Morphine-induced suppression of thymocyte proliferation is mediated by inhibition of IL-2 synthesis.
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Animals↗
Biomedical subjects
R A Barke
Publications and source records attributed to R A Barke.
The role of programmed cell death (apoptosis) in thymic involution following sepsis.
OBJECTIVE: To test the hypothesis that thymic involution following peritoneal sepsis is secondary to thymocyte programmed cell death. DESIGN: We investigated the temporal response of thymic weight and thymic DNA fragmentation following peritoneal sepsis induced by cecal ligation and puncture in a rat model. We investigated the possible role of decreased interleukin (IL)-2 synthesis in the induction of apoptosis using rat thymocytes in primary culture. Finally, we studied IL-2 gene expression and IL-2 protein synthesis in phytohemagglutinin and IL-1 beta-treated thymocytes derived from the cecal ligation and puncture model of sepsis. RESULTS: We demonstrated that (1) there is a significant decrease in thymic weight and an increase in thymic DNA fragmentation with the characteristic apoptotic DNA "ladder" fragmentation pattern on agarose gel electrophoresis following peritoneal sepsis; (2) thymocytes in primary culture sustain a significant increase in thymocyte apoptosis following IL-2 withdrawal; and (3) peritoneal sepsis results in inhibition of phytohemagglutinin and IL-1 beta-induced thymocyte IL-2 messenger RNA accumulation and protein synthesis. CONCLUSIONS: Thymic involution following peritoneal sepsis is associated with increased thymocyte programmed cell death. Thymocyte apoptosis induced by sepsis may be the result, in part, of inhibition of IL-2 gene expression.
Animals↗
Hepatic CCAAT/enhancer binding protein (C/EBP-alpha and C/EBP-beta) expression changes with riboflavin deficiency, diet restriction and starvation in rats.
To study the role of nuclear regulatory proteins in mediating dietary effects, hepatic CCAAT/enhancer binding protein (C/EBP), mRNA and transcription rate were measured for C/EBP-alpha and C/EBP-beta in nutritional states that profoundly alter energy metabolism and growth. Weanling male Sprague-Dawley rats were fed riboflavin-sufficient (R+) or deficient (R-) diets for 4 wk. A diet-restricted, pair-fed (RP) group was maintained concurrently, because riboflavin-deficient rats voluntarily decrease food consumption by approximately 50% compared with controls. Half of each group was deprived of food for 48 h. The 4-wk treatment altered hepatic levels of both proteins (P < 0.05). C/EBP-alpha protein levels were increased -twofold by diet restriction. C/ EBP-beta protein levels were increased nearly threefold by riboflavin deficiency. Starvation had no significant effect on the expression of either protein. We investigated the mechanism responsible for increased protein by measuring steady-state mRNA levels and transcription rates for C/EBP-alpha and C/EBP-beta. In both isoforms, increases in mRNA were parallel to increases in transcription rates. The nutrient-induced changes in protein, mRNA and transcription rates could not be attributed only to alterations in serum glucagon or insulin concentrations. We conclude that 1) C/EBP-alpha and C/EBP-beta expression responds to diet but may involve different dietary signals for diet restriction vs. riboflavin deficiency; 2) the dietary regulation of C/EBP-alpha and C/EBP-beta expression seems to be controlled in part at the level of gene transcription; and 3) C/EBP-alpha and C/EBP-beta nuclear proteins, by virtue of their increased quantities, may participate in regulating altered energy metabolism and growth by influencing hepatic transcription of key metabolic enzymes.
Animals↗
Sepsis-induced release of interleukin-6 may activate the immediate-early gene program through a hypothalamic-hypophyseal mechanism.
BACKGROUND: The immediate-early gene c-fos has been implicated in transcriptional regulation after sepsis. We test the hypothesis that sepsis-induced central nervous system release of interleukin (IL)-6 regulates hepatic c-fos gene expression. METHODS: Using a stereotaxically placed intracerebral-ventricular (ICV) catheter in rats with and without hypophysectomy, we measured hepatic c-fos protein accumulation after treatment with either IL-6 or vehicle control. Using a rat cecal ligation and puncture (CLP) model, we studied the following groups: (1) sham-CLP, (2) CLP, (3) hypophysectomized sham-CLP, and (4) hypophysectomized CLP and measured hepatic c-fos mRNA. RESULTS: ICV IL-6 treatment increased hepatic c-fos protein in the IL-6-treated group compared with the vehicle-treated group, and hypophysectomy inhibited the ICV IL-6-mediated increase in c-fos protein. After peritoneal sepsis, CLP increased hepatic c-fos messenger RNA compared to either the sham-CLP or the hypophysectomized sham-CLP group, and hypophysectomy before CLP inhibited hepatic c-fos mRNA compared with the CLP group. CONCLUSIONS: ICV IL-6 results in an increase in hepatic fos protein that is mediated through a hypothalamic-hypophyseal mechanism. Peritoneal sepsis results in an increase in hepatic c-fos gene expression that may be, in part, mediated by central nervous system release of IL-6 through a hypothalamic-hypophyseal mechanism.
