Characterization of individual bile acids in vivo utilizing a novel low bile acid mouse model.

Bile acids (BAs) are signaling molecules synthesized in the liver initially by CYP7A1 and CYP27A1 in the classical and alternative pathways, respectively. BAs are essential for cholesterol clearance, intestinal absorption of lipids, and endogenous modulators of farnesoid x receptor (FXR). FXR is critical in maintaining BA homeostasis and gut-liver crosstalk. Complex reactions in vivo and the lack of suitable animal models impede our understanding of the functions of individual BAs. In this study, we characterized the in vivo effects of three-day feeding of cholic acid (CA), deoxycholic acid (DCA), or ursodeoxycholic acid (UDCA) at physiological/non-hepatotoxic concentrations in a novel low-BA mouse model (Cyp7a1  -/-/Cyp27a1  -/-, DKO). Liver injury, BA levels and composition and BA signaling by the FXR-fibroblast growth factor 15 (FGF15) axis were determined. Overall, higher basal inflammation and altered lipid metabolism in DKO mice might be associated with low BAs. CA, DCA and UDCA feeding activated FXR signals with tissue specificity. Dietary CA and DCA similarly altered tissue BA profiles to be less hydrophobic, while UDCA promoted a more hydrophobic tissue BA pool with the profiles shifted towards non-12α-OH BAs and secondary BAs. However, UDCA did not offer any overt protective effects as expected. These findings allow us to determine the precise effects of individual BAs in vivo on BA-FXR signaling and overall BA homeostasis in liver physiology and pathologies.

Skin Models Used to Define Mechanisms of Action of Sulfur Mustard.

Sulfur mustard (SM) is a threat to both civilian and military populations. Human skin is highly sensitive to SM, causing delayed erythema, edema, and inflammatory cell infiltration, followed by the appearance of large fluid-filled blisters. Skin wound repair is prolonged following blistering, which can result in impaired barrier function. Key to understanding the action of SM in the skin is the development of animal models that have a pathophysiology comparable to humans such that quantitative assessments of therapeutic drugs efficacy can be assessed. Two animal models, hairless guinea pigs and swine, are preferred to evaluate dermal products because their skin is morphologically similar to human skin. In these animal models, SM induces degradation of epidermal and dermal tissues but does not induce overt blistering, only microblistering. Mechanisms of wound healing are distinct in these animal models. Whereas a guinea pig heals by contraction, swine skin, like humans, heals by re-epithelialization. Mice, rats, and rabbits are also used for SM mechanistic studies. However, healing is also mediated by contraction; moreover, only microblistering is observed. Improvements in animal models are essential for the development of therapeutics to mitigate toxicity resulting from dermal exposure to SM.

Nurse managers' experience during the COVID-19 pandemic in China: A qualitative study.

AIMS: The aim of the study was to explore the experiences of female new nurse managers during the COVID-19 pandemic. DESIGN: This was a phenomenological study, and qualitative descriptive analysis was used. METHODS: New nurse managers were defined as new nurse managers with less than 3 years of management experience in this study. During November and December of 2021, 18 female new nurse managers were interviewed face-to-face with a semi-structured interview guide in three municipal hospitals. The study followed the Consolidated Criteria for Reporting Qualitative Research (COREQ) guidelines for evaluating qualitative research reports. Data analysis was performed using Colaizzi's seven-step method. RESULTS: Four main themes and 10 sub-themes were extracted from the collected data. The four major themes were as follows: (1) a shift in stress; (2) work-related physical and psychological discomfort; (3) reflection on the cause; (4) coping and struggles. CONCLUSIONS: New nurse managers were experiencing great stress and exhaustion in their roles. It is important that they are helped to handle situations. Providing them with readily accessible support, addressing their psychosocial needs and addressing exhaustion is necessary. Considering their short management time, the hospital should provide adequate support in human, financial and material areas and provide training to help new nurse managers better adapt to their new roles. In addition, nurse directors should create a culture of mutual respect, identify workplace bullying and create a harmonious and cooperation-oriented work environment for new nurse managers. PATIENT OR PUBLIC CONTRIBUTION: No patient or public contribution.

