Combined intervention with N-acetylcysteine and desipramine alleviated silicosis development by regulating the Nrf2/HO-1 and ASMase/ceramide signaling pathways.

Silicosis is a systemic disease characterized by diffuse fibrosis of the lung tissue caused by long-term inhalation of large amounts of free silica (SiO2) dust. The pathogenesis of silicosis has not been fully elucidated, and there is a lack of effective treatment methods. N-acetylcysteine (NAC) can potentially treat pulmonary fibrosis by exerting antioxidant effects. Desipramine (DMI) can influence pulmonary fibrosis development by inhibiting acid sphingomyelinase (ASMase) activity and regulating ceramide concentrations. Both can interfere with pulmonary fibrosis through different mechanisms, but the intervention effects of NAC combined with DMI on silicosis fibrosis have not been reported. Therefore, this study established a rat silicosis model using a single tracheal drip of SiO2 dust suspension in Wistar rats to investigate the effect of NAC combined with DMI on SiO2 dust-induced silicosis and its related molecular mechanisms. The histopathological examination of the SiO2 dust-induced silicosis rats suggested that NAC and DMI alone or in combination could decrease the severity of pulmonary fibrosis in rats. The combined intervention had a better effect on reducing fibrosis than the individual interventions. NAC and DMI, alone or in combination, decreased the levels of markers related to pulmonary fibrosis in rats (smooth muscle α-actin (α-SMA), collagen (Col) I, Col III, hydroxyproline (HYP), inflammatory factors (transforming growth factor-ß1 (TGF-ß1) and tumor necrosis factor-α (TNF-α)), and lipid peroxidase malondialdehyde (MDA)). The nuclear factor-erythroid 2-related factor 2 (Nrf2)/heme-oxygenase-1 (HO-1) and ASMase/ceramide pathways were inhibited to some extent by increasing the superoxide dismutase (SOD) levels of antioxidant enzymes and 8-iso-prostaglandin F2α (8-iso-PGF2α) levels of lipid peroxides. The combined intervention and NAC alone inhibited the SiO2 dust-induced elevation of matrix metalloproteinase 1 (MMP-1) and tissue inhibitor matrix metalloproteinase 1 (TIMP-1), but the effect was not significant in the DMI-treated group. Combining DMI and NAC inhibited Col I deposition and reduced HO-1, TIMP-1, and ASMase levels in lung tissues compared to individual treatments. In summary, the SiO2 dust could induce oxidative stress and inflammation in rats, resulting in an imbalance in extracellular matrix (ECM) synthesis/catabolism and ASMase/ceramide signaling pathway activation, leading to silicosis development.The combined intervention of DMI and NAC may synergistically regulate the Nrf2/HO-1 pathway, maintain the anabolic balance of the ECM, inhibit ASMase/ceramide signaling pathway activation by suppressing the inflammatory response and effectively delay silicosis fibrosis progression.

Cytotoxicity of the 42 kDa SMase C sphingomyelinase secreted by Leptospira interrogans serovar Pomona on Vero cells.

Sphingomyelinases produced by the pathogenic members of the genus Leptospira are implicated in the haemorrhagic manifestations seen in the severe form of leptospirosis. With multiple sphingomyelinase genes present in the genome of pathogenic Leptospira, much remains to be understood about these molecules. They include factors regulating their expression, post-translational modifications, and release of the biologically active forms of these molecules. In this study, serovar Pomona was chosen as it is reported to express high levels of sphingomyelinase that explained the haemolytic activity seen in experimental animals infected with this pathogen. Here, we demonstrate the cytotoxicity of a 42 kDa sphingomyelinase secreted by Leptospira interrogans serovar Pomona strain Pomona upon infecting Vero cells. This sphingomyelinase detected using specific anti-sphingomyelinase antibodies, exhibited haemolytic and sphingomyelinase activities that caused host-cell damage evident from the confocal images and scanning electron micrographs. The implications of these findings and the detection of a 42 kDa sphingomyelinase in the urine of human patients with leptospirosis in our earlier study is discussed with an emphasis on the potential of these sphingomyelinases as candidate markers for the early diagnosis of leptospirosis.

Nonclinical safety assessment of recombinant human acid sphingomyelinase (rhASM) for the treatment of acid sphingomyelinase deficiency:the utility of animal models of disease in the toxicological evaluation of potential therapeutics.

