling during membrane ruffling and phagocytosis. IMPORTANCE: Aspergillus fumigatusis the most frequent cause of human infections in theAspergillusgenus. In immunocompromised populations, invasive aspergillosis (IA) is associated with a mortality rate of up to 90%, and current antifungal therapies have failed to prevent or reverse the infection. Therefore, a deeper understanding of the interactions betweenA. fumigatusand its host is required. In healthy humans, alveolar macrophages can ingest and eliminate fungal spores, thus limiting their germination into mycotoxin-producing hyphae. Our studies reveal that gliotoxin-the most abundantAspergillusmycotoxin-undermines the ability of phagocytes to carry out their protective functions. By targeting PtdIns(3,4,5)P3signaling and downregulating phagocytic immune defenses, the toxin could also exacerbate polymicrobial infections. Notably, we were able to reverse gliotoxin toxicity by addition of diacylglycerol analogues, which may provide the basis for therapeutic interventions.

Interplay between Gliotoxin Resistance, Secretion, and the Methyl/Methionine Cycle in Aspergillus fumigatus.

Mechanistic studies on gliotoxin biosynthesis and self-protection in Aspergillus fumigatus, both of which require the gliotoxin oxidoreductase GliT, have revealed a rich landscape of highly novel biochemistries, yet key aspects of this complex molecular architecture remain obscure. Here we show that an A. fumigatus ΔgliA strain is completely deficient in gliotoxin secretion but still retains the ability to efflux bisdethiobis(methylthio)gliotoxin (BmGT). This correlates with a significant increase in sensitivity to exogenous gliotoxin because gliotoxin trapped inside the cell leads to (i) activation of the gli cluster, as disabling gli cluster activation, via gliZ deletion, attenuates the sensitivity of an A. fumigatus ΔgliT strain to gliotoxin, thus implicating cluster activation as a factor in gliotoxin sensitivity, and (ii) increased methylation activity due to excess substrate (dithiol gliotoxin) for the gliotoxin bis-thiomethyltransferase GtmA. Intracellular dithiol gliotoxin is oxidized by GliT and subsequently effluxed by GliA. In the absence of GliA, gliotoxin persists in the cell and is converted to BmGT, with levels significantly higher than those in the wild type. Similarly, in the ΔgliT strain, gliotoxin oxidation is impeded, and methylation occurs unchecked, leading to significant S-adenosylmethionine (SAM) depletion and S-adenosylhomocysteine (SAH) overproduction. This in turn significantly contributes to the observed hypersensitivity of gliT-deficient A. fumigatus to gliotoxin. Our observations reveal a key role for GliT in preventing dysregulation of the methyl/methionine cycle to control intracellular SAM and SAH homeostasis during gliotoxin biosynthesis and exposure. Moreover, we reveal attenuated GliT abundance in the A. fumigatus ΔgliK strain, but not the ΔgliG strain, following exposure to gliotoxin, correlating with relative sensitivities. Overall, we illuminate new systems interactions that have evolved in gliotoxin-producing, compared to gliotoxin-naive, fungi to facilitate their cellular presence.

RNA-seq reveals the pan-transcriptomic impact of attenuating the gliotoxin self-protection mechanism in Aspergillus fumigatus.

BACKGROUND: Aspergillus fumigatus produces a number of secondary metabolites, one of which, gliotoxin, has been shown to exhibit anti-fungal activity. Thus, A. fumigatus must be able to protect itself against gliotoxin. Indeed one of the genes in the gliotoxin biosynthetic gene cluster in A. fumigatus, gliT, is required for self-protection against the toxin- however the global self-protection mechanism deployed is unclear. RNA-seq was employed to identify genes differentially regulated upon exposure to gliotoxin in A. fumigatus wild-type and A. fumigatus ∆gliT, a strain that is hypersensitive to gliotoxin. RESULTS: Deletion of A. fumigatus gliT resulted in altered expression of 208 genes (log2 fold change of 1.5) when compared to A. fumigatus wild-type, of which 175 genes were up-regulated and 33 genes were down-regulated. Expression of 164 genes was differentially regulated (log2 fold change of 1.5) in A. fumigatus wild-type when exposed to gliotoxin, consisting of 101 genes with up-regulated expression and 63 genes with down-regulated expression. Interestingly, a much larger number of genes, 1700, were found to be differentially regulated (log2 fold change of 1.5) in A. fumigatus ∆gliT when challenged with gliotoxin. These consisted of 508 genes with up-regulated expression, and 1192 genes with down-regulated expression. Functional Catalogue (FunCat) classification of differentially regulated genes revealed an enrichment of genes involved in both primary metabolic functions and secondary metabolism. Specifically, genes involved in gliotoxin biosynthesis, helvolic acid biosynthesis, siderophore-iron transport genes and also nitrogen metabolism genes and ribosome biogenesis genes underwent altered expression. It was confirmed that gliotoxin biosynthesis is induced upon exposure to exogenous gliotoxin, production of unrelated secondary metabolites is attenuated in A. fumigatus ∆gliT, while quantitative proteomic analysis confirmed disrupted translation in A. fumigatus ∆gliT challenged with exogenous gliotoxin. CONCLUSIONS: This study presents the first global investigation of the transcriptional response to exogenous gliotoxin in A. fumigatus wild-type and the hyper-sensitive strain, ∆gliT. Our data highlight the global and extensive affects of exogenous gliotoxin on a sensitive strain devoid of a self-protection mechanism and infer that GliT functionality is required for the optimal biosynthesis of selected secondary metabolites in A. fumigatus.

