Corticosterone suppresses mesenteric lymph node T cells by inhibiting p38/ERK pathway and promotes bacterial translocation after alcohol and burn injury

Previous studies showed that alcohol (EtOH) intoxication before burn injury suppresses mesenteric lymph node (MLN) T cell functions and increases gut bacterial translocation. In this study, we examined whether corticosterone (Cort) plays any role in suppressing MLN T cell function and bacterial accumulation after EtOH intoxication and burn injury. Rats were gavaged with EtOH to achieve a blood EtOH level of approximately 100 mg/dl before receiving 25% total body surface area burn or sham injury. A group of rats was treated with the Cort synthesis inhibitor metyrapone (25 mg/kg) at the time of injury and on day 1 after injury. Two days after injury, a significant increase in blood Cort levels and suppression of MLN T cell proliferation and IL-2 production was observed in rats receiving combined insult of EtOH intoxication and burn injury compared with rats receiving EtOH intoxication or burn injury alone. There was no change in T cell apoptosis after combined insult of EtOH and burn injury. Furthermore, T cell suppression was accompanied by a significant decrease in p38 and ERK1/2 activation (phosphorylation). There was no difference in JNK activation after EtOH and burn injury. Treatment of rats with metyrapone prevented the suppression of MLN T cell proliferation, IL-2 production, and p38 and ERK1/2 phosphorylation. Restoration of T cell function in metyrapone-treated animals was also associated with the decrease in bacterial accumulation in MLN. These findings suggest that EtOH intoxication before burn injury augments Cort release, which suppresses MLN T cell function by inhibiting p38 and ERK1/2 activation and promotes bacterial accumulation in MLN after EtOH and burn injury.     

Mouse genetic background influences severity of immune responses following trauma-hemorrhage

Studies have shown that following bacterial infection or endotoxin administration, immune functions are regulated differently in mice of different genetic background. Since the susceptibility to sepsis following trauma-hemorrhage is dependant on the severity of injury, it is important to determine whether genetic background of the animal influence immune functions after trauma-hemorrhage. The aim of our studies, therefore, was to assess differences in the immune functions in genetically different strains of age-matched C3H/HeN and C57BL/6 male mice following trauma-hemorrhage. The analysis for immune functions included: proliferation of splenocyte and bone-marrow cells, IL-2 and IFN-gamma release by splenocytes, and TNF-alpha and IL-10 release by splenic, peritoneal, liver (Kupffer cell), and bone-marrow macrophages. The results show significant differences in splenocyte and bone-marrow functions, and in the release of the mediators of immune function by immune competent cells: (a) between the two genetic strains, and (b) in each mouse strain following trauma-hemorrhage. Thus, genetic background appears to significantly influence the severity of immune responses in males following trauma-hemorrhage.     

Gender differences in the inflammatory response and survival following haemorrhage and subsequent sepsis

Studies have shown gender dimorphism in cell-mediated immune responses following haemorrhage, with depressed responses in young males and maintained or enhanced responses in proestrus females. However, it remains unknown whether or not the sexually dimorphic immune response to haemorrhage provides any protection against a subsequent in vivo polymicrobial septic challenge. To study this, male and proestrus female C3H/HeN mice were subjected to haemorrhage (35+/-5 mmHg for 90 min followed by fluid resuscitation) or sham operation. Twenty-four hours thereafter, all mice were subjected to polymicrobial sepsis by cecal ligation and puncture (CLP) and survival was assessed over a 10 day period. Haemorrhage prior to CLP significantly increased mortality in males as compared to shams. In contrast, mortality in females following CLP was comparable between the sham and haemorrhage groups. Plasma levels of interleukin (IL-)6, tumour necrosis factor (TNF)-alpha and prostaglandin E(2)(PGE(2)) at 5 h after CLP were significantly increased in males subjected to prior haemorrhage. In contrast, plasma levels of IL-6 and TNF-alpha in females did not increase under such conditions. PGE(2)levels were comparable in males and females following CLP, however prior haemorrhage significantly reduced PGE(2)levels in females, whereas no change was observed in males. Liver and splenic expression of cyclooxygenase-2 protein paralleled the changes in plasma PGE(2). Female sex hormones, therefore, appear to play an important role not only in maintaining immune function following haemorrhage, but also provide a survival advantage against subsequent septic challenge.     