Recombinant human acid sphingomyelinase (rhASM) is being developed as an enzyme replacement therapy for patients with acid sphingomyelinase deficiency (Niemann-Pick disease types A and B), which causes sphingomyelin to accumulate in lysosomes. In the acid sphingomyelinase knock-out (ASMKO) mouse, intravenously administered rhASM reduced tissue sphingomyelin levels in a dose-dependent manner. When rhASM was administered to normal rats, mice, and dogs, no toxicity was observed up to a dose of 30mg/kg. However, high doses of rhASM≥10mg/kg administered to ASMKO mice resulted in unexpected toxicity characterized by cardiovascular shock, hepatic inflammation, adrenal hemorrhage, elevations in ceramide and cytokines (especially IL-6, G-CSF, and keratinocyte chemoattractant [KC]), and death. The toxicity could be completely prevented by the administration of several low doses (3mg/kg) of rhASM prior to single or repeated high doses (≥20mg/kg). These results suggest that the observed toxicity involves the rapid breakdown of large amounts of sphingomyelin into ceramide and/or other toxic downstream metabolites, which are known signaling molecules with cardiovascular and pro-inflammatory effects. Our results suggest that the nonclinical safety assessment of novel therapeutics should include the use of specific animal models of disease whenever feasible.

Inhibition of endothelial interleukin-8 production and neutrophil transmigration by Staphylococcus aureus beta-hemolysin.

Neutrophils play a crucial role in the host response to infection with Staphylococcus aureus, which is a major human pathogen capable of causing life-threatening disease. Interleukin-8 (IL-8) is a potent chemoattractant and activator of neutrophils. We previously reported that S. aureus secretes a factor that suppresses IL-8 production by human endothelial cells. Here we isolated an inhibitor of IL-8 production from the supernatant and identified it as staphylococcal beta-hemolysin. Beta-hemolysin reduced IL-8 production without cytotoxicity to endothelial cells. Pretreatment with beta-hemolysin decreased the expression of both IL-8 mRNA and protein induced by tumor necrosis factor alpha (TNF-alpha). Migration of neutrophils across TNF-alpha-activated endothelium was also inhibited by beta-hemolysin. In contrast, beta-hemolysin had no effect on intercellular adhesive molecule 1 expression in activated endothelial cells. These results showed that beta-hemolysin produced by S. aureus interferes with inflammatory signaling in endothelial cells and may help S. aureus evade the host immune response.

Identification of factors contributing to T-cell toxicity of Staphylococcus aureus clinical isolates.

We examined the ability of 206 clinical isolates of Staphylococcus aureus to lyse T cells and found differences between Agr groups. We found that the beta and delta hemolysins are involved and that methicillin-resistant S. aureus strains are less toxic than methicillin-susceptible S. aureus strains.

Inhibition of CFTR Cl- channel function caused by enzymatic hydrolysis of sphingomyelin.

Numerous mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR, a Cl(-) channel) disrupt salt and fluid transport and lead to the formation of thick mucus in patients' airways. Obstruction by mucus predisposes CF patients to chronic infections and inflammation, which become gradually harder to control and eventually fatal. Aggressive antibiotic therapy and supportive measures have dramatically lengthened CF patients' lives. Here, we report that sphingomyelinases (SMase) from human respiratory pathogens strongly inhibit CFTR function. The hydrolysis of sphingomyelin by SMase makes it more difficult to activate CFTR by phosphorylation of its regulatory domain. By inhibiting CFTR currents, SMase-producing respiratory tract bacteria may not only aggravate pulmonary infection in some CF patients but may also elicit a condition, analogous to CFTR deficiency, in non-CF patients suffering from bacterial lung infection.

Tetracycline protects against dermonecrosis induced by Loxosceles spider venom.

Envenomation by spiders belonging to the Loxosceles genus (brown spider) often results in local dermonecrotic lesions. We have previously shown that Loxosceles sphingomyelinase D (SMase D), the venom component responsible for all the pathological effects, induced the expression of matrix metalloproteinases (MMPs) in rabbits and in human keratinocytic cells. We also showed that the SMase D-induced apoptosis and MMP expression of keratinocytes was inhibited by tetracyclines. We have further investigated the ability of tetracyclines to inhibit or prevent the dermonecrotic lesion induced by Loxosceles venom in vivo and in vitro models. Primary cultures of rabbit fibroblasts incubated with increasing concentrations of venom or SMase D showed a decrease in cell viability, which was prevented by tetracyclines. In vivo experiments showed that topical treatments with tetracycline of rabbits, inoculated with crude Loxosceles intermedia venom or recombinant SMase D, significantly reduced the progression of the dermonecrotic lesion. Furthermore, tetracyclines also reduced the expression of MMP-2 and prevented the induction of MMP-9. Our results suggest that tetracycline may be an effective therapeutic agent for the treatment of cutaneous loxoscelism.