Sulfur mustard corneal injury is associated with alterations in the epithelial basement membrane and stromal extracellular matrix.

Sulfur mustard (SM; bis(2-chloroethyl) sulfide) is a highly reactive bifunctional alkylating agent synthesized for chemical warfare. The eyes are particularly sensitive to SM where it causes irritation, pain, photophobia, and blepharitis, depending on the dose and duration of exposure. In these studies, we examined the effects of SM vapor on the corneas of New Zealand white male rabbits. Edema and hazing of the cornea, signs of acute injury, were observed within one day of exposure to SM, followed by neovascularization, a sign of chronic or late phase pathology, which persisted for at least 28 days. Significant epithelial-stromal separation ranging from ~8-17% of the epithelial surface was observed. In the stroma, there was a marked increase in CD45+ leukocytes and a decrease of keratocytes, along with areas of disorganization of collagen fibers. SM also disrupted the corneal basement membrane and altered the expression of perlecan, a heparan sulfate proteoglycan, and cellular fibronectin, an extracellular matrix glycoprotein. This was associated with an increase in basement membrane matrix metalloproteinases including ADAM17, which is important in remodeling of the basement membrane during wound healing. Tenascin-C, an extracellular matrix glycoprotein, was also upregulated in the stroma 14-28 d post SM, a finding consistent with its role in organizing structural components of the stroma necessary for corneal transparency. These data demonstrate that SM vapor causes persistent alterations in structural components of the cornea. Further characterization of SM-induced injury in rabbit cornea will be useful for the identification of targets for the development of ocular countermeasures.

Characterization of the rabbit conjunctiva: Effects of sulfur mustard.

Sulfur mustard (SM; bis (2-chloroethyl) sulfide) is a potent vesicant which causes irritation of the conjunctiva and damage to the cornea. In the present studies, we characterized the ocular effects of SM in New Zealand white rabbits. Within one day of exposure to SM, edema and hazing of the cornea were observed, followed by neovascularization which persisted for at least 28 days. This was associated with upper and lower eyelid edema and conjunctival inflammation. The conjunctiva is composed of a proliferating epithelium largely consisting of stratified columnar epithelial cells overlying a well-defined dermis. Superficial layers of the conjunctival epithelium were found to express keratin 1, a marker of differentiating squamous epithelium, while in cells overlying the basement membrane expressed keratin 17, a marker of stratified squamous epithelium. SM exposure upregulated keratin 17 expression. Mucin 5 ac producing goblet cells were interspersed within the conjunctiva. These cells generated both acidic and neutral mucins. Increased numbers of goblet cells producing neutral mucins were evident after SM exposure; upregulation of expression of membrane-associated mucin 1 and mucin 4 in the superficial layers of the conjunctival epithelium were also noted. These data demonstrate that ocular exposure of rabbits to SM causes significant damage not only to the cornea, but to the eyelid and conjunctiva, suggesting multiple targets within the eye that should be assessed when evaluating the efficacy of potential countermeasures.

SM1997 downregulates mustard-induced enzymes that disrupt corneal epithelial attachment.

Amino-Plex (SM1997) is a spray or liquid cosmeceutical that has been used for skin dryness, aging, or sun exposure. Its formulation includes electrolytes, trace minerals, amino acids, peptides, nucleosides and nucleotides, all substances that are <10 kDa. It is designed to increase oxygen levels in cells, improve glucose transport, stimulate ATP synthesis, and stimulate collagen formation, actions that can help facilitate repair of damaged cells. It also supports collagen synthesis and formation of healthy granulation tissue, accelerating reepithelization of damaged skin. Here, SM1997 has been tested as an agent to improve the healing of mustard injury to the cornea. The results indicate that SM1997 facilitates the retention of corneal epithelial attachment when applied to corneal organ cultures after nitrogen mustard exposure. In addition, it reduces the activation of enzymes that lead to epithelial-stromal separation, namely, ADAM17 and MMP-9. Therefore, SM1997 should be further investigated as a potential therapy sulfur mustard and nitrogen mustard exposure.