Opposed effects of enzymatic gliotoxin N- and S-methylations.

Gliotoxin (1), a virulence factor of the human pathogenic fungus Aspergillus fumigatus, is the prototype of epipoly(thiodioxopiperazine) (ETP) toxins. Here we report the discovery and functional analysis of two methyl transferases (MTs) that play crucial roles for ETP toxicity. Genome comparisons, knockouts, and in vitro enzyme studies identified a new S-adenosyl-l-methionine-dependent S-MT (TmtA) that is, surprisingly, encoded outside the gli gene cluster. We found that TmtA irreversibly inactivates ETP by S-alkylation and that this detoxification strategy appears to be not only limited to ETP producers. Furthermore, we unveiled that GliN functions as a freestanding amide N-MT. GliN-mediated amide methylation confers stability to ETP, damping the spontaneous formation of tri- and tetrasulfides. In addition, enzymatic N-alkylation constitutes the last step in gliotoxin biosynthesis and is a prerequisite for the cytotoxicity of the molecule. Thus, these specialized alkylating enzymes have dramatic and fully opposed effects: complete activation or inactivation of the toxin.

Gliotoxin-induced swelling of astrocytes hinders diffusion in brain extracellular space via formation of dead-space microdomains.

One of the hallmarks of numerous life-threatening and debilitating brain diseases is cellular swelling that negatively impacts extracellular space (ECS) structure. The ECS structure is determined by two macroscopic parameters, namely tortuosity (λ) and volume fraction (α). Tortuosity represents hindrance imposed on the diffusing molecules by the tissue in comparison with an obstacle-free medium. Volume fraction is the proportion of tissue volume occupied by the ECS. From a clinical perspective, it is essential to recognize which factors determine the ECS parameters and how these factors change in brain diseases. Previous studies demonstrated that dead-space (DS) microdomains increased λ during ischemia and hypotonic stress, as these pocket-like structures transiently trapped diffusing molecules. We hypothesize that astrocytes play a key role in the formation of DS microdomains because their thin processes have concave shapes that may elongate as astrocytes swell in these pathologies. Here we selectively swelled astrocytes in the somatosensory neocortex of rat brain slices with a gliotoxin DL-α-Aminoadipic Acid (DL-AA), and we quantified the ECS parameters using Integrative Optical Imaging (IOI) and Real-Time Iontophoretic (RTI) diffusion methods. We found that α decreased and λ increased during DL-AA application. During recovery, α was restored whereas λ remained elevated. Increase in λ during astrocytic swelling and recovery is consistent with the formation of DS microdomains. Our data attribute to the astrocytes an important role in determining the ECS parameters, and indicate that extracellular diffusion can be improved not only by reducing the swelling but also by disrupting the DS microdomains.

Individual and combined effects of mycotoxins from typical indoor moulds.