Loxosceles sphingomyelinase induces complement-dependent dermonecrosis, neutrophil infiltration, and endogenous gelatinase expression.

Envenomation by the spider Loxosceles can result in dermonecrosis and severe ulceration. Our aim was to investigate the role of the complement system and of the endogenous metalloproteinases in the initiation of the pathology of dermonecrosis. Histological analysis of skin of rabbits injected with Loxosceles intermedia venom and purified or recombinant sphingomyelinases showed a large influx of neutrophils, concomitant with dissociation of the collagenous fibers in the dermis. Decomplementation, using cobra venom factor, largely prevented the influx of neutrophils, while influx of neutrophils was also reduced in genetically C6-deficient rabbits, suggesting roles for both C5a and the membrane attack complex in the induction of dermonecrosis. However, C-depletion and C6 deficiency did not prevent the haemorrhage and the collagen injury. Zymography analysis of skin extracts showed the induction of expression of the endogenous gelatinase MMP-9 in the skin of envenomated animals. Rabbit neutrophils contained high levels of MMP-9, expression of which was further increased after incubation with venom, suggesting that these cells may be a source of the MMP-9 found in the skin of envenomated animals. Furthermore, skin fibroblasts also secreted MMP-9 and MMP-2 upon incubation with venom, suggesting that locally produced MMPs can also contribute to proteolytic tissue destruction.

Analysis of the toxic potential of venom from Loxosceles adelaida, a Brazilian brown spider from karstic areas.

Loxosceles adelaida spiders (Araneae, Sicariidae) are found near and inside the caves in the Parque Estadual Turistico do Alto Ribeira (PETAR), Sao Paulo, Brazil, which are visited by thousands of tourists every year. Several Loxosceles species are a public health problem in many regions of the world, by causing severe dermonecrosis and/or complement dependent haemolysis upon envenomation. The aim of this study was to characterize the biochemical and biological properties of L. adelaida venom and evaluate the toxic potential of envenomation by this non-synanthropic Loxosceles species. The biological activities of the L. adelaida venom was compared to that of Loxosceles gaucho, a synanthropic species of medical importance in Brazil. L. adelaida venom showed a similar potential to induce haemolysis, dermonecrosis and lethality as L. gaucho venom. L. adelaida crude venom was purified, yielding a 31 kDa component endowed with haemolytic and dermonecrotic activities. In conclusion, we show here that the troglophile Loxosceles species, L. adelaida, commonly found in the complex of caves from PETAR, is potentially able to cause envenomation with the same gravity of those produced by synanthropic species.

Molecular cloning and expression of a functional dermonecrotic and haemolytic factor from Loxosceles laeta venom.

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement-dependent haemolysis. The aim of this study was to generate recombinant proteins from the Loxosceles spider gland to facilitate structural and functional studies in the mechanisms of loxoscelism. Using "Expressed Sequencing Tag" strategy of aleatory clones from, L. laeta venom gland cDNA library we have identified clones containing inserts coding for proteins with significant similarity with previously obtained N-terminus of sphingomyelinases from Loxosceles intermedia venom [1]. Clone H17 was expressed as a fusion protein containing a 6x His-tag at its N-terminus and yielded a 33kDa protein. The recombinant protein was endowed with all biological properties ascribed to the whole L. laeta venom and sphingomyelinases from L. intermedia, including dermonecrotic and complement-dependent haemolytic activities. Antiserum raised against the recombinant protein recognised a 32-kDa protein in crude L. laeta venom and was able to block the dermonecrotic reaction caused by whole L. laeta venom. This study demonstrates conclusively that the sphingomyelinase activity in the whole venom is responsible for the major pathological effects of Loxosceles spider envenomation.

SmcL, a novel membrane-damaging virulence factor in Listeria.

We describe here the fourth listerial membrane-damaging virulence factor, a sphingomyelinase C (SMase) that is produced specifically by the ruminant pathogen Listeria ivanovii. Its coding gene, smcL, is a monocistron expressed independently of PrfA. The smcL product, SmcL, is highly similar to the staphylococcal beta-toxin and is responsible for the differential hemolytic properties of L. ivanovii (bizonal hemolysis and CAMP-like reaction with R. equi). The role of SmcL in virulence was assessed by gene disruption and complementation. Our data show that SmcL mediates disruption of the membrane of primary phagosomes, thereby promoting bacterial intracellular proliferation. They also suggest that SmcL may play a role in host tropism. smcL is located in LIPI-2, a novel 18-kb pathogenicity island which also contains a cluster of internalin genes. LIPI-2 is unstable, L. ivanovii-specific and required for full virulence in mice and lambs.