Expression of Laminin γ2 Proteolytic Fragments in Murine Skin Following Exposure to Sulfur Mustard.

Laminin-332 is a basement membrane protein composed of three genetically distinct polypeptide chains that actively promote both skin epidermal cell adhesion and migration. Proteolytic fragments of the laminin γ2 chain stimulate migration and scattering of keratinocytes and cancer cells. Sulfur mustard (SM) is a bifunctional alkylating agent that induces separation of basal keratinocytes from the dermal-epidermal junction and invokes a strong inflammatory response leading to delayed wound repair. In the present studies, the role of laminin γ2 in SM-induced skin injury and wound repair was investigated using the mouse ear vesicant model. We found that laminin γ2 chain mRNA was preferentially upregulated in mouse ear skin exposed to SM. In situ hybridization confirmed overexpression of laminin γ2 transcript. Western blot analysis showed increased protein expression of the full-length proform of laminin γ2 and smaller processed fragments of laminin γ2 in skin exposed to SM. Dual immunofluorescence labeling indicated that laminin γ2 fragments are prevalent in suprabasal keratinocytes behind the leading edge in areas of hyperplasia in injured skin. In addition, co-expression of laminin γ2 and the senescent marker, p16-INK4a was found to overlap with the hyperplastic migratory epithelial sheet. This observation is similar to hypermotile keratinocytes reported in invasive carcinoma cells. Overall, our studies indicate that laminin γ2 is preferentially expressed in skin post SM exposure and that protein expression appears to become progressively more fragmented. The laminin γ2 fragments may play a role in regulating SM-induced skin wound repair. Anat Rec, 2020. © 2020 American Association for Anatomy.

Long non-coding RNA H19 regulates proliferation and doxorubicin resistance in MCF-7 cells by targeting PARP1.

Chemoresistance is a major obstacle to effective breast cancer chemotherapy. However, the underlying molecular mechanisms remain unclear. The long noncoding RNA H19 (H19) is involved in various stages of tumorigenesis, however, its role in doxorubicin resistance remains unknown. The goal of this study was to evaluate the role of H19 in the development of doxorubicin-resistant breast cancer. Quantitative real-time PCR (qRT-PCR) analyzed H19 expression in chemotherapy-resistant and sensitive breast cancer tissues. Both knockdown and overexpression of H19 were used to assess the sensitivity to doxorubicin in breast cancer cells in vitro and in vivo. qRT-PCR and Western blot were used to explore the doxorubicin resistance mechanism of H19. We observed that the H19 expression was significantly upregulated in chemotherapy-resistant breast cancer tissues and doxorubicin-resistant breast cancer cell lines. Knockdown of H19 enhanced the sensitivity to doxorubicin both in vitro and in vivo. While H19 overexpression developed doxorubicin-resistant in breast cancer cells both in vitro and in vivo. Furthermore, it was revealed that H19 negatively regulated PARP1 expression in breast cancer cells following doxorubicin treatment. Knockdown of H19 sensitized breast cancer cells to doxorubicin by promoting PARP1 upregulation. H19 overexpression could recapitulate doxorubicin resistance by PARP1 downregulation. Our findings revealed that H19 plays a leading role in breast cancer chemoresistance development, mediated mainly through a H19-PARP1 pathway.

An Engineered Human Fibroblast Growth Factor-1 Derivative, TTHX1114, Ameliorates Short-term Corneal Nitrogen Mustard Injury in Rabbit Organ Cultures.