The mycotoxins patulin, gliotoxin and sterigmatocystin can be produced by common indoor moulds and enter the human body via inhalation of mycotoxin containing spores and particulates. There are various studies about the individual effects of these mycotoxins, but a lack of knowledge about their effects in mixtures. The aim of this study was to evaluate combined effects on the singe celled organism Tetrahymena pyriformis. Using the MIXTOX model (EU project NOMIRACLE) synergistic or antagonistic effects with dose level deviation or dose ratio dependent deviation were analyzed. The most toxic compound gliotoxin (EC50 0.37 µM) and patulin (EC50 9.3 µM) as shown by the MIXTOX model acted synergistic, caused by similar mode of actions. Within the combination with sterigmatocystin (maximum inhibition of 45% at 12.5 µM) antagonistic effects were observed with switch to synergism if the toxicity of the mixture is mainly caused by sterigmatocystin. In the end the MIXTOX model was applicable for the prediction of combined effects of toxic compounds.

Gliotoxin-induced changes in rat liver regeneration after partial hepatectomy.

BACKGROUND: Hepatic non-parenchymal cells (NPCs), encompassing hepatic stellate cells (HSCs), macrophages and endothelial cells, synthesize new hepatocyte growth factor (HGF) during liver regeneration (LR), and also play an important function in matrix production at the end of regeneration. AIMS: The aim of this study was to determine whether ablating NPCs either during hepatocyte proliferation or during matrix resynthesis will have any effect on LR. METHODS: Rats were injected with either gliotoxin (which induces NPC apoptosis) or vehicle control at various stages during partial hepatectomy (PH). NPCs and hepatocytes were also treated in vitro with gliotoxin. RESULTS: Proliferating cells were abundant in control livers 24 h after PH, while in gliotoxin-treated rats, mitosis was absent, apoptotic NPCs were apparent and HGF was decreased. In vitro studies demonstrated a > 50% decrease in cell viability in NPC cultures, while hepatocyte viability and proliferation were unaffected. Chronic elimination of NPCs over a period of 5 days after PH led to increased desmin-positive HSCs and fewer alpha smooth muscle actin-expressing HSCs. Finally, there was continued proliferation of hepatocytes and decreased collagen I and TGF-ß when HSCs, the matrix-producing NPCs, were ablated during later stages of LR. CONCLUSIONS: Ablation of NPCs at early time points after PH interferes with liver regeneration, while their ablation at late stages causes impairment in the termination of LR, demonstrating a time-dependent regulatory role of NPCs in the regenerative process.

Identity of endogenous NMDAR glycine site agonist in amygdala is determined by synaptic activity level.

Mechanisms of N-methyl-D-aspartate receptor-dependent synaptic plasticity contribute to the acquisition and retention of conditioned fear memory. However, synaptic rules which may determine the extent of N-methyl-D-aspartate receptor activation in the amygdala, a key structure implicated in fear learning, remain unknown. Here we show that the identity of the N-methyl-D-aspartate receptor glycine site agonist at synapses in the lateral nucleus of the amygdala may depend on the level of synaptic activation. Tonic activation of N-methyl-D-aspartate receptors at synapses in the amygdala under low activity conditions is supported by ambient D-serine, whereas glycine may be released from astrocytes in response to afferent impulses. The release of glycine may decode the increases in afferent activity levels into enhanced N-methyl-D-aspartate receptor-mediated synaptic events, serving an essential function in the induction of N-methyl-D-aspartate receptor-dependent long-term potentiation in fear conditioning pathways.

VeA regulates conidiation, gliotoxin production, and protease activity in the opportunistic human pathogen Aspergillus fumigatus.

Invasive aspergillosis by Aspergillus fumigatus is a leading cause of infection-related mortality in immunocompromised patients. In this study, we show that veA, a major conserved regulatory gene that is unique to fungi, is necessary for normal morphogenesis in this medically relevant fungus. Although deletion of veA results in a strain with reduced conidiation, overexpression of this gene further reduced conidial production, indicating that veA has a major role as a regulator of development in A. fumigatus and that normal conidiation is only sustained in the presence of wild-type VeA levels. Furthermore, our studies revealed that veA is a positive regulator in the production of gliotoxin, a secondary metabolite known to be a virulent factor in A. fumigatus. Deletion of veA resulted in a reduction of gliotoxin production with respect to that of the wild-type control. This reduction in toxin coincided with a decrease in gliZ and gliP expression, which is necessary for gliotoxin biosynthesis. Interestingly, veA also influences protease activity in this organism. Specifically, deletion of veA resulted in a reduction of protease activity; this is the first report of a veA homolog with a role in controlling fungal hydrolytic activity. Although veA affects several cellular processes in A. fumigatus, pathogenicity studies in a neutropenic mouse infection model indicated that veA is dispensable for virulence.