Norepinephrine-induced hepatocellular dysfunction in early sepsis is mediated by activation of alpha2-adrenoceptors

Gut-derived norepinephrine (NE) has been shown to play a critical role in producing hepatocellular dysfunction in early sepsis, but it is not known whether alpha2-adrenoceptor activation mediates this dysfunction. We infused normal male adult rats with NE, NE plus the specific alpha2-adrenergic antagonist rauwolscine (RW), or vehicle (normal saline) for 2 h. Hepatocellular function was determined by in vivo indocyanine green (ICG) clearance. An isolated perfused liver preparation was also used to assess hepatocellular function by in vitro ICG clearance; NE alone or with RW was added to the perfusate. Rats were subjected to sepsis by cecal ligation and puncture (CLP). At 1 h after CLP, RW was infused for 15 min. At 5 h after CLP, we measured hepatocellular function and serum tumor necrosis factor-alpha (TNF-alpha) levels. Intraportal NE infusion in normal rats produced hepatocellular dysfunction, which was prevented by RW and NE infusion. This is confirmed by findings with the isolated perfused liver preparation. RW administration in early sepsis maintained hepatocellular function and downregulated TNF-alpha production at 5 h after CLP. These results suggest that NE-induced hepatocellular dysfunction in early sepsis is mediated by alpha2-adrenoceptor activation, which appears to upregulate TNF-alpha production. Modulation of hepatic responsiveness to NE by alpha2-adrenergic antagonists should provide a novel approach for maintaining cell and organ functions during sepsis.     

Resistance of macrophages to the suppressive effect of interleukin-10 following thermal injury

The activation of a macrophage(M)-dependent proinflammatory cascade following thermal injuryplays an important role in the development of immunosuppression andincreased susceptibility to subsequent sepsis in burn patients. Incontrast, although interleukin (IL)-10, an anti-inflammatory cytokinethat can downregulate M activity, has also been implicated inpostburn immune dysfunction, its role in the regulation of Mfunction postburn remains unclear. To study this, C57BL/6 female micewere subjected to a 25% total body surface area third-degree scaldburn, and splenic Ms were isolated 7 days later. Lipopolysaccharide(LPS)-stimulated IL-10, IL-6, tumor necrosis factor (TNF)-, andnitric oxide (NO) production were significantly increased in the burngroup compared with shams. Blockade of endogenous IL-10 activityenhanced IL-6 and TNF- release, but not NO release, in both groups.The addition of exogenous IL-10 to the M cultures dose dependentlysuppressed production of these inflammatory mediators in both groups.The timing of IL-10 addition to the cultures in relation to LPSstimulation, however, was critical. The suppressive effect of exogenousIL-10 was attenuated in both groups when the cells were exposed toIL-10 at 4-6 h after LPS stimulation; however, Ms from injuredmice were significantly better able to maintain inflammatorymediator-productive capacity. The resistance of Ms from injured miceto IL-10-mediated suppression correlated with decreased IL-10 receptor(IL-10R) expression and increased CD11b expression. These findingssuggest that Ms, following thermal injury, display resistance tosuppression by IL-10 due in part to downregulation of IL-10R expression.

     

Decomposition of cocoa procyanidins in the gastric milieu

There is considerable interest in the bioavailability of flavonoids and phenolic components of the diet and their bioactivity in vivo. However, little is known of pre-absorption events in the gastric lumen. The effects of the acidic environment, as found in the gastric milieu, on procyanidin oligomers of catechin polyphenols has been investigated. The results show that under these conditions the procyanidin oligomers (trimer to hexamer) are hydrolysed to mixtures of epicatechin monomer and dimer, thus enhancing the potential for their absorption in the small intestine.     

Does ATP cross the cell plasma membrane.

Although there is an abundance of evidence which indicates that ATP is released as well as taken up by cells, the concept that ATP cannot cross the cell membrane has tended to prevail. This article reviews the evidence for the release as well as uptake of ATP by cells. The evidence presented by various investigators clearly indicates that ATP can cross the cell membrane and suggests that the release and uptake of ATP are physiological processes.     