Purpose: Organ cultures of rabbit corneas have been used to ascertain the effectiveness of a human fibroblast growth factor (FGF)-1 derivative (TTHX1114), lacking cysteine residues, to protect against and/or repair epithelial lesions following exposure to nitrogen mustard (NM). Methods: Rabbit corneas were exposed to NM and cultured for up to 14 days, with or without drug (TTHX1114). At specified times, tissue was examined by histopathology and graded by a novel composite scale. Proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) incorporation, and the expression of native FGF-1 and ADAM-17 after NM exposure was determined by immunofluorescence. Results: Rabbit corneas, exposed to a single dose of NM, showed a nearly complete loss of epithelial cells by day 6 but were significantly regenerated by day 14. When treated continuously with TTHX1114 following vesicant exposure, the losses remained at day 2 levels. The loss of keratocytes in the stroma was not affected by TTHX1114. EdU incorporation over the same time course showed a steady increase in tissue that had not been treated with TTHX1114, while corneas that were treated with the drug showed a higher percent incorporation initially, which then decreased, indicating the strong proliferative response to TTHX1114. ADAM-17 was not significantly altered by TTHX1114 treatment. Corneal epithelial FGF-1 disappeared after only 1 day following exposure to NM. Conclusions: TTHX1114 is protective against NM-induced damage of the corneal epithelium, possibly by supplying an NM-resistant source of trophic support and by stimulating regeneration of new epithelial cells. These responses underscore the potential value of TTHX1114 as an anti-vesicant therapeutic.

Lipopolysaccharides increase Kir2.1 expression in lung endothelial cells.

Kir2.1 is an inwardly rectifying K+ channel that modulates membrane potential. It is expressed widely in smooth muscle, neurons, and endothelial cells. Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe clinical syndromes, often causing the damage of epithelial and endothelial cells. Lipopolysaccharides (LPS) usually cause ALI/ARDS, directly or indirectly, and are used to reproduce the model in vivo. Here, we used differentially expressed gene analysis to find increasing Kir2.1 channel expression in human pulmonary microvascular endothelial cells cultured with LPS. Primary cultured mice pulmonary microvascular endothelial cells were verified by immunofluorescence. LPS incubation increased Kir2.1 channel expression in cultured mice pulmonary microvascular endothelial cells. A whole-cell voltage clamp was used to record the K+ current in cultured endothelial cells, showing increased whole-cell current in LPS treatment compared with controls. Additionally, the application of Ba2+, as an inhibitor of Kir2.1 channel, inhibited K+ current in both groups. We demonstrated that LPS may increase Kir2.1 channel expression in mice pulmonary microvascular endothelial cells to increase K+ flux, maintain hyperpolarization, and cause vasodilation, which may increase blood flow in pulmonary vessel bed, leading to pulmonary congestion contributing pneumonemia and ALI/ARDS.

The molecules in the corneal basement membrane zone affected by mustard exposure suggest potential therapies.

Mustard exposures result in epithelial-stromal separations in the cornea and epidermal-dermal separations in the skin. Large blisters often manifest in skin, while the cornea develops microblisters, and, when enough form, the epithelium sloughs. If the exposure is severe, healing can be imperfect and can result in long-term adverse consequences. For the cornea, this could manifest as recurrent corneal erosions. Since the corneal epithelial-stromal separations are in the region identified by electron microscopy as the lamina lucida, the same region affected by the blistering disease junctional epidermolysis bullosa (JEB), we postulated that the molecules that are defective in JEB would be the same ones cleaved by mustard compounds. These molecules are α6ß4 integrin and collagen XVII, which can be cleaved by matrix metalloproteinase-9 (MMP-9) and ADAM17, respectively. Therefore, our laboratory has tested MMP-9 and ADAM17 inhibitors as potential therapies to attenuate corneal mustard injury. Our results demonstrated that inhibiting MMP-9 and ADAM17 resulted in less epithelial-stromal separation in the corneas at 24 h postexposure, as compared with using only medium as a therapy.

