Protective effect of black relative to white race against non-alcoholic fatty liver disease in patients with severe obesity, independent of type 2 diabetes.

Severe obesity (body mass index ⩾35 kg m-2) and type 2 diabetes (T2D) are potent and additive risk factors for non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH). Scant available evidence indicates that black relative to white patients with severe obesity are less susceptible to NAFLD, but it is unclear if T2D abolishes this apparent racial disparity. Therefore, we compared biopsy-proven NAFLD and its progression between black (n=71) and white (n=155) patients with severe obesity stratified by presence or absence of T2D. Although prevalence of T2D was similar between races (37%, P>0.9), whites were significantly more likely than blacks to have NAFLD, NASH and advanced fibrosis (defined as bridging fibrosis and/or cirrhosis). Importantly, T2D was associated with increased odds of NAFLD, NASH and advanced fibrosis (defined as bridging fibrosis or cirrhosis) in whites only (P<0.05). In turn, a higher proportion of blacks than whites with T2D were free of NAFLD (58 versus 22%, P<0.01). These preliminary findings question translation of the powerful interconnection between T2D and NAFLD in whites with severe obesity to blacks and point to an important role of race in the pathophysiology and treatment of these diseases.

Comparing Normothermic Machine Perfusion Preservation With Different Perfusates on Porcine Livers From Donors After Circulatory Death.

The utilization of normothermic machine perfusion (NMP) may be an effective strategy to resuscitate livers from donation after circulatory death (DCD). There is no consensus regarding the efficacy of different perfusates on graft and bile duct viability. The aim of this study was to compare, in an NMP porcine DCD model, the preservation potential of three different perfusates. Twenty porcine livers with 60 min of warm ischemia were separated into four preservation groups: cold storage (CS), NMP with Steen solution (Steen; XVIVO Perfusion Inc., Denver, CO), Steen plus red blood cells (RBCs), or whole blood (WB). All livers were preserved for 10 h and reperfused to simulate transplantation for 24 h. During preservation, the NMP with Steen group presented the highest hepatocellular injury. At reperfusion, the CS group had the lowest bile production and the worst hepatocellular injury compared with all other groups, followed by NMP with Steen; the Steen plus RBC and WB groups presented the best functional and hepatocellular injury outcomes, with WB livers showing lower aspartate aminotransferase release and a trend toward better results for most parameters. Based on our results, a perfusate that contains an oxygen carrier is most effective in a model of NMP porcine DCD livers compared with Steen solution. Specifically, WB-perfused livers showed a trend toward better outcomes compared with Steen plus RBCs.

Novel CPR with periodic Gz acceleration.

The effects of periodic Gz acceleration (pGz) on cardiovascular function and hemodynamics were determined in a pig model of acute cardiopulmonary resuscitation (CPR). The application of pGz (horizontal head-to-foot oscillations) at 2 Hz increased cardiac output in fibrillated animals proportional to the amplitude of the applied acceleration force that plateaued at 0.7 G. Cardiac output in fibrillating animals was restored to 20% of the values obtained before fibrillation with pGz-CPR and arterial blood gas values were normal during this period. The central vascular pressure gradient driving blood flow was only about 6 mmHg, suggesting low vascular resistance during pGz-CPR. In another study, capillary blood flow was determined before and after pGz-CPR using colored microspheres. Capillary perfusion was detected in all tissue beds studied during pGz-CPR. Significant capillary blood flow was detected in the endocardium and brain stem during pGz-CPR that represented 39 and 197% of control values before fibrillation, respectively. Thus, the cardiac output during pGz-CPR was preferentially distributed to the myocardial and brain tissues. In a final group, animals were successfully resuscitated with return of spontaneous circulation (ROSC) after pGz-CPR for 15 min following cardiac fibrillation with a 3-min non-intervention period. Following ROSC, blood pressure was maintained at pre-arrest values for 2 h without any pharmacological or mechanical support. Arterial blood gases during the pGz-CPR and the ROSC periods were normal and not different from values obtained before fibrillation. None of the control animals (18 min of fibrillation without pGz-CPR) survived the experimental protocol and only two of these six animals briefly returned to spontaneous circulation (<20 min). In conclusion, experimental pGz-CPR produces cardiac output, capillary blood flow, and ventilation sufficient to maintain fibrillating animals for 18 min with ROSC for 2 h without support.

