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The risk of idiosyncratic drug-induced agranulocytosis (IDIAG) markedly constrains the use of clozapine, a neuroleptic with unparalleled efficacy. Most clozapine patients experience an early inflammatory response, likely a necessary step in IDIAG onset. However, most patients do not progress to IDIAG, presumably because of the requirement of specific human leukocyte antigen (HLA) haplotypes, T cell receptors, and other unknown factors. We established that clozapine activates inflammasomes and that myeloperoxidase bioactivation of clozapine generates neoantigens, but the connection between these early mechanistic events remained unknown and, thus, was the aim of this work. We found that the myeloperoxidase inhibitor PF-1355 attenuated myeloperoxidase activity in phorbol myristate acetate (PMA)-differentiated THP-1 macrophages, and it also attenuated clozapine-induced release of inflammatory mediators (e.g., IL-1β, CXCL1, and C-reactive protein). In vivo, pretreatment of Sprague Dawley rats with PF-1355 significantly attenuated clozapine-induced increases in neutrophil mobilization from the bone marrow to the blood and spleen, as determined using differential blood counts and flow cytometry. Moreover, the clozapine-triggered release of inflammatory mediators (e.g., IL-1β, calprotectin, CXCL1, and α-1-acid glycoprotein) from the liver, spleen, and bone marrow was dampened by myeloperoxidase inhibition. These data support the working hypothesis that oxidation of clozapine to a reactive metabolite by myeloperoxidase is critical for induction of the inflammatory response to clozapine. Ultimately, a better mechanistic understanding of the early events involved in the immune response to clozapine may elucidate ways to prevent IDIAG, enabling safer, more frequent therapeutic use of this and potentially other highly efficacious drugs. [ABSTRACT FROM AUTHOR]

The risk of idiosyncratic drug-induced agranulocytosis (IDIAG) markedly constrains the use of clozapine, a neuroleptic with unparalleled efficacy. Most clozapine patients experience an early inflammatory response, likely a necessary step in IDIAG onset. However, most patients do not progress to IDIAG, presumably because of the requirement of specific human leukocyte antigen (HLA) haplotypes, T cell receptors, and other unknown factors. We established that clozapine activates inflammasomes and that myeloperoxidase bioactivation of clozapine generates neoantigens, but the connection between these early mechanistic events remained unknown and, thus, was the aim of this work. We found that the myeloperoxidase inhibitor PF-1355 attenuated myeloperoxidase activity in phorbol myristate acetate (PMA)-differentiated THP-1 macrophages, and it also attenuated clozapine-induced release of inflammatory mediators (e.g., IL-1β, CXCL1, and C-reactive protein). In vivo, pretreatment of Sprague Dawley rats with PF-1355 significantly attenuated clozapine-induced increases in neutrophil mobilization from the bone marrow to the blood and spleen, as determined using differential blood counts and flow cytometry. Moreover, the clozapine-triggered release of inflammatory mediators (e.g., IL-1β, calprotectin, CXCL1, and α-1-acid glycoprotein) from the liver, spleen, and bone marrow was dampened by myeloperoxidase inhibition. These data support the working hypothesis that oxidation of clozapine to a reactive metabolite by myeloperoxidase is critical for induction of the inflammatory response to clozapine. Ultimately, a better mechanistic understanding of the early events involved in the immune response to clozapine may elucidate ways to prevent IDIAG, enabling safer, more frequent therapeutic use of this and potentially other highly efficacious drugs.