ADAM17 Inhibitors Attenuate Corneal Epithelial Detachment Induced by Mustard Exposure.

PURPOSE: Sulfur mustard, nitrogen mustard (NM), and 2-chloroethyl ethyl sulfide all cause corneal injury with epithelial-stromal separation, differing only by degree. Injury can resolve in a few weeks or develop into chronic corneal problems. These vesicants induce microbullae at the epithelial-stromal junction, which is partially caused by cleavage of transmembranous hemidesmosomal collagen XVII, a component anchoring the epithelium to the stroma. ADAM17 is an enzyme involved in wound healing and is able to cleave collagen XVII. The activity of ADAM17 was inhibited in vesicant-exposed corneas by four different hydroxamates, to evaluate their therapeutic potential when applied 2 hours after exposure, thereby allowing ADAM17 to perform its early steps in wound healing. METHODS: Rabbit corneal organ cultures exposed to NM for 2 hours were washed, then incubated at 37°C for 22 hours, with or without one of the four hydroxamates (dose range, 0.3-100 nmol in 20 µL, applied four times). Corneas were analyzed by light and immunofluorescence microscopy, and ADAM17 activity assays. RESULTS: Nitrogen mustard-induced corneal injury showed significant activation of ADAM17 levels accompanying epithelial-stromal detachment. Corneas treated with hydroxamates starting 2 hours post exposure showed a dose-dependent ADAM17 activity inhibition up to concentrations of 3 nmol. Of the four hydroxamates, NDH4417 (N-octyl-N-hydroxy-2-[4-hydroxy-3-methoxyphenyl] acetamide) was most effective for inhibiting ADAM17 and retaining epithelial-stromal attachment. CONCLUSIONS: Mustard exposure leads to corneal epithelial sloughing caused, in part, by the activation of ADAM17 at the epithelial-stromal junction. Select hydroxamate compounds applied 2 hours after NM exposure mitigated epithelial-stromal separation.

Expression of type XXIII collagen mRNA and protein.

Collagen XXIII is a member of the transmembranous subfamily of collagens containing a cytoplasmic domain, a membrane-spanning hydrophobic domain, and three extracellular triple helical collagenous domains interspersed with non-collagenous domains. We cloned mouse, chicken, and humanalpha1(XXIII) collagen cDNAs and showed that this non-abundant collagen has a limited tissue distribution in non-tumor tissues. Lung, cornea, brain, skin, tendon, and kidney are the major sites of expression. In contrast, five transformed cell lines were tested for collagen XXIII expression, and all expressed the mRNA. In vivo the alpha1(XXIII) mRNA is found in mature and developing organs, the latter demonstrated using stages of embryonic chick cornea and mouse embryos. Polyclonal antibodies were generated in guinea pig and rabbit and showed that collagen XXIII has a transmembranous form and a shed form. Comparison of collagen XXIII with its closest relatives in the transmembranous subfamily of collagens, types XIII and XXV, which have the same number of triple helical and non-collagenous regions, showed that there is a discontinuity in the alignment of domains but that striking similarities remain despite this.

Extracellular matrix changes in the umbilical arteries of growth-restricted fetuses.

OBJECTIVE: This study was undertaken to determine whether the expression of extracellular matrix components (ECM) is altered in the umbilical arteries from preterm fetal growth-restricted (FGR) pregnancies. STUDY DESIGN: Preterm pregnancies with FGR were compared with appropriately grown preterm pregnancies. Umbilical artery messenger RNA (mRNA) levels for fibrillar collagens I and III, nonfibrillar collagen XIV, and decorin were determined by using relative reverse transcriptase polymerase chain reaction (RT-PCR). The mRNA expression was compared between cases and controls by using the Student t test. P < or = .05 was considered significant. RESULTS: Eight FGR cases and 5 control pregnancies were analyzed. Mean counts per minute (cpm) +/- SEM for collagen I and collagen III were increased in FGR pregnancies. There were no differences in mRNA expression of collagen XIV or decorin. CONCLUSION: Increased mRNA expression of collagen I and III, but not collagen XIV or decorin, is found in the umbilical arteries of preterm FGR pregnancies.