Regional blood flow during periodic acceleration.

OBJECTIVE: To determine whether a motion platform that imparts noninvasive periodic acceleration (pGz) forces to the body causes systemic vasodilation and changes local organ blood flow. DESIGN: Prospective paired blocked design. SETTING: Medical center research laboratory. SUBJECTS: Juvenile Yorkshire pigs. INTERVENTIONS: Juvenile pigs (12 kg) were anesthetized, paralyzed, and placed on a motion platform that oscillated at a frequency of 4 Hz and a force of approximately 0.4 G. MEASUREMENTS AND MAIN RESULTS: Regional blood flows, as assessed by colored microspheres, increased during pGz relative to values obtained before pGz. Blood flow (mL.min-1.100 g-1) significantly increased to the epicardium (71%), endocardium (93%), cerebrum (183%), brain stem (177%), renal cortex (53%), ileal mucosa (69%), gastric antral mucosa (72%), and liver (86%). Spleen and skeletal muscle blood flow increased without statistical significance, 38% and 158% with pGz, relative to paired control values. Regional blood flows returned to baseline 10 mins after discontinuation of pGz, except in the myocardial layers, where blood flow remained significantly elevated. There was no difference compared with baseline in heart rate, arterial blood gases, and blood pressure, but serum nitrite concentration was significantly higher (58%) during pGz. In another series of animals, pGz increased pulmonary artery blood flow directly proportional to the magnitude of the applied acceleration force with frequency held constant. CONCLUSIONS: Periodic sinusoidal inertial forces in the spinal axis increase blood flow to tissues. The increased blood flow is reversible and may be caused by vasodilation secondary to local mediator release. These effects may be desirable in clinical conditions of low tissue oxygen delivery and perfusion.

Noninvasive motion ventilation (NIMV): a novel approach to ventilatory support.

A motion platform was developed that oscillates an animal in a foot-to-head direction (z-plane). The platform varies the frequency and intensity of acceleration, imparting periodic sinusoidal inertial forces (pG(z)) to the body. The aim of the study was to characterize ventilation produced by the noninvasive motion ventilator (NIMV) in animals with healthy and diseased lungs. Incremental increases in pG(z) (acceleration) with the frequency held constant (f = 4 Hz) produced almost linear increases in minute ventilation (VE). Frequencies of 2-4 Hz produced the greatest VE and tidal volume (VT) for any given acceleration between +/-0.2 and +/-0.8 G. Increasing the force due to acceleration produced proportional increases in both transpulmonary and transdiaphragmatic pressures. Increasing transpulmonary pressure by increasing pG(z) produced linear increases in VT, similar to spontaneous breathing. NIMV reversed deliberately induced hypoventilation and normalized the changes in arterial blood gases induced by meconium aspiration. In conclusion, a novel motion platform is described that imparts periodic sinusoidal acceleration forces at moderate frequencies (4 Hz) to the whole body in the z-plane. These forces, when properly adjusted, are capable of highly effective ventilation of normal and diseased lungs. Such noninvasive ventilation is accomplished at airway pressures equivalent to atmospheric or continuous positive airway pressure, with acceleration forces less than +/-1 G(z).

Hemodynamic effects of periodic G(z) acceleration in meconium aspiration in pigs.

The hemodynamic effects of periodic acceleration (pG(z)), induced in the spinal axis with noninvasive motion ventilation (NIMV), were studied in a piglet model of pulmonary hypertension associated with meconium aspiration. Animals (n = 12) were anesthetized, paralyzed, intubated, and supported by conventional mechanical ventilation (CMV). Thirty minutes after tracheal instillation of meconium solution (6 ml/kg), either CMV (n = 6) was continued or NIMV (n = 6) was initiated. Changes in systemic and pulmonary hemodynamics and arterial blood gases were tracked for 2 h after aspiration. Thermodilution, cardiac output, and heart rate were not significantly different after meconium aspiration in the pG(z) group relative to the CMV controls. Aortic pressure and systemic vascular resistance were significantly lower (approximately 30%) after meconium aspiration in NIMV animals relative to CMV animals. Pulmonary arterial pressure and pulmonary vascular resistance were also significantly lower, by 100%, after aspiration of meconium in the NIMV animals compared with the CMV controls. Meconium aspiration significantly decreased total respiratory compliance by approximately 50% and increased total respiratory resistance by approximately 100% in both CMV and NIMV animals, but such alterations did not differ between the two groups. Both CMV and NIMV satisfactorily supported ventilation in these paralyzed animals. In conclusion, NIMV through pG(z) in the spinal axis decreased systemic and pulmonary vascular resistance in piglets after meconium aspiration.

