Zinc injection as a novel castration method in beef bulls: effects on performance, behavior, and testosterone and haptoglobin concentration.

Crossbred beef bulls (n = 180) were blocked by initial BW (337 ± 10.9 kg; six blocks) and assigned randomly to one of three treatments on day 0: 1) INJ; received 1 mL (100 mg Zn) of a Zn solution in each testis, 2) BAN; received blood- restrictive rubber band placed around the dorsal aspect of the scrotum, 3) BUL; bulls with testicles remaining intact in a randomized complete block design (three treatment pens per block and 10 cattle per pen). A subset of 54 animals (n = 3 per pen) was fitted with accelerometers on day 0 to quantify behavior variables continuously for 28 d. Testis width and scrotal circumference, and serum haptoglobin (days 0, 1, 3, 5, 7, and 14) and testosterone concentrations (every 28 d until slaughter) were also determined for the subset. During the slaughter process, testes from INJ and BUL were collected to assess final testes weight and for histopathological evaluation. Data were analyzed using a mixed model (α = 0.05); pen served as the experimental unit for all dependent variables. Final BW was greater (P < 0.01) for INJ and BUL compared to BAN (672, 686, and 611 kg, respectively; SEM = 4.4). Overall ADG and G:F were greater (P ≤ 0.03) in INJ and BUL than BAN; whereas, DMI was similar between treatments for the study duration (P = 0.46). Histopathological evaluation (n = 13; INJ = 7; BUL = 6) indicated that INJ testes were degenerative and reproductively nonviable whereas BUL testes were normal. Serum testosterone concentrations on day 168 were similar (P = 0.14) between INJ and BUL whereas after day 14, BAN was nondetectable; however, initial serum testosterone concentrations were similarly low across treatments. Serum haptoglobin concentration was greater (P < 0.01) in INJ than BUL and BAN on days 1, 3, 5, and 7. Scrotal circumference (P = 0.08) and testis width (P = 0.07) on day 168 tended to be greater for BUL than INJ. Motion index (P ≤ 0.02) and step count (P = 0.04) was greater in BUL and INJ compared to BAN cattle during the 28 d monitoring period. No difference in standing time (P ≥ 0.85) or lying bouts (P = 0.35) occurred. Zinc injection resulted in sterilization but did not cause complete cessation of testicular function evidenced by testosterone concentrations more similar to BUL than BAN. This resulted in overall increased BW and G:F for INJ vs. BAN, yet the acute phase response was markedly greater directly after Zn injection. Collectively, Zn injection resulted in outcomes more similar to BUL than BAN, implying minimal efficacy of INJ as a castration method in older bulls arriving to the feedlot.

Paraoxonases and infectious diseases.

The paraoxonases (PON1, PON2 and PON3) are an enzyme family with a high structural homology. All of them have lactonase activity and degrade lipid peroxides in lipoproteins and cells. As such, they play a role in protection against oxidation and inflammation. Infectious diseases are often associated with oxidative stress and an inflammatory response. Infection and inflammation trigger a cascade of reactions in the host, known as the acute-phase response. This response is associated with dramatic changes in serum proteins and lipoproteins, including a decrease in serum PON1 activity. These alterations have clinical consequences for the infected patient, including an increased risk for cardiovascular diseases, and an impaired protection against the formation of antibiotic-resistant bacterial biofilms. Several studies have investigated the value of serum PON1 measurement as a biomarker of the infection process. Low serum PON1 activities are associated with poor survival in patients with severe sepsis. In addition, preliminary studies suggest that serum PON1 concentration and/or enzyme activity may be useful as markers of acute concomitant infection in patients with an indwelling central venous catheter. Investigating the associations between paraoxonases and infectious diseases is a recent, and productive, line of research.

Acute lung injury induced by whole gastric fluid: hepatic acute phase response contributes to increase lung antiprotease protection.