Exaggerated hepatotoxicity of acetaminophen in mice lacking tumor necrosis factor receptor-1. Potential role of inflammatory mediators.

Transgenic mice with a targeted disruption of the tumor necrosis factor receptor 1 (TNFR1) gene were used to analyze the role of TNF-alpha in pro- and anti-inflammatory mediator production and liver injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was correlated with expression of inducible nitric oxide synthase (NOS II) and nitrotyrosine staining of the liver. Expression of macrophage chemotactic protein-1 (MCP-1), KC/gro, interleukin-1beta (IL-1beta), matrix metalloproteinase-9 (MMP-9), and connective tissue growth factor (CTGF), inflammatory mediators known to participate in tissue repair, as well as the anti-inflammatory cytokine, interleukin-10 (IL-10), also increased in the liver following acetaminophen administration. TNFR1(-/-) mice were found to be significantly more sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This was correlated with more rapid and prolonged induction of NOS II in the liver and changes in the pattern of nitrotyrosine staining. Acetaminophen-induced expression of MCP-1, IL-1beta, CTGF, and MMP-9 mRNA was also delayed or reduced in TNFR1(-/-) mice relative to wild-type mice. In contrast, increases in IL-10 were more rapid and more pronounced. These data demonstrate that signaling through TNFR1 is important in inflammatory mediator production and toxicity induced by acetaminophen.

Collagen XXIV, a vertebrate fibrillar collagen with structural features of invertebrate collagens: selective expression in developing cornea and bone.

Tissue-specific assembly of fibers composed of the major collagen types I and II depends in part on the formation of heterotypic fibrils, using the quantitatively minor collagens V and XI. Here we report the identification of a new fibrillar-like collagen chain that is related to the fibrillar alpha1(V), alpha1(XI), and alpha2(XI) collagen polypeptides and which is coexpressed with type I collagen in the developing bone and eye. The new collagen was designated the alpha1(XXIV) chain and consists of a long triple helical domain flanked by typical propeptide-like sequences. The carboxyl propeptide is classic, with 8 conserved cysteine residues. The amino-terminal peptide contains a thrombospodin-N-terminal-like (TSP) motif and a highly charged segment interspersed with several tyrosine residues, like the fibril diameter-regulating collagen chains alpha1(V) and alpha1(XI). However, a short imperfection in the triple helix makes alpha1(XXIV) unique from other chains of the vertebrate fibrillar collagen family. The triple helical interruption and additional select features in both terminal peptides are common to the fibrillar chains of invertebrate organisms. Based on these data, we propose that collagen XXIV is an ancient molecule that may contribute to the regulation of type I collagen fibrillogenesis at specific anatomical locations during fetal development.

Collagen messenger RNA expression in the human amniochorion in premature rupture of membranes.