Dietary supplementation with polyunsaturated fatty acids (PUFA) n-3 can affect cutaneous wound healing; however, recent findings demonstrate the variable extent of their influence on the quality of healing. Here, we compare the effect of several dietary oils, containing different levels of PUFA n-3 and PUFA n-6, on wound healing in the rat model. Rats were fed the feed mixture with 8% palm oil (P), safflower oil (S), fish oil (F) or Schizochytrium microalga extract (Sch) and compared to the animals fed by control feed mixture (C). Dorsal full-thickness cutaneous excisions were performed after 52 days of feeding and skin was left to heal for an additional 12 days. Histopathological analysis of skin wounds was performed, including immune cells immunolabeling and the determination of hydroxyproline amount as well as gene expression analyses of molecules contributing to different steps of the healing. Matrix-assisted-laser-desorption-ionization mass-spectrometry-imaging (MALDI-MSI) was used to determine the amount of collagen α-1(III) chain fragment in healing samples. Treatment by Schizochytrium extract resulted in decrease in the total wound area, in contrast to the safflower oil group where the size of the wound was larger when comparing to control animals. Diet with Schizochytrium extract and safflower oils displayed a tendency to increase the number of new vessels. The number of MPO-positive cells was diminished following any of oil treatment in comparison to the control, but their highest amount was found in animals with a fish oil diet. On the other hand, the number of CD68-positive macrophages was increased, with the most significant enhancement in the fish oil and safflower oil group. Hydroxyproline concentration was the highest in the safflower oil group but it was also enhanced in all other analyzed treatments in comparison to the control. MALDI-MSI signal intensity of a collagen III fragment decreased in the sequence C > S > Sch > P > F treatment. In conclusion, we observed differences in tissue response during healing between dietary oils, with the activation of inflammation observed following the treatment with oil containing high eicosapentaenoic acid (EPA) level (fish oil) and enhanced healing features were induced by the diet with high content of docosahexaenoic acid (DHA, Schizochytrium extract).

N-acetylcysteine (NAC), often used as an antioxidant-scavenging reactive oxygen species (ROS) in vitro, was recently shown to increase the cytotoxicity of other compounds through ROS-dependent and ROS-independent mechanisms. In this study, NAC itself was found to induce extensive ROS production in human leukemia HL-60 and U937 cells. The cytotoxicity depends on ROS-modulating enzyme expression. In HL-60 cells, NAC activated NOX2 to produce superoxide (O2•−). Its subsequent conversion into H2O2 by superoxide dismutase 1 and 3 (SOD1, SOD3) and production of ClO− from H2O2 by myeloperoxidase (MPO) was necessary for cell death induction. While the addition of extracellular SOD potentiated NAC-induced cell death, extracellular catalase (CAT) prevented cell death in HL-60 cells. The MPO inhibitor partially reduced the number of dying HL-60 cells. In U937 cells, the weak cytotoxicity of NAC is probably caused by lower expression of NOX2, SOD1, SOD3, and by the absence of MOP expression. However, even here, the addition of extracellular SOD induced cell death in U937 cells, and this effect could be reversed by extracellular CAT. NAC-induced cell death exhibited predominantly apoptotic features in both cell lines. Conclusions: NAC itself can induce extensive production of O2•− in HL-60 and U937 cell lines. The fate of the cells then depends on the expression of enzymes that control the formation and conversion of ROS: NOX, SOD, and MPO. The mode of cell death in response to NAC treatment bears apoptotic and apoptotic-like features in both cell lines. [ABSTRACT FROM AUTHOR]