Oxygen-carrying capacity during 10 hours of hypercapnia in ventilated dogs.

OBJECTIVE: To test if a relatively long-term exogenous hypercapnia, equivalent to those maintained during permissive hypercapnia, can persistently increase oxygen-carrying capacity in ventilated dogs. DESIGN: Prospective study. SETTING: Research laboratory in a hospital. SUBJECTS: Six mongrel dogs (3 males; 3 females). INTERVENTIONS: The dogs were anesthetized (30 mg/kg pentobarbital, i.v.), intubated, and cannulated in one femoral artery, one femoral vein, and the right jugular vein. The mean arterial blood pressure, heart rate, and mean pulmonary artery pressure were continuously recorded. Anesthesia, fluid balance, and normothermia were maintained. Arterial hypercapnia was generated by the addition of 60 torr dry CO2 (8 kPa) to the inspired air for 10 hrs, continuously. All subjects were paralyzed (vecuronium bromide) and ventilated with room air, while the ventilator settings were kept constant. MEASUREMENTS AND MAIN RESULTS: Arterial and venous gas exchange profiles, hemoglobin concentration, oxygen saturation, oxygen content, cardiac output, and oxygen consumption were determined, before, during, and after 10 hrs of hypercapnia, periodically. Both hemoglobin concentration and oxygen content were gradually increased during hypercapnia and reached significant levels at 8 and 10 hrs of hypercapnia, respectively. These increases continued up to 2 hrs after termination of hypercapnia. The PaO2/FIO2, as an index of arterial oxygenation, was significantly increased during the first 3 hrs of hypercapnia and then remained at the normoxic level up to 10 hrs of hypercapnia. No significant changes occurred in the mean arterial blood pressure and oxygen consumption. The heart rate and cardiac output were significantly reduced at 4 and 8 hrs of hypercapnia, respectively. The mean pulmonary artery pressure was increased throughout the hypercapnic trial. CONCLUSIONS: A relatively long-term exogenous hypercapnia can significantly increase oxygen-carrying capacity in normal ventilated dogs. Whether this effect can occur during permissive hypercapnia because of controlled ventilation in patients warrants investigation.

Acute hypercapnia increases the oxygen-carrying capacity of the blood in ventilated dogs.

OBJECTIVE: To test the hypothesis that PaCO2 levels generated during permissive hypercapnia may enhance arterial oxygenation, when ventilation is maintained. DESIGN: Prospective study. SETTING: Research laboratory in a hospital. SUBJECTS: One group of eight mongrel dogs (four male; four female). INTERVENTIONS: The dogs were anesthetized (30 mg/kg iv pentobarbital), intubated, and cannulated in one femoral artery and vein. While paralyzed with 0.1 mg/kg/hr iv vecouronium bromide, all subjects were ventilated with room air. Anesthesia was maintained, using 2 to 3 mg/kg/hr iv pentobarbital. Arterial hypercapnia at the levels generated during permissive hypercapnia was produced by stepwise increases in the dry, inspired Pco2 (PiCO2) (0, 30, 45, 60 and 75 torr [0, 4, 6, 8, and 10 kPa]; 15 mins each). MEASUREMENTS AND MAIN RESULTS: Blood gas profiles were determined at each level of hypercapnia. The minute volume was maintained at the baseline level during all exposures. Arterial hypercapnia produced gradual and significant increases in the hemoglobin concentration. These increases were approximately 6%, 7%, 11%, and 14% at PiCO2 of 30, 45, 60, and 75 torr (4, 6, 8, and 10 kPa), respectively (p < .05; repeated analysis of variance followed by Dunnett multiple comparisons test). In parallel, the oxygen content increased by approximately 6%, 7%, 11%, and 13%, respectively. During hypercapnic trials, the PaO2 remained at the normal range, whereas the dry, inspired PO2 (PiO2) was reduced from 150 to 138 torr (20 to 18.4 kPa). The average PaO2 at the highest investigated level of arterial hypercapnia was at a normal range. The hemoglobin concentration and oxygen content returned to baseline values 30 mins after hypercapnic trials. The PaCO2 and pH became normalized 15 mins after hypercapnic trials. Indirect evidence for a similar response to hypercapnia in humans is presented. CONCLUSIONS: Permissive hypercapnia due to inhaled CO2 increases oxygen-carrying capacity in dogs. The PaO2 remains at normal range even at a PiCO2 of 75 torr (10 kPa). The benefits of these effects during permissive hypercapnia, due to controlled hypoventilation, warrants investigation.