BACKGROUND: Gastric contents aspiration in humans is a risk factor for severe respiratory failure with elevated mortality. Although aspiration-induced local lung inflammation has been studied in animal models, little is known about extrapulmonary effects of aspiration. We investigated whether a single orotracheal instillation of whole gastric fluid elicits a liver acute phase response and if this response contributes to enrich the alveolar spaces with proteins having antiprotease activity. METHODS: In anesthetized Sprague-Dawley rats receiving whole gastric fluid, we studied at different times after instillation (4 h -7 days): changes in blood cytokines and acute phase proteins (fibrinogen and the antiproteases alpha1-antitrypsin and alpha2-macroglobulin) as well as liver mRNA expression of the two antiproteases. The impact of the systemic changes on lung antiprotease defense was evaluated by measuring levels and bioactivity of antiproteases in broncho-alveolar lavage fluid (BALF). Markers of alveolar-capillary barrier derangement were also studied. Non-parametric ANOVA (Kruskall-Wallis) and linear regression analysis were used. RESULTS: Severe peribronchiolar injury involving edema, intra-alveolar proteinaceous debris, hemorrhage and PMNn cell infiltration was seen in the first 24 h and later resolved. Despite a large increase in several lung cytokines, only IL-6 was found elevated in blood, preceding increased liver expression and blood concentration of both antiproteases. These changes, with an acute phase response profile, were significantly larger for alpha2-macroglobulin (40-fold increment in expression with 12-fold elevation in blood protein concentration) than for alpha1-antitrypsin (2-3 fold increment in expression with 0.5-fold elevation in blood protein concentration). Both the increment in capillary-alveolar antiprotease concentration gradient due to increased antiprotease liver synthesis and a timely-associated derangement of the alveolar-capillary barrier induced by aspiration, contributed a 58-fold and a 190-fold increase in BALF alpha1-antitrypsin and alpha2-macroglobulin levels respectively (p < 0.001). CONCLUSIONS: Gastric contents-induced acute lung injury elicits a liver acute phase response characterized by increased mRNA expression of antiproteases and elevation of blood antiprotease concentrations. Hepatic changes act in concert with derangement of the alveolar capillary barrier to enrich alveolar spaces with antiproteases. These findings may have significant implications decreasing protease burden, limiting injury in this and other models of acute lung injury and likely, in recurrent aspiration.

Investigation into the role of Cu/Zn-SOD delivery system on its antioxidant and antiinflammatory activity in rat model of peritonitis.

BACKGROUND: The current study evaluated the role of delivery system (solution, conventional liposomes and PEG-ylated liposomes) on superoxide dismutase (SOD) antioxidant and antiinflammatory properties in a rat model of lipopolysaccharide (LPS)-induced peritonitis. METHODS: Fifty male albino rats (Wistar-Bratislava) were divided into five groups (n=10). Control group received saline and the other four groups received intraperitoneal injections of LPS (5mg/kg). Among the LPS-injected groups, one was LPS control group and the other three groups received the endotoxin injection 30min after receiving the same dose of SOD (500U/kg, ip) in different delivery systems: saline solution (SOD-S), conventional liposomes (SOD-L) or PEG-ylated liposomes (SOD-PL). The animals were euthanized 6h after LPS injection, blood samples were collected and acute phase response (total and differential leukocytes count; tumor necrosis factor α), antioxidants (total antioxidants; reduced glutathione), oxidative stress (total oxidants; lipid peroxidation) and nitrosative stress (nitric oxide metabolites; nitrotyrosine) were evaluated. RESULTS: Intraperitoneal administration of LPS to rats induced a marked inflammatory and oxidative response in plasma. On the other hand, all SOD formulations had protective effect against endotoxin-induced inflammation and oxidative/nitrosative stress, but PEG-ylated liposomes had the most significant activity. Thus, SOD-PL administration significantly reduced the effects of LPS on bone marrow acute phase response, the oxidative status and production of nitric oxide metabolites, while increasing the markers of antioxidant response in a significant manner. CONCLUSION: SOD supplementation interferes both with inflammatory and oxidative pathways involved in LPS-induced acute inflammation, PEG-ylated liposomal formulation being of choice among the tested delivery systems.

Regulation of urea synthesis during the acute phase response in rats.