Dietary supplementation with polyunsaturated fatty acids (PUFA) n-3 can affect cutaneous wound healing; however, recent findings demonstrate the variable extent of their influence on the quality of healing. Here, we compare the effect of several dietary oils, containing different levels of PUFA n-3 and PUFA n-6, on wound healing in the rat model. Rats were fed the feed mixture with 8% palm oil (P), safflower oil (S), fish oil (F) or Schizochytrium microalga extract (Sch) and compared to the animals fed by control feed mixture (C). Dorsal full-thickness cutaneous excisions were performed after 52 days of feeding and skin was left to heal for an additional 12 days. Histopathological analysis of skin wounds was performed, including immune cells immunolabeling and the determination of hydroxyproline amount as well as gene expression analyses of molecules contributing to different steps of the healing. Matrix-assisted-laser-desorption-ionization mass-spectrometry-imaging (MALDI-MSI) was used to determine the amount of collagen α-1(III) chain fragment in healing samples. Treatment by Schizochytrium extract resulted in decrease in the total wound area, in contrast to the safflower oil group where the size of the wound was larger when comparing to control animals. Diet with Schizochytrium extract and safflower oils displayed a tendency to increase the number of new vessels. The number of MPO-positive cells was diminished following any of oil treatment in comparison to the control, but their highest amount was found in animals with a fish oil diet. On the other hand, the number of CD68-positive macrophages was increased, with the most significant enhancement in the fish oil and safflower oil group. Hydroxyproline concentration was the highest in the safflower oil group but it was also enhanced in all other analyzed treatments in comparison to the control. MALDI-MSI signal intensity of a collagen III fragment decreased in the sequence C > S > Sch > P > F treatment. In conclusion, we observed differences in tissue response during healing between dietary oils, with the activation of inflammation observed following the treatment with oil containing high eicosapentaenoic acid (EPA) level (fish oil) and enhanced healing features were induced by the diet with high content of docosahexaenoic acid (DHA, Schizochytrium extract). [ABSTRACT FROM AUTHOR]

The aim of this study is to examine the use of an inflammasome competitor as a preventative agent. Coronaviruses have zoonotic potential due to the adaptability of their S protein to bind receptors of other species, most notably demonstrated by SARS-CoV. The binding of SARS-CoV-2 to TLR (Toll-like receptor) causes the release of pro-IL-1β, which is cleaved by caspase-1, followed by the formation and activation of the inflammasome, which is a mediator of lung inflammation, fever, and fibrosis. The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome is implicated in a variety of human diseases including Alzheimer's disease (AD), prion diseases, type 2 diabetes, and numerous infectious diseases. By examining the use of 4,4′-diaminodiphenyl sulfone (DDS) in the treatment of patients with Hansen's disease, also diagnosed as Alzheimer's disease, this study demonstrates the diverse mechanisms involved in the activation of inflammasomes. TLRs, due to genetic polymorphisms, can alter the immune response to a wide variety of microbial ligands, including viruses. In particular, TLR2Arg677Trp was reported to be exclusively present in Korean patients with lepromatous leprosy (LL). Previously, mutation of the intracellular domain of TLR2 has demonstrated its role in determining the susceptibility to LL, though LL was successfully treated using a combination of DDS with rifampicin and clofazimine. Of the three tested antibiotics, DDS was effective in the molecular regulation of NLRP3 inflammasome activators that are important in mild cognitive impairment (MCI), Parkinson's disease (PD), and AD. The specific targeting of NLRP3 itself or up-/downstream factors of the NLRP3 inflammasome by DDS may be responsible for its observed preventive effects, functioning as a competitor. [ABSTRACT FROM AUTHOR]

Gout is an inflammatory disease caused by monosodium urate (MSU) crystals. The role of neutrophils in gout is less clear, although several studies have shown neutrophil extracellular trap (NET) formation in acutely inflamed joints of gout patients. MSU crystals are known to induce the production of reactive oxygen species (ROS) and NET formation in neutrophils isolated from blood, but there is inconclusive knowledge on the localization of ROS production as well as whether the ROS are required for NET formation. In this report we demonstrate that MSU crystals activate human neutrophils to produce ROS exclusively in intracellular compartments. Additionally, in vivo transmigrated neutrophils derived from experimental skin chambers displayed markedly increased ROS production as compared to resting blood neutrophils. We also confirmed that MSU stimulation potently induced NET formation, but this response was not primed in in vivo transmigrated neutrophils. In line with this we found that MSU-triggered NET formation was independent of ROS production and proceeded normally in neutrophils from patients with dysfunctional respiratory burst (chronic granulomatous disease (CGD) and complete myeloperoxidase (MPO) deficiency). Our data indicate that in vivo transmigrated neutrophils are markedly primed for oxidative responses to MSU crystals and that MSU triggered NET formation is independent of ROS production. [ABSTRACT FROM AUTHOR]