Animals↗
The regulation of mitochondrial fatty acid oxidation and hepatic gene expression by catecholamine.
We hypothesized that dopamine or dobutamine may alter hepatic mitochondrial fatty acid oxidation secondary to an effect on hepatic gene expression. We investigated the effect of dopamine or dobutamine on hepatic fat oxidation and gene transcription by studying the enzyme carnitine palmitoyltransferase (CPT), the rate-limiting step in hepatic mitochondrial long-chain fat oxidation. We incubated either H4IIE rat hepatoma cells or rat hepatocytes in primary cell culture with either dopamine (1, 0.1, 0.01 microgram/ml), dobutamine (1, 0.1, 0.01 microgram/ml), or vehicle control for 1, 2, 3, or 4 hr. We investigated the effect on (1) CPT mRNA (Northern or dot blotting) and the possible regulatory mechanism by incubating dopamine (0.1 microgram/ml) or dobutamine (0.1 microgram/ml) with propranolol or phentolamine, (2) CPT translation (CPT [35S]methionine incorporation), and (3) hepatic mitochondrial fatty acid oxidation ([1-14C]-palmitate oxidation to acid-soluble products). We conclude that (1) dopamine or dobutamine increases both hepatic CPT mRNA and CPT protein translation, (2) the effect on CPT mRNA is mediated by the beta-receptor, (3) the increase in hepatic mitochondrial fat oxidation induced by dopamine or dobutamine may be, in part, secondary to increased CPT transcription and translation, and (4) the significant difference in hepatic fat oxidation induced by dopamine as compared with that by dobutamine is secondary to factors other than transcriptional or translational mechanisms. We speculate that dopamine treatment in the critically ill may increase hepatic mitochondrial fatty acid oxidation and ketogenesis and that this increase in beta-oxidation may be, in part, secondary to increased CPT gene expression.
Animals↗
The possible inhibitory role of the leucine-zipper DNA binding protein c-fos in the regulation of hepatic gene expression after sepsis.
BACKGROUND: The leucine-zipper c-fos has been implicated in the regulation of gene expression. We investigated the possible role of c-fos in the regulation of hepatic gene expression after sepsis. Based on previous data demonstrating that sepsis inhibits hepatic gene expression of carnitine palmitoyltransferase (CPT), we hypothesized that c-fos may play a role in the inhibition of CPT gene expression after sepsis. METHODS: We studied c-fos gene expression after peritoneal sepsis induced by cecal ligation and puncture (CLP) or sham-CLP. To investigate the possible inhibitory role of c-fos on CPT gene transcription, we investigated the effect of c-fos on c-jun-driven CPT promoter-chloramphenicol acyltransferase reporter gene expression in a HepG2 hepatoma cell cotransfection model. To investigate the possible role of cyclic adenosine monophosphate (cAMP) in the regulation of c-fos in vivo, we treated either the sham-CLP group or the CLP group with either vehicle or cAMP. RESULTS: Peritoneal sepsis in the rat model resulted in a four-fold increase in hepatic c-fos mRNA and c-fos protein. In the cotransfection model, c-fos significantly inhibited c-jun-induced chloramphenicol acyltransferase activity. Treatment with cAMP resulted in a 50% decrease in c-fos protein in either the sham-CLP or CLP group. CONCLUSIONS: We conclude that (1) sepsis increases hepatic c-fos transcription and translation, (2) c-fos inhibits c-jun-induced CPT gene expression, and (3) cAMP probably does not directly mediate the increase in c-fos after sepsis.
Animals↗
The Ca2+ second messenger system and interleukin-1-alpha modulation of hepatic gene transcription and mitochondrial fat oxidation.