Catabolism is a serious clinical problem in patients with active inflammation. Under such stressful conditions, the catabolism and loss of tissue nitrogen (N) result from proteolysis and are augmented by an up-regulation of the hepatic capacity to eliminate amino-N via urea-N. Our earlier studies suggest that this is part of the acute phase response to inflammation despite the increased need for amino-N for incorporation into acute phase proteins synthesised by the liver. It is, therefore, patho-physiologically and potentially therapeutically important to identify regulators of urea synthesis which, in this way, aggravate the inflammatory loss of body N; this study aimed at identifying such mediators, quantifying their effects, and unravelling their mode of action. The cytokines tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) play key roles in inflammation, and they both induce protein breakdown and catabolism. Therefore, they are both potential mediators of the up-regulation of urea synthesis. Our first experiments showed that TNF-α administration in rats acutely, i.e. after 3 h, up-regulated the in vivo capacity of urea-N synthesis (CUNS) by 30%, whereas IL-6 was observed not to acutely change CUNS. Furthermore, our experiments aimed at characterising the regulation of hepatic N elimination via urea during different phases of the TNF-α-induced acute phase response and to identify the steps between gene expression and physiological function that might be involved. We did so by using four different methods 1, 3, 24, and 72 h after TNF-α injection in rats: examination of urea cycle enzyme mRNA levels in liver tissue, the hepatocyte urea cycle enzyme proteins, CUNS, and known hormonal regulators of CUNS. The major serum acute phase proteins and their liver mRNA levels were also measured. Despite a progressive down-regulation of the urea cycle genes and a fully established acute phase response 24 h after TNF-α administration, no change in the in vivo capacity for the disposal of amino-N by urea synthesis was observed 1, 24, and 72 h after TNF-α injection. Moreover, TNF-α actually up-regulated urea synthesis 3 h after administration (cf. above). The dissociation of the effects of TNF-α on the urea genes and on physiological functions remains unexplained. The lack of down-regulation of whole body urea synthesis may promote the loss of N from the body and contribute towards inflammatory catabolism. This indicates the presence of an independent hepatic component of the inflammation response that is of primary importance for the stress-catabolic state.

Erythrocyte selenium concentration as a marker of selenium status.

BACKGROUND & AIMS: Plasma selenium concentration and glutathione peroxidase (GPx) activity are commonly used as markers of selenium nutritional status. However, plasma selenium concentrations fall independently of selenium status during the acute phase response and GPx is analytically problematic. The assay for erythrocyte selenium is robust and concentrations are unaffected by the systemic inflammatory response. This study was performed to investigate the validity of erythrocyte selenium measurement in assessing selenium status. METHODS: C-reactive protein (CRP), plasma and erythrocyte selenium concentrations and GPx activity were measured in 96 women from two regions of Malawi with low and high selenium dietary intakes. CRP and plasma and erythrocyte selenium was measured in 91 critically ill patients with a systemic inflammatory response. RESULTS & CONCLUSIONS: The median CRP value of all subjects from Malawi was 4.2 mg/L indicating no inflammation. The median CRP value for the critically ill patients was 126 mg/L indicating this group was inflamed. In the non-inflamed population there was a strong positive correlation (r = 0.95) between erythrocyte and plasma selenium and a strong positive correlation (r = 0.77) between erythrocyte selenium and erythrocyte GPx up to 6.10 nmol/g Hb after which maximal activity was reached. In the inflamed population, plasma selenium was low, erythrocyte selenium was normal and there was a weak correlation (r = 0.30) between selenium concentrations in plasma and erythrocytes. This demonstrates that plasma selenium is affected by the inflammatory response while erythrocyte selenium concentration is unaffected and can be used to reliably assess selenium status across a wide range of selenium intakes.

Lipopolysaccharide-induced anti-inflammatory acute phase response is enhanced in spermidine/spermine N1-acetyltransferase (SSAT) overexpressing mice.

Bacterial lipopolysaccharide (LPS) is an effective activator of the components of innate immunity. It has been shown that polyamines and their metabolic enzymes affect the LPS-induced immune response by modulating both pro- and anti-inflammatory actions. On the other hand, LPS causes changes in cellular polyamine metabolism. In this study, the LPS-induced inflammatory response in spermidine/spermine N(1)-acetyltransferase overexpressing transgenic mice (SSAT mice) was analyzed. In liver and kidneys, LPS enhanced the activity of the polyamine biosynthetic enzyme ornithine decarboxylase and increased the intracellular putrescine content in both SSAT overexpressing and wild-type mice. In survival studies, the enhanced polyamine catabolism and concomitantly altered cellular polyamine pools in SSAT mice did not affect the LPS-induced mortality of these animals. However, in the acute phase of LPS-induced inflammatory response, the serum levels of proinflammatory cytokines interleukin-1ß and interferon-γ were significantly reduced and, on the contrary, anti-inflammatory cytokine interleukin-10 was significantly increased in the sera of SSAT mice compared with the wild-type animals. In addition, hepatic acute-phase proteins C-reactive protein, haptoglobin and α(1)-acid glycoprotein were expressed in higher amounts in SSAT mice than in the wild-type animals. In summary, the study suggests that SSAT overexpression obtained in SSAT mice enhances the anti-inflammatory actions in the acute phase of LPS-induced immune response.

Effect of splenectomy and autologous spleen transplantation on the serum platelet-activating factor acetylhydrolase (PAF-AH) activity and acute phase response (APR) in a porcine model.