Effects of the 5-lipoxygenase inhibitor A-64077 on intestinal hypothermic organ preservation injury.

The effects of the orally active selective 5-lipoxygenase inhibitor Zileuton (A-64077, (N-1(1-benzo{b}thien-2-ylethyl)-N-hydroxyurea) were studied in a canine model of hypothermic intestinal organ ischemia-reperfusion (I/R) injury (transplant preservation injury). Forty-eighty hours of hypothermic intestinal ischemia utilizing Collin's flush, followed by 1 hr of reperfusion (transplantation) in A-64077-treated animals, resulted in a 3-fold increase in intestinal oxygen uptake and blood flow relative to the untreated controls. The postreperfusion movement of fluid from the microcirculation into the intestinal lumen significantly increased in the control animals at reperfusion, and A-64077 treatment dramatically exacerbated this phenomenon. Mucosal neutrophil infiltration, or the processes leading to infiltration, significantly increased after 48 hr of cold ischemia and 1 hr of normothermic reperfusion in the untreated animals. A similar response was observed in A-64077-treated dogs, but the absolute levels of MPO were 10-fold less relative to untreated animals, including intestinal tissue obtained before I/R. Hypothermic I/R injury in this model resulted in severe histologic injury. A-64077-treated dogs, however, demonstrated significant improvements in histologic injury. Mucosal synthesis of LTB4 rose significantly after cold I/R injury and was abrogated by A-64077 treatment. The synthesis of PGE2 significantly increased after cold I/R in both untreated and A-64077-treated dogs. The increase in PGE2 production after hypothermic I/R in the A-64077-treated animals was higher relative to the untreated control animals. In conclusion, this study indicates that arachidonic acid metabolism via the 5-lipoxygenase pathway plays a significant role in the pathophysiology of hypothermic intestinal I/R injury. Furthermore, the 5-lipoxygenase inhibitor A-64077 possesses favorable pharmacologic and biologic responses in this intestinal injury and should be considered in the clinical amelioration of intestinal transplantation preservation injury.

Arachidonic acid metabolism in intestinal hypothermic preservation injury.

The effect of hypothermic intestinal ischemia and short-term reperfusion on mucosal arachidonic acid metabolism was studied in a dog model of intestinal preservation injury. Canine intestinal segments were flushed with cold Collins solution, cold stored (4 degrees C) for either 24 or 48 h, and subsequently reperfused in the donor for 1 h. Samples of intestinal mucosa obtained before ischemia, after the ischemia period, and after the reperfusion period were placed into tissue culture, and arachidonic acid metabolites were measured in the tissue incubation media. Prostaglandin E2 (PGE2) and prostacyclin (PGI2) production significantly increased after 24 h of cold ischemia and after 1 h of reperfusion, respectively. Intestines cold stored for 48 h and after 1 h of reperfusion produced significantly elevated quantities of thromboxane B2, PGI2, PGE2, and leukotriene B4, relative to the production rates from nonischemic control tissue or tissue subjected to 48 h of hypothermic ischemia without reperfusion. Mucosal production of thiol ether leukotrienes (LTC4, LTD4, LTE4) was not altered by ischemia or reperfusion at any time of cold ischemia. The synthesis of the lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE) was not altered by hypothermic ischemia or reperfusion, but this arachidonate metabolite was produced by small intestinal mucosa in the greatest quantities. Specifically, nanogram quantities of 12-HETE were produced by intestinal mucosa compared to picogram quantities of the other metabolites measured. Significant synthesis of the delta lactone derivative of 5-hydroxyeicosatetraenoic acid was detected by HPLC in many tissue samples undergoing 48 h of ischemia and reperfusion, relative to nonischemic tissue samples. In conclusion, significant increases in arachidonate cyclooxygenase and lipoxygenase metabolites have been identified in intestinal mucosa subjected to long-term hypothermic ischemia and short-term reperfusion. Synthesis of these products increases with the duration of cold ischemia and may play a role in intestinal preservation injury.