Cytokines have been implicated in the modulation of fat metabolism after sepsis. Carnitine palmitoyltransferase (CPT), the regulatory enzyme of hepatic mitochondrial long-chain fatty-acid oxidation, is involved in the control of hepatic fat oxidation in sepsis. Using either H4IIe rat hepatoma cells or rat hepatocytes in primary culture, we tested the hypothesis that interleukin-1-alpha (IL-1 alpha) would modulate CPT transcription (CPT mRNA), CPT translation (35S-methionine CPT protein incorporation), and hepatic mitochondrial oxidation of 1-Carbon 14-labeled (14C) palmitate to ketone bodies (acid soluble products). We showed that IL-1 alpha significantly increased CPT mRNA, 35S-methionine incorporation CPT protein, and hepatic mitochondrial oxidation of 1-14C-palmitate to acid soluble products. We further hypothesized that the Ca2+ second messenger system may play a role in the IL-1 alpha induction of hepatic CPT gene transcription. We showed that either calcium ionophore (A23187) or phorbol myristate acetate increased CPT gene transcription and that either calcium chelation, protein kinase C inhibition (acridine orange), or chronic exposure to phorbol myristate acetate significantly inhibited IL-1 alpha induction of CPT mRNA. We conclude that the IL-1 alpha increases in hepatic mitochondrial fatty-acid oxidation may be, in part, secondary to increased CPT gene transcription and translation and that the Ca2+ second messenger system may play an important role in IL-1 alpha induction of CPT gene transcription.
Animals↗
Enterococcal sepsis and lung microvascular injury in sheep.
In a common bile duct contamination model, we studied the effect of Streptococcus faecalis compared with Escherichia coli in sheep with chronic lymph fistulas to investigate the role of enterococcus in acute lung injury and acute sepsis. Early pulmonary hypertension in the E coli group was not expressed in the S faecalis group, probably due to a failure of S faecalis to illicit a thromboxane A2 response. In the late period, E coli was associated with significantly greater lung microvascular damage compared with S faecalis. The lack of difference between groups with respect to complement activation suggests the action of chemotactic factors, in addition to complement, mediating granulocyte aggregation, and neutropenia. In this model, S faecalis demonstrated limited pathogenicity as expressed in lung microvascular injury compared with E coli.
6-Ketoprostaglandin F1 alpha↗
The association of Escherichia coli virulence and pulmonary microvascular damage.
Bacterial virulence indicates the degree of pathogenicity of a given strain of microbe for a given host. The effect of Escherichia coli virulence on lung microvascular permeability was studied in sheep with chronic pulmonary lymph fistulas following peritoneal contamination. The study was divided into four groups: (1) wild-type E coli (WT group, 2.5 x 10(9) colony-forming units [CFUs]/kg); (2) virulent E coli (PV group, 2.3 x 10(9) CFUs/kg); (3) nonvirulent E coli (PNV group, 2.6 x 10(9) CFUs/kg); (4) high-inoculum wild-type E coli (HIWT group, 6.1 x 10(9) CFUs/kg). In the late period (two to six hours), the increase in lung lymph flow in the PV group was significantly greater than the WT, PNV, and HIWT groups, with no difference noted among groups with respect to the pulmonary artery pressure, pulmonary wedge pressure, or albumin lymph/plasma ratio. It was concluded that (1) increased E coli virulence results in increased lung microvascular damage and (2) increased lung microvascular damage as a function of E coli virulence may not be solely due to increased bacterial numbers as a function of time.
Albumins↗
Effects of biliary obstruction on hepatic clearance of bacteria.
High surgical mortality in patients with obstructive jaundice and sepsis have been attributed to reticuloendothelial system (RES) depression. The purpose of this study was to clarify the effects of mechanical biliary obstruction on RES clearance of pathogenic bacteria by comparing the phagocytic index (K) with the directly measured hepatic uptake of indium 111-labeled bacteria injected into the portal vein of normal dogs and dogs with partial (PBO) or complete biliary obstruction (CBO). No significant difference was observed between the K in normal dogs (0.19 +/- 0.08; n = 6) and that in dogs with PBO (0.24 +/- 0.06; n = 5) or CBO (0.21 +/- 0.03; n = 4). There was no significant difference in uptake of radiolabel by the liver among the three groups of dogs. In our model, biliary obstruction had no effect on hepatic RES function and may not represent a significant determinant of mortality in patients with obstructive jaundice.