The aim of this study was to evaluate the inflammatory response after total splenectomy and spleen autotransplantation in a porcine model by measuring serum platelet-activating factor acetylhydrolase activity, C-reactive protein and albumin concentrations. Nineteen piglets were used in the experiment. After induction of anesthesia, animals were randomly divided into three groups: sham-operation with spleens intact (n = 6), total splenectomy (n = 6), and splenic autotransplantation (n = 7) with small fragments of the spleen autotransplanted into the greater omentum. The blood samples were taken just before surgery and on day 1st, 5th, 12th, 26th and 40th postoperatively. PAF-AH activity, CRP and albumin concentrations were assayed in the sera. After total splenectomy, PAF-AH activity was significantly increased on day 5th, while there was no significant increase after spleen autotransplantation or the sham-operation. CRP was significantly increased after surgery in all experimental groups. Albumin was significantly decreased after surgery from day 5th until day 40th in splenectomized and autotransplanted pigs. Increased PAF-AH activity after splenectomy and spleen autotransplantation might be attributed to inflammatory conditions due to the loss of splenic tissue and trauma. Time-course increase of CRP, in all groups after surgery suggests post-injury inflammatory response due to tissue lesion during operation.

Equine laminitis: membrane type matrix metalloproteinase-1 (MMP-14) is involved in acute phase onset.

REASONS FOR PERFORMING STUDY: Enzymatic separation at the hoof lamellar dermal-epidermal interface may play a role in the development of laminitis and characterising and locating matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of MMPs or TIMPs) in lamellar tissues may further understanding of pathogenesis. OBJECTIVES: To clone and sequence the cDNA encoding lamellar MMP-14 and TIMP-2, and quantify their transcription in normal and laminitic tissue; and to develop antibody to locate MMP-14 in lamellar tissues. METHODS: Tissue samples were obtained from an oligofructose induced model of laminitis. Total RNA was isolated, amplified by RT-PCR, cloned into a vector and sequenced. Real-time PCR was used to quantify MMP-14 and TIMP-2 expression. Rabbit anti-equine MMP-14 antibody was developed to analyse MMP-14 proteins from hoof tissues. RESULTS: Immunohistochemistry detected MMP-14 in the cytoplasm of normal lamellar basal and parabasal cells in close proximity to the lamellar basement membrane. In laminitis affected tissue MMP-14 immunostaining was depleted in lamellar basal cells. Quantitative real-time PCR showed MMP-14 and TIMP-2 expression significantly (P<0.05) elevated and lowered respectively in laminitis affected tissues. CONCLUSION: MMP-14, located in the cytoplasm of normal lamellar basal cells, disappears during laminitis development. The pathology of laminitis is associated with increased and lowered transcription of MMP-14 and TIMP-2, respectively. POTENTIAL RELEVANCE: Enzymes have a role in laminitis pathology and inhibition of their activity may prevent laminitis.

Upregulation of heme oxygenase-1 gene by turpentine oil-induced localized inflammation: involvement of interleukin-6.

Heme oxygenase-1 (HO-1) is the inducible isoform of an enzyme family responsible for heme degradation and was suggested to be involved in the acute phase response in the liver. However, the mechanisms of the HO-1 regulation under inflammatory conditions are poorly understood. Therefore, the purpose of the current work was to study the expression of HO-1 in the liver and other organs of rats with a localized inflammation after intramuscular injection of turpentine oil (TO). Since interleukin-6 (IL-6) is known to be a principal mediator of inflammation, the levels of this cytokine were also estimated in the animal model used. HO-1 and IL-6 expression was evaluated by Northern blot, in situ hybridization, Western blot, immunohistochemistry and enzyme-linked immunosorbent assay. In the liver and injured muscle, the HO-1 mRNA levels were dramatically increased 4-6 h after TO administration. HO-1 protein levels in the liver were elevated starting from 6-12 h after the treatment. In other internal organs such as the heart, kidney and large intestine, only a slight induction of HO-1 mRNA was observed. IL-6-specific transcripts appeared only in the injured muscle and were in accordance with serum levels of IL-6. In turn, temporal expression of IL-6 in the muscle and circulatory IL-6 levels correlated well with HO-1 expression in the liver and injured muscle. In the liver of control rats HO-1 protein was detected in Kupffer cells, while in TO-injected rats also hepatocytes became strongly HO-1 positive. Conversely, in the injured muscle, HO-1 immunoreactivity was attributed only to macrophages. Our data demonstrate that during localized inflammation HO-1 expression was rapidly and strongly induced in macrophages of injured muscle and in hepatocytes, and IL-6 derived from injured muscle seems to be responsible for the HO-1 induction in the liver.