Characterization of hypothermic intestinal ischemia-reperfusion injury in dogs. Effects of glycine.

The effects of 48 hr of hypothermic (4 degrees C ischemia) and short-term reperfusion. (I-R) on intestinal function and metabolism were studied in dogs utilizing Collins flush alone or with the putative cytoprotectant amino acid, glycine. Intestinal blood flow after hypothermic ischemia in Collins-flushed segments briefly rose at reperfusion, rapidly declined after 5 min, and plateaued over the 60-minute reperfusion period. Paired intestinal segments flushed with 5 mM glycine demonstrated parallel changes in blood flow over the reperfusion period, but the blood flow values were significantly higher (100-300%), relative to the Collins segments. Intestinal oxygen consumption (VO2) was about 50% of normal nonischemic intestinal segments at all times after reperfusion. The glycine-flushed intestinal segments significantly consumed about 100% more oxygen, relative to the paired control intestines. Intestinal fluid and protein flux into the lumen significantly increased after I-R in both glycine- and Collins-flushed segments. Mucosal tissue myeloperoxidase (MPO) activity, a biochemical marker of neutrophils, significantly increased after 48 hr of cold ischemia with Collins flush and 1 hr of reperfusion, relative to tissue obtained before ischemia. The reperfusion-induced increase in MPO activity was abolished in intestinal segments flushed with glycine. Mucosal synthesis of the chemoattractant leukotriene B4 (LTB4) significantly increased after I-R and glycine flush abolished these increases. Nitric oxide synthesis by mucosal tissue in Collins-flushed segments subjected to 48 hr of hypothermic ischemia and 1 hr of reperfusion was significantly higher, compared with nonischemic tissue or mucosal tissue subjected to cold ischemia without reperfusion. Glycine flush did not alter this pattern of NO synthesis. Light microscopic analysis in both Collins- and glycine-flushed segments revealed that intestinal hypothermic ischemia and reperfusion caused significant morphologic changes characterized by loss of villus epithelium, decreased villus height, and venous congestion. These data indicate that glycine significantly improve oxygenation after hypothermic ischemia and reperfusion and prevented the I-R-induced increase in tissue neutrophil infiltration and leukotriene synthesis.

The Kupffer cell in endotoxin tolerance: mechanisms of protection against lethal endotoxemia.

Kupffer cells (KC) of the hepatic sinusoid respond to endotoxemia by producing mediators which promote or inhibit systemic inflammatory responses. Sublethal lipopolysaccharide (LPS) pretreatment confers tolerance to the lethality of a subsequent LPS exposure. However, the precise role of the KC in endotoxin tolerance (ET) remains unclear. This study evaluated the effect of ET induction upon the rat KC production of the mediators tumor necrosis factor-alpha (TNF-alpha), prostaglandin E2 (PGE2), and interleukin-6 (IL-6), and upon the in vivo phagocytic capacity of the KCs. 3 days prior to KC isolation, age-matched rats received either 5 mg/kg LPS (ET) or normal saline (nontolerant, NT), which protected 100% of the ET rats against an LPS dose 3 days later which was lethal in 72% of NT rats. On an in vitro LPS rechallenge, ET KC produced significantly lower amounts of TNF than NT KC (p < .01). In contrast, the ET KC produced significantly more PGE2 (p < .05) and IL-6 (p < .001) than the NT KC. The percentage of KC phagocytosing fluorescent latex spheres in vivo was increased 7-fold in the ET rats. Thus, ET induction, which protects rats against subsequent lethal endotoxemia, selectively alters KC mediator production and phagocytic capacity. These findings strongly implicate the KC in the mediation of early endotoxin tolerance.

Autoregulation by eicosanoids of human Kupffer cell secretory products. A study of interleukin-1, interleukin-6, tumor necrosis factor-alpha, transforming growth factor-beta, and nitric oxide.