Alkaline Phosphatase↗
Macrophages and translymphatic absorption represent the first line of host defense of the peritoneal cavity.
To quantitate the host defenses of the rat peritoneal cavity, nonviable radiolabeled Escherichia coli were injected intraperitoneally and clearance, leukocyte influx, and phagocytosis were examined. Macrophages (MCs) were present initially and remained relatively constant in number. The polymorphonuclear leukocyte (PMN) response began at one to two hours and was maximal at 24 to 72 hours. A previously unidentified inoculum-dependent PMN response was defined. Clearance and phagocytosis were extremely rapid, and few (less than 3%) free bacteria were present after two hours. Phagocytic activity of MCs and PMNs was identical, but MCs were numerically predominant initially and thus accounted for the majority of early phagocytosis. Thus, MC phagocytosis and clearance represent the primary line of host peritoneal defenses. We hypothesize that the subsequent inoculum-dependent PMN response may have evolved to cope with those larger inocula for which this initial response is inadequate.
Absorption↗
Effects of Escherichia coli and Bacteroides fragilis on peritoneal host defenses.
Escherichia coli and Bacteroides fragilis are common copathogens in clinical intra-abdominal sepsis, yet it is unclear how they interact synergistically in vivo. We sought to determine whether E. coli and B. fragilis, in combination but not alone, could exert a detrimental effect on the peritoneal host defenses of translymphatic absorption and bacterial phagocytosis. Our data indicated that nonviable E. coli (O18ab:K56/K7:- and O111:B4), Klebsiella pneumoniae, B. fragilis, and Bacteroides thetaiotaomicron were handled in a similar fashion by both host defenses of the peritoneal cavity. The use of 2 X 10(8) nonviable radiolabeled E. coli as a tracer and either 2 X 10(9) B. fragilis or 2 X 10(9) E. coli (either viable or nonviable) as a competing agent to inhibit host defenses demonstrated that although clearance and phagocytosis could be inhibited, the inhibition occurred to a similar degree with either E. coli or B. fragilis. Thus, B. fragilis did not compete to any greater extent than E. coli did for peritoneal clearance or opsonization and phagocytosis in vivo. These data indicate that bacterial synergy probably does not occur on the basis of reduced peritoneal clearance or by a reduction in the opsonization and phagocytosis of either organism by the copathogen. These results provide indirect support for the hypothesis that in bacterial synergy, one organism directly stimulates the growth of the other, perhaps by providing a growth factor.
Animals↗
Role of resident macrophages, peripheral neutrophils, and translymphatic absorption in bacterial clearance from the peritoneal cavity.
Microbial pathogens within the peritoneal cavity are thought to encounter three categories of host defense mechanisms: (i) removal mechanisms, which occur via diaphragmatic lymphatic absorption; (ii) killing mechanisms, in which host phagocytes act as effector cells; and (iii) sequestration mechanisms due to fibrin trapping and the formation of adhesions between visceral surfaces. We sought to define and quantitate the relative role of the first two components in an experimental rat model of Escherichia coli peritonitis in which fibrinous adhesions do not form. Intraperitoneal challenge with greater than or equal to 2 X 10(8) CFU of viable E. coli led to an initial decline in bacterial numbers followed by ongoing proliferation and greater than 50% mortality. With inocula of less than or equal to 5 X 10(7) CFU, elimination of bacteria occurred after moderate initial proliferation, and no mortality ensued. Nonviable, radiolabeled E. coli organisms were utilized to examine bacterial clearance via translymphatic absorption and phagocytosis. Both processes were extremely rapid, serving to eliminate free bacteria rapidly within the peritoneal cavity. Although macrophages and polymorphonuclear leukocytes within the peritoneal cavity demonstrated similar phagocytic capacities, the predominance of macrophages at the time of the initial bacterial insult led to the conclusion that these cells, in addition to translymphatic absorption, represent the first line of host defenses, acting to eliminate bacteria in the incipient stages of infection.
Animals↗
Comparative pulmonary effects of intraperitoneal inoculation of live v dead Escherichia coli.