Suppression of DHEA sulfotransferase (Sult2A1) during the acute-phase response.

The acute-phase response (APR) induces alterations in lipid metabolism, and our data suggest that this is associated with suppression of type II nuclear hormone receptors that are key regulators of fatty acid, cholesterol, and bile acid metabolism. Recently, the farnesoid X receptor (FXR), constitutive androstane receptor (CAR), and pregnane X receptor (PXR) were found to regulate DHEA sulfotransferase (Sult2A1), which plays an important role in DHEA sulfation and detoxification of bile acids. Because FXR, PXR, and CAR are suppressed during the APR, we hypothesized that Sult2A1 is downregulated during the APR. To induce the APR, mice were treated with LPS, which will then trigger the release of various cytokines, and the mRNA levels of Sult2A1 and the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate synthase 2 (PAPSS2), as well as the enzyme activity of Sult2A1, were determined in the liver. We found that mRNA levels of Sult2A1 decrease in a time- and dose-dependent manner during the LPS-induced APR. Similar changes were observed in the mRNA levels of PAPSS2, the major synthase of PAPS in the liver. Moreover, hepatic Sult2A1 activity and serum levels of DHEA-sulfate (DHEA-S) were significantly decreased in LPS-treated animals. These results suggest that decreased levels or activities of FXR, PXR, and CAR during the APR could contribute to decreases in Sult2A1, resulting in decreased sulfation of DHEA and lower circulating level of DHEA-S. Finally, we found that both TNF and IL-1 caused a significant decrease in the mRNA level of Sult2A1 in Hep3B human hepatoma cells, suggesting that the proinflammatory cytokines TNF and IL-1 mediate the inhibitory effect of LPS on Sult2A1 mRNA level. Our study provides a possible mechanism by which infection and inflammation are associated with altered steroid metabolism and cholestasis.

Decreased expression levels of rat liver glutathione S-transferase A2 and albumin during the acute phase response are mediated by HNF1 (hepatic nuclear factor 1) and IL6DEX-NP.

The acute phase response is characterized by positive and negative regulation of many liver proteins including GSTs (glutathione S-transferases) and albumin. The expression of albumin and some GSTs are dependent on HNF1 (hepatic nuclear factor 1). Interleukin 6 plus dexamethasone induce a nuclear protein (IL6DEX-NP) in rat hepatocytes in vitro that binds to a promoter element adjacent to the HNF1 site of rGSTA2 and decreases its expression. We determined how HNF1 and IL6DEX-NP regulate rGSTA2 and albumin expression in rats during the acute phase response after LPS (lipopolysaccharide) treatment. Expression of rGSTA2 and albumin mRNA decreased 3 h after LPS treatment and remained low for 48 h. Transcription rates showed a similar pattern but albumin transcription was less affected. HNF1 and IL6DEX-NP binding to the rGSTA2 promoter was present in control livers but was absent at 3 and 6 h after LPS. By 12 h, HNF1 and IL6DEX-NP binding to the rGSTA2 promoter reappeared and increased to above normal at 48 h. The patterns of HNF1 and IL6DEX-NP binding to the albumin promoter were similar. Affinity of IL6DEX-NP for the albumin promoter was less than that for the rGSTA2 promoter and changes in the transcription rates were consistent with the difference. Early decreases in rGSTA2 and albumin during the acute phase response are due to decreased binding of HNF1. Later persistent decreases in transcriptional rate of rGSTA2 and to a lesser extent albumin are due to increased IL6DEX-NP binding. IL6DEX-NP appears to be an important negative regulator of gene expression in vitro and in vivo.

Characterization of the mouse olfactory glutathione S-transferases during the acute phase response.

The acute phase response (APR) has been shown to alter expression and activity of biotransformation enzymes, such as the phase I cytochromes p450 and phase II glutathione S-transferases (GSTs). The cytochromes p450 and GSTs are expressed abundantly and colocalized to non-neuronal cells of the olfactory mucosa. Previous studies indicate that olfactory cytochromes p450 expression and activity is altered during periods of localized inflammation and infection. Little is understood, however, about the influence of the APR on olfactory GST enzymes. This study investigated effects of the APR on olfactory GST isozymes expression and activity in mouse olfactory mucosa after 24-hr treatment with the acute phase inducer, polyinosinic: polycytidylic acid (polyIC). Western blot analysis using antibodies directed against specific GST isoforms alpha (A1-1), micro (M1-1), and pi (P1-1) demonstrated that their expression was unaltered by polyIC treatment. In contrast, olfactory p450 2E1 expression was significantly decreased. Enzymatic activity of the olfactory GSTs toward the general substrate, 1-chloro-2,4-dinitrobenzene (CDNB) was unchanged during the APR. Analysis of olfactory glutathione content during the APR showed that it was also unaffected by polyIC. The insensitivity of these olfactory GST isoforms during the APR may play a significant role toward limiting the impact of infection and inflammation on the olfactory system.