Molecular and chemical characterization of plants regenerated from Ri-mediated hairy root cultures of Rauwolfia serpentina

Hairy root lines were induced from leaf explants of Rauwolfia serpentina known to contain high levels of reserpine (0.0882 % DW) content. Out of five high yielding hairy root lines, three (R1, R14 and R15) exhibited spontaneous regeneration of shoots after 6–8 weeks in liquid B5 medium. Excised regenerated shoots underwent robust shoot proliferation when cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l naphthanleneacetic acid (NAA) and 1.0 mg/l 6-benzyladenine. When shoots were transferred to a root induction medium, consisting of MS basal medium and 1.0 mg/l NAA, all rooted within 2–3 weeks. Of a total of 45 plants developed from three different hairy root lines, 30 were successfully acclimatized and transferred to the green house. Almost 90 % of these plants grown in the green house showed no observed phenotypic differences, while 10 % were stunted and grew poorly, in comparison to non-transformed plants. Phenotypic assessment of regenerated plants for plant length, number of nodes and intermodal lengths, number of leaves per node, leaf color, leaf size, number of flowering shoots, flower size, fruit size, lateral root branching and root biomass was conducted. Polymerase chain reaction and Southern blot hybridization revealed that all plants derived from hairy roots carried the Ri T_L-DNA fragment. Moreover for plants derived from transgenic hairy root line R14, presence of more than a single transgene copy number was observed, and this might have contributed to observed abnormal phenotypes. Analysis of reserpine content revealed that roots of regenerated plants had similar levels (0.0889 % DW) to those of their corresponding hairy roots.     

The discovery of potent and long-acting oral factor Xa inhibitors with tetrahydroisoquinoline and benzazepine P4 motifs

The discovery and evaluation of potent and long-acting oral sulfonamidopyrrolidin-2-one factor Xa inhibitors with tetrahydroisoquinoline and benzazepine P4 motifs are described. Unexpected selectivity issues versus tissue plasminogen activator in the former series were addressed in the later, delivering a robust candidate for progression towards clinical studies.     

Role of PPARγ in the salutary effects of 17β‐estradiol on kupffer cell cytokine production following trauma‐hemorrhage

Studies have shown that administration of 17β‐estradiol prevents trauma‐hemorrhage‐induced increase in proinflammatory cytokine production by Kupffer cells and associated multiple organ injury. Since activation of peroxisome proliferator‐activated receptor γ (PPARγ) following ischemic conditions has been shown to be protective, we examined if PPARγ plays any role in the salutary effects of 17β‐estradiol on Kupffer cell cytokine production following trauma‐hemorrhage. Male mice underwent trauma‐hemorrhage (mean blood pressure 40 mmHg for 90 min, then resuscitation). 17β‐estradiol (50 µg/kg) or vehicle with or without PPARγ antagonist GW9662 was injected subcutaneously at the middle of resuscitation. At 2 h after trauma‐hemorrhage, plasma interleukin (IL)‐6 and tumor necrosis factor (TNF)‐α levels, Kupffer cell IL‐6 and TNF‐α production and mRNA expression, and PPARγ, nuclear factor (NF)‐κB and activator protein (AP)‐1 DNA binding activity were determined. Kupffer cell IL‐6 and TNF‐α production, as well as plasma IL‐6 and TNF‐α levels, increased following trauma‐hemorrhage. Moreover, NF‐κB and AP‐1 DNA binding activity and IL‐6 and TNF‐α mRNA expression were also enhanced under such conditions. However, 17β‐estradiol administration normalized all these parameters. Although PPARγ activity decreased after trauma‐hemorrhage, administration of 17β‐estradiol following trauma‐hemorrhage elevated PPARγ activity above the normal level. Inhibition of PPARγ by co‐administration of GW9662, however, abolished the salutary effects of 17β‐estradiol on plasma cytokine and Kupffer cells. Thus, activation of PPARγ appears to play an important role in mediating the salutary effects of 17β‐estradiol on plasma cytokine levels and Kupffer cell cytokine production after trauma‐hemorrhage, which are likely mediated via NF‐κB and AP‐1. J. Cell. Physiol. 226: 205–211, 2010. © 2010 Wiley‐Liss, Inc.