We studied the effect of 2.5 X 10(9) live Escherichia coli per kilogram v 2.7 X 10(9) dead E coli per kilogram injected into the peritoneal cavity of sheep with chronic pulmonary lymph fistulas. The effects of dead E coli were compared with those of live E coli, with respect to (1) pulmonary hypertension, (2) hemodynamic failure, (3) damage to the pulmonary microvasculature, (4) systemic arterial hypoxemia, (5) neutropenia and lymphopenia, (6) thrombocytopenia and platelet aggregation, (7) plasma fibrinogen concentration, and (8) classic- and alternative-pathway hemolytic complement. The time after injection of the bacteria was divided into an early period (zero to two hours) and a late period (two to seven hours). We made two conclusions: (1) The early period effects, with the exception of the absolute neutrophil count and Pao2, were independent of bacterial viability, whereas the late period effects were strongly dependent on bacterial viability. (2) The early notable difference between the live v dead groups, with respect to the absolute neutrophil count and Pao2, could not be explained on the basis of an increase in bacterial numbers alone.
Animals↗
The adjuvant effect of peritoneal fluid in experimental peritonitis. Mechanism and clinical implications.
At laparotomy, many surgeons routinely instill crystalloid solutions into the peritoneal cavity, presumably to dilute out necrotic debris, bacteria, and adjuvant substances which foster bacterial growth. We examined the effect on mortality, bacterial growth, clearance, and phagocytosis of various volumes of saline instilled into the peritoneal cavity of rats during Escherichia coli peritonitis. Minimal intraperitoneal bacterial growth was seen after the introduction of a nonlethal inoculum of viable E. coli in 1 ml of saline, while administration of an identical inoculum in 30 ml of saline intraperitoneally (i.p.) led to increased 48-hour mortality (p less than 0.01), and associated rapid bacterial proliferation (p less than 0.01). Clearance of nonviable radiolabelled E. coli from the peritoneal cavity was delayed, bacterial association with host peritoneal leukocytes was decreased, and blood uptake of radiolabelled bacteria was diminished in animals receiving 30 ml of saline i.p., compared to controls which received the identical inoculum in 1 ml of saline i.p. The clinical relevance of these studies is manifold: (1) they provide a possible explanation why patients with ascites due to cirrhosis or the nephrotic syndrome, or those patients undergoing peritoneal dialysis are more susceptible to primary and secondary bacterial peritonitis, possibly on the basis of impaired peritoneal clearance or diminished phagocytosis and, (2) although irrigation of the peritoneal cavity with crystalloid solution would seem prudent during laparotomy, these solutions must be removed prior to closure to prevent interference with normal peritoneal host defense mechanisms.
Adjuvants, Immunologic↗
Quantitative evaluation of clinical course in surgical ICU patients: the data conform to catastrophe theory.
A response surface for critically ill patients is described. The coordinates of the three-dimensional response surface are two control variables, or state variables, related to aerobic and anaerobic metabolism, and a response variable, the A-VO2 difference. The data conform to a cusp catastrophe manifold. Cardiac insufficiency, adaptive response to stress, and sepsis may be distinguished by this model. The distinction between control and response variables is discussed.
Aerobiosis↗
Mechanisms of the adjuvant effect of hemoglobin in experimental peritonitis. VII. Hemoglobin does not inhibit clearance of Escherichia coli from the peritoneal cavity.
Hemoglobin has been shown to be a potent adjuvant in experimental Escherichia coli peritonitis, although a satisfactory mechanistic rationale is still obscure. Hemoglobin has been thought to impair intraperitoneal neutrophil function, delay clearance of bacteria from the peritoneal cavity by the normal absorptive mechanisms, or directly enhance bacterial growth. Using highly purified stroma-free hemoglobin (SFHgb), we have largely discounted any direct effect of hemoglobin on peritoneal white blood cell function. In the present study, we confirmed that uncontrolled proliferation of bacteria takes place in the presence of hemoglobin in the peritoneal cavity. Nonviable 5-iododeoxyuridine 125I-labelled bacteria were then used to directly study peritoneal clearance kinetics, eliminating the problem of bacterial growth. SFHgb had no influence on the removal of intraperitoneal bacteria. The rate of bloodstream appearance of radiolabel was similar with or without intraperitoneal SFHgb. Thus, SFHgb does not prevent clearance of bacteria from the peritoneal cavity by interfering with normal host clearance mechanisms. SFHgb may act as a bacterial growth adjuvant, either by serving as a bacterial nutrient or by suitably modifying the environment so that extensive bacterial proliferation can occur. The latter hypothesis appears to be an area in which investigation concerning the adjuvant effect of hemoglobin may prove most fruitful.
Animals↗