Biologic contribution of P1 promoter-mediated expression of ST6Gal I sialyltransferase.

The synthesis of the common and well-documented Siaalpha 2,6 to Galbeta 1,4GlcNAc structure (Sia6LacNAc) is principally mediated by the sialyltransferase ST6Gal I, which is particularly highly expressed in liver, lactating mammary gland, intestinal epithelia of newborn animals, and B cells. Multiple independent promoters govern the expression of Siat1, the ST6Gal I gene. In liver, elevation of hepatic and serum ST6Gal is part of the acute phase reaction, the hepatic response to systemic trauma, and is governed by the inducible, liver-specific promoter-regulatory region, P1. A constitutive and nontissue-specific promoter, P3, mediates low-level, basal hepatic Siat1 transcription. We generated a mouse specifically unable to use the transcriptional initiation site uniquely used in P1-mediated ST6Gal I expression. These animals, Siat1deltaP1, are viable and display reduced ST6Gal I mRNA in liver with concomitantly reduced sialyltransferase activities in liver and in serum. Siat1deltaP1 animals are unable to elevate hepatic Siat1 mRNA as part of the inflammatory response induced by turpentine. Surprisingly, serum glycoprotein components exhibit normal extent of sialylation, with no noticeable difference in binding to SNA, the alpha2,6-sialyl-specific lectin. Siat1deltaP1 animals also exhibit an outwardly normal B cell response. On intraperitoneal challenge with the pathogen Salmonella typhimurium, a significantly greater accumulation of neutrophils within the peritoneal space was observed in Siat1deltaP1 animals compared to wild-type mice. Siat1deltaP1 mice also exhibit a greater bacterial burden in liver and spleen, accompanied by more pronounced spleno-/hepatomegaly and greater leukocyte infiltration into affected organs than their wild-type counterparts.

Human secretory phospholipase A2 mediates decreased plasma levels of HDL cholesterol and apoA-I in response to inflammation in human apoA-I transgenic mice.

OBJECTIVE: Plasma levels of high density lipoprotein (HDL) cholesterol and apolipoprotein (apo)A-I are decreased in inflammatory states. Secretory phospholipase A2 (sPLA2), an acute-phase protein, may play a key role in the pathophysiology of this phenomenon. METHODS AND RESULTS: To investigate the effects of sPLA2 on human-like HDL particles in vivo, we generated transgenic mice overexpressing human apoA-I and human sPLA2 (apoA-I/sPLA2 mice). Compared with apoA-I mice, apoA-I/sPLA2 mice had significantly lower plasma levels of phospholipids, HDL cholesterol, and apoA-I (each P<0.01). HDL from apoA-I/sPLA2 mice was significantly depleted in phospholipids and cholesteryl esters (each P<0.001) but was enriched in protein and triglycerides (each P<0.001). As assessed by gel filtration and nondenaturing gel electrophoresis, sPLA2 overexpression in apoA-I mice resulted in a dramatic shift of the HDL particle size toward smaller particles. Furthermore, virtually all plasma sPLA2 in apoA-I/sPLA2 mice was found in association with the HDL fraction. The acute-phase response was induced in apoA-I/sPLA2 double-transgenic and apoA-I single-transgenic mice by intraperitoneal lipopolysaccharide (LPS) injection. Plasma sPLA2 was significantly increased after LPS injection in apoA-I/sPLA2 mice. Twelve hours after LPS administration, plasma total cholesterol, HDL cholesterol, apoA-I, and phospholipids were unchanged in apoA-I transgenic control mice but had decreased significantly in the apoA-I/sPLA2 mice (-57%, -62%, and -54%, -61%, respectively; each P<0.001). Both groups of mice had increased plasma levels of serum amyloid A (SAA) in response to LPS. To test the hypothesis that SAA may be an in vivo activator of sPLA2, we specifically overexpressed SAA in apoA-I/sPLA2 mice by means of liver-directed gene transfer. Despite high plasma levels of SAA, plasma lipid and lipoprotein profiles were not different than those in control mice. CONCLUSIONS: These results in a mouse model of human-like HDL indicate that sPLA2 expression significantly influences HDL particle size and composition and demonstrate that an induction of sPLA2 is required for the decrease in plasma HDL cholesterol in response to inflammatory stimuli in mice and that this effect is independent of SAA.