OBJECTIVE: Methods employed previously to analyze the secretory behavior of rodent Kupffer cells (KC) were used to examine the human KC's secretory response to lipopolysaccharide (LPS). SUMMARY BACKGROUND DATA: As the resident hepatic macrophage, the KC resides at the interface between the portal and systemic circulations. Consequently, this cell may play an integral role in the immune response to antigens and bacteria in the sinusoid. Study of cytokine production by the KC has relied predominantly on the rat as the source of these cells. Whether human KCs respond similarly to rat KCs after LPS stimulation has been a matter of speculation. METHODS: Kupffer cells obtained from seven human livers were tested under conditions identical to those used to study rat KCs. Kupffer cells rested for 12 hours after isolation were stimulated with LPS (2.5 micrograms/mL). Arginine concentration in the culture medium varied from 0.01 to 1.2 mM. To examine the role of eicosanoids, parallel culture wells received indomethacin (10 microM). Culture supernatants were assayed for interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), prostaglandin E2 (PGE2), and nitric oxide. RESULTS: Similar to the rat KC, LPS-stimulated human KCs released IL-1, IL-6, TNF-alpha, TGF-beta, and PGE2. However, unlike rat KCs, nitric oxide could not be detected, regardless of whether the human KCs were exposed to LPS, interferon-gamma (INF-gamma), or LPS + IFN-gamma. Similar to rat KCs, indomethacin prevented PGE2 release while significantly upregulating TNF-alpha, IL-1, and IL-6, but not TGF-beta, consistent with an autoregulatory control of eicosanoids over proinflammatory cytokines. As has been shown in the rat, physiologic levels of L-arginine (0.01 mM) significantly enhanced LPS-induced PGE2 secretion relative to the response in medium containing standard L-arginine concentration (1.2 mM); however, unlike the rat KC, the human's cytokine response to LPS was not downregulated by this enhanced PGE2 release. CONCLUSIONS: Although many functional features are shared by rat and human KCs, significant differences do exist. Such discrepancies reinforce the need to proceed with caution when generalizing from the results obtained in other species to human physiology.

Effects of the arachidonate 5-lipoxygenase synthesis inhibitor A-64077 in intestinal ischemia-reperfusion injury.

This study was designed to characterize the role of arachidonate 5-lipoxygenase metabolism during experimental intestinal ischemia-reperfusion (I/R) injury. Canines were subjected to 3 hr of intestinal ischemia followed by 1 hr of normobaric reperfusion. Intestinal ischemia followed by 1 hr of normobaric reperfusion. Intestinal mucosal leukotriene B4 and leukotriene C4 synthesis tripled after ischemia and ischemia-reperfusion, relative to non-ischemic intestinal mucosa. The flux of fluid and protein from the capillary to the lumen also increased 3-fold after I/R. The selective 5-lipoxygenase synthesis inhibitor A-64077 (Ziluten, 5 mg/kg, p.o.) abolished I/R-induced leukotriene synthesis and reduced transluminal protein flux (50%) but did not influence the lumenal accumulation of fluid after I/R. In animals treated with the leukotriene synthesis inhibitor, intestinal vascular resistance significantly declined during the imposed ischemia period and after 60 min of reperfusion. Mucosal myeloperoxidase activity, a biochemical marker for tissue neutrophils, rose significantly after I/R, and these increases were prevented with the 5-lipoxygenase synthesis inhibitor. In other experiments, the lipoxygenase inhibitor nondihydroguaretic acid produced similar results to those of A64077. In an attempt to determine the source of mucosal leukotrienes during intestinal I/R, we imposed in vitro ischemia and reperfusion on normal mucosal tissue in a blood-free environment. Mucosal tissue was incubated in Krebs buffer under oxygen for 3 hr to simulate the control condition, under nitrogen for 3 hr to simulate ischemia and under nitrogen for 2 hr followed by oxygen for 1 hr to simulate I/R.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet activating factor synthesis and metabolism in intestinal ischaemia-reperfusion injury.