OBJECTIVE: It has been suggested that there is a decrease in the collagen content of the fetal membranes when there is premature rupture of the membranes before the onset of labor. This study was designed to determine whether decreased amniochorion collagen production (as measured by reduced amounts of messenger RNA) or alterations in relative production of different fibrillar and nonfibrillar collagens are associated with premature rupture of the membranes. STUDY DESIGN: Fetal membranes were collected after preterm (24-36 weeks of gestation) and term (> or =37 weeks of gestation) deliveries both with and without premature rupture of the membranes. Specimens with evidence of histologic chorioamnionitis were excluded. The messenger RNA levels for fibrillar collagen types I, III, and V and fibril-associated collagens with interrupted triple-helices types XII and XIV were measured with relative quantitative reverse transcriptase-polymerase chain reaction. RESULTS: The messenger RNA levels for fibrillar collagens decreased with advancing gestational age. Preterm premature rupture of membranes was associated with increased messenger RNA levels for fibrillar collagens and fibril-associated collagens with interrupted triple-helices collagen XII, but not type XIV. The greatest change in relative amounts of collagen messsenger RNA was demonstrated by an increased type I/XIV ratio, which was due to the up-regulation of type I levels, but not type XIV levels. CONCLUSION: A rise in fibrillar collagen production (messenger RNA) for types I, III, and V and fibril-associated collagens with interrupted triple-helices collagen type XII is observed with preterm premature rupture of the membranes. There is no evidence for a similar up-regulation of messenger RNA for fibril-associated collagens with interrupted triple-helices collagen type XIV. The rise in the collagen I/XIV messenger RNA ratio in preterm premature rupture of the membranes may result in collagen fibrils without enough stabilizing fibril-associated collagens with interrupted triple-helices type XIV on the fibril surface to maintain structural integrity.

Reduced hepatotoxicity of acetaminophen in mice lacking inducible nitric oxide synthase: potential role of tumor necrosis factor-alpha and interleukin-10.

Macrophage-derived inflammatory mediators have been implicated in tissue injury induced by a number of hepatotoxicants. In the present studies, we used transgenic mice with a targeted disruption of the gene for inducible nitric oxide synthase (NOS II) to analyze the role of nitric oxide in inflammatory mediator production in the liver and in tissue injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis, which was evident within 3 h and reached a maximum at 18 h. This was correlated with NOS II expression and nitrotyrosine staining of the liver, which was most prominent after 6 h. Expression of mRNA for tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), matrix metalloproteinase-9, and connective tissue growth factor (CTGF) also increased in the liver following acetaminophen treatment of wild-type mice. NOS II knockout mice were found to be less sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This did not appear to be due to differences in acetaminophen-induced glutathione depletion or adduct formation. In NOS II knockout mice treated with acetaminophen, hepatic expression of TNF-alpha, as well as CTGF, was significantly increased compared to wild-type mice. In contrast, IL-10 expression was reduced. These data demonstrate that nitric oxide is important in hepatotoxicity induced by acetaminophen. Moreover, some of its effects may be mediated by altering production of pro- and antiinflammatory cytokines and proteins important in tissue repair.

Activation of adherent vascular neutrophils in the lung during acute endotoxemia.

BACKGROUND: Neutrophils constitute the first line of defense against invading microorganisms. Whereas these cells readily undergo apoptosis under homeostatic conditions, their survival is prolonged during inflammatory reactions and they become biochemically and functionally activated. In the present study, we analyzed the effects of acute endotoxemia on the response of a unique subpopulation of neutrophils tightly adhered to the lung vasculature. METHODS: Rats were treated with 5 mg/kg lipopolysaccharide (i.v.) to induce acute endotoxemia. Adherent neutrophils were isolated from the lung vasculature by collagenase digestion and sequential filtering. Agarose gel electrophoresis, RT-PCR, western blotting and electrophoretic mobility shift assays were used to evaluate neutrophil activity. RESULTS: Adherent vascular neutrophils isolated from endotoxemic animals exhibited decreased apoptosis when compared to cells from control animals. This was associated with a marked increase in expression of the anti-apoptotic protein, Mcl-1. Cells isolated 0.5-2 hours after endotoxin administration were more chemotactic than cells from control animals and expressed increased tumor necrosis factor-alpha and cyclooxygenase-2 mRNA and protein, demonstrating that they are functionally activated. Endotoxin treatment of the animals also induced p38 and p44/42 mitogen activated protein kinases in the adherent lung neutrophils, as well as nuclear binding activity of the transcription factors, NF-kappaB and cAMP response element binding protein. CONCLUSION: These data demonstrate that adherent vascular lung neutrophils are highly responsive to endotoxin and that pathways regulating apoptosis and cellular activation are upregulated in these cells.