Cytochrome P450 and antioxidant activity in interleukin-6 knockout mice after induction of the acute-phase response.

Hepatic cytochrome P450 (CYP) expression and antioxidant activity have been shown to decrease following endotoxin (lipopolysaccharide [LPS]) or proinflammatory cytokine administration. Using mice deficient in interleukin-6 (IL-6), the role of IL-6 in the regulation of hepatic CYP activity, glutathione (GSH) metabolism, and catalase (CAT) activity was analyzed after LPS administration. Administration of LPS produced comparable decreases in hepatic CYP3A activity in WT B6x129 (WT) mice and IL-6 knockout mice. No decrease was observed for CYP2D9 activity after LPS administration in either WT or IL-6 knockout mice. LPS administration significantly increased hepatic and renal CYP2E1 and CYP4A activity in WT mice, with no effect in IL-6 knockout mice. CYP2A12 activity increased in IL-6 knockout, mice with no change in WT mice after LPS administration. LPS administration had no significant effect on hepatic GSH reductase, GST peroxidase, GSH-S-transferase (GST), or total GSH in either WT or IL-6 knockout. However, hepatic CAT activity was significantly reduced in WT mice after LPS administration, with no effect in IL-6 knockout mice. These results support IL-6 as a critical mediator of the effects of LPS on specific hepatic and renal CYP activities and hepatic CAT activity.

Role of reactive oxygen intermediates in the decrease of hepatic cytochrome P450 activity by serum of humans and rabbits with an acute inflammatory reaction.

Serum of rabbits with a turpentine-induced acute inflammatory reaction (RS(INFLA)) and serum of humans with a viral infection (HS(INF)) were previously shown to diminish hepatic cytochrome P450 (P450) content and activity. To document the role of reactive oxygen intermediates in the serum-mediated decrease in P450 content and activity, hepatocytes of rabbits with an acute inflammatory reaction (H(INFLA)) were incubated with RS(INFLA) and HS(INF) for 4 h, and total P450 content (spectrally measurable P450), P450 activity (assessed by estimating the formation of theophylline metabolites), and amount of CYP1A1, CYP1A2, and CYP3A6 proteins were measured. RS(INFLA) or HS(INF) decreased P450 content and activity without affecting the amount of CYP1A1 and -1A2 H(INFLA). Exposure of H(CONT) or H(INFLA) to hydrogen peroxide (0.01-1.0 mM) and sodium nitroprusside (0.01-1.0 mM) produced a dose-dependent decrease in P450 content and in the formation of theophylline metabolites without modifying the amount of CYP1A1 and CYP1A2, whereas lipid peroxidation increased. Incubation of L-NAME (0.05-1.0 mM), dimethylthiourea (6.25-50 mM), or N-acetylcysteine (0.01-1.0 mM) with H(INFLA) partially prevented the decrease in P450 content and activity and the increased lipid peroxidation induced by RS(INFLA) and HS(INF). On the other hand, 3-amino-1,2,4-triazole (10-100 mM) or diethyldithiocarbamate (1.0-10 mM) potentiated RS(INFLA)- and HS(INF)-mediated decreases in P450 content and activity and the increase in lipid peroxidation, without affecting the amount of CYP1A1 or -1A2; DL-buthionine-(S,R)-sulfoximine (2.5-25 mM) potentiated only the inhibition of 1,3-dimethyluric acid formation. It is concluded that reactive oxygen intermediates are implicated in the decrease of H(INFLA) P450 content and activity induced by 4 h of exposure to RS(INFLA) or HS(INF).

The hepatic clearance of recombinant tissue-type plasminogen activator decreases after an inflammatory stimulus.

We have shown that tissue-type plasminogen activator (tPA) and plasma kallikrein share a common pathway for liver clearance and that the hepatic clearance rate of plasma kallikrein increases during the acute-phase (AP) response. We now report the clearance of tPA from the circulation and by the isolated, exsanguinated and in situ perfused rat liver during the AP response (48-h ex-turpentine treatment). For the sake of comparison, the hepatic clearance of a tissue kallikrein and thrombin was also studied. We verified that, in vivo, the clearance of 125I-tPA from the circulation of turpentine-treated rats (2.2 +/- 0.2 ml/min, N = 7) decreases significantly (P = 0.016) when compared to normal rats (3.2 +/- 0.3 ml/min, N = 6). The AP response does not modify the tissue distribution of administered 125I-tPA and the liver accounts for most of the 125I-tPA (>80%) cleared from the circulation. The clearance rate of tPA by the isolated and perfused liver of turpentine-treated rats (15.5 +/- 1.3 microg/min, N = 4) was slower (P = 0.003) than the clearance rate by the liver of normal rats (22. 5 +/- 0.7 microg/min, N = 10). After the inflammatory stimulus and additional Kupffer cell ablation (GdCl3 treatment), tPA was cleared by the perfused liver at 16.2 +/- 2.4 microg/min (N = 5), suggesting that Kupffer cells have a minor influence on the hepatic tPA clearance during the AP response. In contrast, hepatic clearance rates of thrombin and pancreatic kallikrein were not altered during the AP response. These results contribute to explaining why the thrombolytic efficacy of tPA does not correlate with the dose administered.