The object of this study was to characterize the synthesis and metabolism of platelet activating factor (PAF) by intestinal mucosa subjected to ischaemia-reperfusion injury. Canine intestinal mucosa produced 16:0-PAF, 18:0-PAF, and high levels of the corresponding lyso- PAF metabolites. Three h of intestinal ischaemia and ischaemia followed by 1 h of reperfusion did not affect the synthesis or metabolism of PAF by intestinal mucosa. Intestinal mucosa elaborated a factor that rapidly hydrolyzes PAF to lyso-PAF. The observed hydrolysis rate was not altered by ischaemia or ischaemia and reperfusion. In conclusion, this study suggests that intestinal mucosa produces PAF and rapidly hydrolyzes PAF. The PAF synthesis and metabolism rates of intestinal mucosa is not altered by ischaemia reperfusion in this model under the imposed conditions.

Characterization of platelet-activating factor receptors on canine T lymphocytes.

The ability of purified canine T lymphocytes to selectively bind platelet activating factor (PAF) was characterized. Authentic radiolabeled PAF rapidly and selectively bound to T lymphocytes and reached saturation within 1 min. This binding was reversible and highly selective for (R) PAF because (S) PAF, lyso-PAF, and diacyl PAF did not displace the bound (R) PAF probe. Only increasing quantities of chemically pure (R) PAF displaced the radiolabeled (R) PAF probe. The binding maximum of PAF was determined to be 35 pM per 2 x 10(6) lymphocytes. Competitive radioligand binding studies and Scatchard analysis indicated a single class of high affinity receptors with a dissociation constant of 0.077 nM and a receptor density of 6419 receptors per cell. The ability of purified canine T lymphocytes to hydrolyze PAF to the biologically inactive metabolite lyso-PAF was also studied. Over a 30-min incubation period, about 5% of PAF was metabolized to lyso PAF. This rate of PAF hydrolysis was the same as the rate observed with the media without cells, suggesting a small degree of nonenzymatic hydrolysis. The effects of varying concentrations of authentic PAF on intracellular free Ca2+ release in purified T lymphocytes was evaluated using the fluorescent probe Fura-2 and excitation-emission spectrofluorometry. PAF below the concentration of 1.0 nM did not significantly increase intracellular Ca2+ in T lymphocytes. More than 1 nM PAF, intracellular-free Ca2+ modestly, but significantly, increased in T lymphocytes. In other experiments, canine PBMC proliferated in response to Con A and in the one way MLR. These proliferative responses were abolished when the selective PAF receptor antagonist SC-47014A was added to the culture medium. In the MLR, this inhibitory effect was dependent on the length of time that the antagonist was in the culture. Specifically, inhibition of proliferation was incrementally reversed when the PAF antagonist was introduced progressively later into the 7-day MLR stimulation period, suggesting that PAF receptor blockade prevents an MLR response from occurring, but is unable to suppress an existing MLR response. Although the Con A-induced mononuclear cell proliferation was abolished with PAF receptor antagonists, the addition of authentic biologically active PAF or PAF analogs did not alter the proliferative response to Con A. In conclusion, canine T lymphocytes possess high affinity receptors for PAF. These binding sites are highly selective and reversible. PAF binding slightly increases intracellular free Ca2+ in T lymphocytes and appears to be involved in lymphocyte proliferation in response to soluble plant mitogen and alloantigen.

Lanthanide "blockade" of antigen-presenting cells suppresses lymphocyte proliferation by inducing nitric oxide synthesis.

Introduction of antigen into the portal venous circulation can induce a state of antigen-specific tolerance. Reversal of this phenomenon by the administration of gadolinium chloride (GD) before portal venous inoculation with antigen has been thought to result from the inhibition of macrophage phagocytosis by GD, thereby permitting passage of antigen through the hepatic sinusoids into the systemic circulation where sensitization can occur. We recently demonstrated that i.v. GD also greatly enhances the ability of the hepatic macrophage, or Kupffer cell (KC), to suppress lymphocyte proliferation in vitro. In this study, lymphocytes from ovalbumin (OVA)-immunized Lewis rats were cocultured with OVA plus KC or adherent splenocytes from syngeneic rats injected i.v. with GD or saline. Whereas small numbers of either hepatic or splenic untreated macrophages enhanced lymphocyte proliferation, both macrophage populations treated with GD were markedly inhibitory. Neither the presence of lymph node macrophages nor of normal KC was able to prevent the effect of GD-treated KC indicating a mechanism of active suppression rather than defective macrophage antigen uptake, processing, and presentation. While cyclooxygenase inhibition had no effect, the addition of inhibitors of nitric oxide synthase, i.e., aminoguanidine and NG-methyl-L-arginine, fully reversed the suppression of proliferation by GD-treated macrophages. Nitrite, but not PGE2, levels correlated with the degree of inhibition by the GD-treated macrophages. The suppression of lymphocyte proliferation and elevated supernatant nitrite levels associated with the presence of GD-treated macrophages were reversed when the macrophages were preincubated with EDTA indicating that GD's effect was directly on macrophages and due to its persistence in cultured cells. The findings of this study suggest that GD's in vivo prevention of portal venous tolerance may be due to enhanced nitric oxide synthesis whose effect on lymphocytes could be to inhibit adhesion or replication in the hepatic sinusoid.

Lymphocyte suppression by Kupffer cells prevents portal venous tolerance induction: a study of macrophage function after intravenous gadolinium.

Ag administration into the portal vein can induce specific tolerance to that Ag, known as portal venous tolerance. Because intrahepatic mechanisms of tolerance induction are still largely undefined, we studied the in vitro response of OVA-sensitized Lewis rat lymphocytes to OVA presented by normal syngeneic rat Kupffer cells (KC) or KC that had been treated in vivo with gadolinium chloride (GD), a rare earth metal, which prevents the induction of portal venous tolerance. KC (2.5 x 10(4)) were able to present OVA to 5 x 10(5) OVA-sensitized APC-depleted lymphocytes as effectively as could lymph node APC. However, the use of GD-treated KC was associated with a significantly (P < 0.001) impaired response of OVA-sensitized APC-depleted lymphocytes to OVA. Although GD nearly abrogated in vivo phagocytosis of fluorescent latex beads by both KC and adherent splenocytes, expression of the class II MHC molecule (Ia) by KC was only slightly reduced by GD treatment. Unresponsiveness of OVA-sensitized lymphocytes to OVA was not related to enhanced PGE2 release by GD-treated KC, as determined both by PGE2 levels in culture supernatants and by cyclooxygenase inhibition. However, the marked ability of GD-treated KC to inhibit the response to OVA by primed lymph node populations containing lymphocytes and APCs supports an active suppressive mechanism. Prevention of the induction of portal venous tolerance by GD, the lack of in vitro KC Ag presentation by GD-treated KC, and active immunosuppression by GD-treated KC support a model of tolerance induction within the liver wherein Ag presentation and lymphocyte proliferation are necessary for the development of tolerance.

Prostaglandin E2 downregulates Kupffer cell production of IL-1 and IL-6 during hepatic regeneration.

The mammalian liver possesses the ability to regenerate to its original size after a 70% partial hepatectomy (PHx). The capacity of rat Kupffer cells (KC) isolated at specific intervals after PHx to produce interleukin (IL)-1, IL-6, and prostaglandin E2 (PGE2) in response to endotoxin [lipopolysaccharide (LPS)] stimulation was evaluated in standard RPMI 1640 (1,200 microM L-arginine) and arginine-depleted RPMI 1640 (< 10 microM L-arginine) media. Because KC function in an environment in which high arginase activity results in negligible L-arginine levels, the 10 microM L-arginine RPMI 1640 was used to simulate the hepatic microenvironment. Regenerating liver KC 12-120 h after PHx responded to LPS with a significantly greater (P < 0.05) production of IL-1 and IL-6 in standard RPMI 1640. This enhancement of regenerating liver KC to produce IL-1 and IL-6 was increased (P < 0.05) by placing these same KC in 10 microM arginine RPMI 1640 culture media. During the same time period, regenerating liver KC produced significantly elevated (P < 0.01) PGE2, again with greater differences in the low-arginine media. In vivo KC PGE2 blockade by indomethacin (5 mg/kg) significantly (P < 0.05) inhibited hepatic regeneration. When the cyclooxygenase inhibitor indomethacin (10 microM) was added to cultures, the production of PGE2 by KC was prevented, and in arginine-depleted cultures, IL-1 and IL-6 production was upregulated (P < 0.05). We conclude that during hepatic regeneration, KC IL-1 and IL-6 production is elevated and is controlled in an autoregulatory fashion by elevated KC PGE2 production.