The hepatic clearance of recombinant tissue-type plasminogen activator decreases after an inflammatory stimulus

We have shown that tissue-type plasminogen activator (tPA) and plasma kallikrein share a common pathway for liver clearance and that the hepatic clearance rate of plasma kallikrein increases during the acute-phase (AP) response. We now report the clearance of tPA from the circulation and by the isolated, exsanguinated and in situ perfused rat liver during the AP response (48-h ex-turpentine treatment). For the sake of comparison, the hepatic clearance of a tissue kallikrein and thrombin was also studied. We verified that, in vivo, the clearance of 125I-tPA from the circulation of turpentine-treated rats (2.2 + or - 0.2 ml/min, N = 7) decreases significantly (P = 0.016) when compared to normal rats (3.2 + or - 0.3 ml/min, N = 6). The AP response does not modify the tissue distribution of administered 125I-tPA and the liver accounts for most of the 125I-tPA (>80 percent) cleared from the circulation. The clearance rate of tPA by the isolated and perfused liver of turpentine-treated rats (15.5 + or - 1.3 µg/min, N = 4) was slower (P = 0.003) than the clearance rate by the liver of normal rats (22.5 + or - 0.7 µg/min, N = 10). After the inflammatory stimulus and additional Kupffer cell ablation (GdCl3 treatment), tPA was cleared by the perfused liver at 16.2 + or - 2.4 µg/min (N = 5), suggesting that Kupffer cells have a minor influence on the hepatic tPA clearance during the AP response. In contrast, hepatic clearance rates of thrombin and pancreatic kallikrein were not altered during the AP response. These results contribute to explaining why the thrombolytic efficacy of tPA does not correlate with the dose administered

Hepatic acute phase induction of murine beta-galactoside alpha 2,6 sialyltransferase (ST6Gal I) is IL-6 dependent and mediated by elevation of exon H-containing class of transcripts.

Hepatic expression of CMP-NeuAc:Gal beta 1,4GlcNAc alpha 2,6-sialyltransferase (ST6Gal I) is induced as part of the acute phase response in mammals by mechanisms that remain poorly understood. Previous work suggests that murine liver ST6Gal I mRNA contains an additional and novel region that is not found on ST6Gal I mRNA from human HepG2 hepatoma cells and from rat liver. This novel region, residing 5' of the common Exon I sequence, is encoded by a discrete upstream exon, Exon H. Here we provide evidence that the Exon H-containing transcript is the murine counterpart of the human and rat ST6Gal I mRNAs transcribed from the hepatic-specific promoter, P1. Exon H-containing ST6Gal I mRNA is expressed in all three mice strains examined: balb/c, C57B46, and 129Sv. Furthermore, murine RNA tissue survey indicates that presence of Exon H-containing transcripts is restricted to the liver. When mice are subjected to subcutaneous injection of turpentine to elicit the hepatic acute phase response, greater than 4-fold elevation in liver ST6Gal I mRNA was observed. Consistent with the view that Exon H-containing transcripts is regulated by the murine P1 promoter, 5'-RACE analysis indicates that the majority of these transcripts contains the Exon H sequence. This is consistent with the view that Exon H-containing transcripts are regulated by the murine P1 region. To assess the mechanism of ST6Gal I response in the hepatic acute phase reaction, mice harboring lesions in both alleles of the IL-6 gene were examined. IL-6(-/-) animals expressed normal levels of ST6Gal I mRNA in liver, with Exon H-containing transcripts remaining the predominant mRNA isoform. However, hepatic ST6Gal I is not elevated upon turpentine injection in the IL-6(-/-) animals. These results indicate that ST6Gal I induction in mouse liver during the acute phase reaction is mediated predominantly by the IL-6 pathway, and results in the induction of the Exon H-containing class of ST6Gal I mRNA that is specific to the liver.