Is circulating endotoxin the trigger for the systemic inflammatory response syndrome seen after injury?

OBJECTIVE: Patients with severe traumatic or burn injury and a mouse model of burn injury were studied early after injury to determine the relation of plasma endotoxin (lipopolysaccharide [LPS]) to the production of proinflammatory cytokines and subsequent resistance to infection. SUMMARY BACKGROUND DATA: Elevated levels of plasma LPS have been reported in patients after serious injury. It has been suggested that circulating LPS may be a trigger for increased proinflammatory cytokine production and may play a role in the septic syndromes seen in a substantial portion of such patients. Yet, despite multiple reports of leakage of LPS from the gut and bacterial translocation after injury in animal models, there is little direct evidence linking circulating LPS with production of inflammatory mediators. METHODS: The authors studied serial samples of peripheral blood from 10 patients with 25% to 50% surface area burns and 8 trauma patients (injury Severity Score, 25-57). Patients were compared with 18 healthy volunteers. The study was focused on the first 10 days after injury before the onset of sepsis or the systemic inflammatory response syndrome. Plasma samples were assayed for LPS, and adherent cells from the blood were studied for basal and LPS-stimulated production of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and interleukin-6 (IL-6). The correlation of increased plasma LPS with TNF-alpha production was studied as was the association of increased plasma LPS and increased TNF-alpha production with subsequent septic complications. We also studied a mouse model of 25% burn injury. Burn mice were compared with sham burn control subjects. Plasma samples were assayed at serial intervals for LPS, and adherent cells from the spleens were studied for basal- and LPS-stimulated production of TNF-alpha, IL-1 beta, and IL-6. Expression of the messenger RNAs for IL-1 beta and TNF-alpha also was measured. The relation of increased TNF-alpha production with mortality from a septic challenge, cecal ligation and puncture (CLP), was determined. Finally, the effect of administration of LPS to normal mice on subsequent mortality after CLP and on TNF-alpha production was studied. RESULTS: Elevated plasma LPS (> 1 pg/mL) was seen in 11 of the 18 patients within 10 days of injury and in no normal control subjects. In this period, patients as compared with control subjects showed increased stimulated production of TNF-alpha, IL-1 beta, and IL-6. Increased TNF-alpha production was not correlated with elevated plasma LPS in the same patients. Neither increased plasma LPS nor increased TNF-alpha production early after injury was correlated with subsequent development of systemic inflammatory response syndrome or sepsis in the patients. Burn mice, as compared with sham burn control subjects, showed elevated plasma LPS levels chiefly in the first 3 days after injury. Increased stimulated production of proinflammatory cytokines by adherent splenocytes from the burn mice also was seen at multiple intervals after injury and did not correlate with mortality from CLP. Increased production of TNF-alpha and IL-1 beta was associated with increased expression of messenger RNAs for these cytokines. Finally, two doses of 1 ng LPS administered 24 hours apart to normal mice had no effect on mortality from CLP performed 7 days later nor on the production of TNF-alpha at the time of CLP. CONCLUSIONS: These findings call into question the idea that circulating LPS is the trigger for increased proinflammatory cytokine production, systemic inflammatory response syndrome, and septic complications in injured patients.     

Differential regulation of monocyte/macrophage cytokine production by pressure

BACKGROUND: Cytokine production by macrophages is essential for the inflammatory response. Normal human interstitial tissue pressure is 20 to 30 mm Hg, but generally decreases in acute inflammation. METHODS: We compared the effect of 20 mm Hg increased pressure (approximating normal interstitial tissue pressure) with that of ambient pressure (resembling pressure in inflamed tissues) on tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta production by undifferentiated (monocytic) and PMA (phorbol 12-, myristate 13-acetate)-differentiated (macrophage-like) THP-1 cells with or without lipopolysaccharide (LPS) (10 ng/mL). RESULTS: Pressure stimulated spontaneous macrophage TNF-alpha secretion (30.5 +/- 6.3 vs. 49.1 +/- 2.8 pg/mL, P <.02), but not monocyte TNF-alpha secretion. Pressure did not stimulate IL-1beta release. As expected, LPS increased basal cytokine release. After LPS stimulation, pressure still tended to stimulate macrophage TNF-alpha, but inhibited monocyte TNF-alpha secretion (P <.05). In contrast, pressure inhibited IL-1beta release by both LPS-treated monocytes (986 +/- 134 vs. 595 +/- 226 pg/mL, P <.02) and macrophages (3,112 +/- 229 vs. 979 +/- 61 pg/mL, P <.01). CONCLUSIONS: Extracellular pressure may regulate TNF-alpha and IL-1beta secretion differentially by monocytes and macrophages.     

Ketamine suppresses proinflammatory cytokine production in human whole blood in vitro.

The production of proinflammatory cytokines, such as tumor necrosis factor (TNF) a, interleukin (IL)-6, and IL-8, increases in patients with sepsis; marked production causes organ failure and septic shock. We previously reported that ketamine suppressed lipopolysaccharide (LPS)-induced TNF-alpha production in mice. However, there are no reports on the effect of ketamine on cytokine production in human whole blood. Therefore, in this study, we investigated the efficacy of ketamine on LPS-induced TNF-alpha, IL-6, and IL-8 production and recombinant human (rh) TNF-a-induced IL-6 and IL-8 production in human whole blood. After adding different doses of ketamine to whole blood, the blood was stimulated with LPS or rhTNF. After incubation, the plasma TNF-alpha activity and IL-6 and IL-8 concentrations were measured using the L929 cell cytotoxic assay or an enzyme-linked immunoassay. Ketamine significantly suppressed LPS-induced TNF-alpha production at concentrations >20 microg/mL. At concentrations >100 microg/mL, ketamine also significantly suppressed both LPS-induced and rhTNF-induced IL-6 and IL-8 production. In this study, we demonstrated that ketamine directly inhibits the production of proinflammatory cytokines such as TNF-alpha, IL-6, and IL-8 in human whole blood. IMPLICATIONS: We found that ketamine suppressed lipopolysaccharide-induced tumor necrosis factor alpha, interleukin (IL)-6, and IL-8 production and recombinant human tumor necrosis factor-induced IL-6 and IL-8 production in human whole blood. Ketamine directly suppresses proinflammatory cytokine production.     

Activation of cytokine synthesis by systemic infusions of lipopolysaccharide and peptidoglycan in a porcine model in vivo and in vitro

BACKGROUND: The incidence of gram-positive and mixed bacterial infections in surgical patients has increased, and there has been an alarming rise in the number of drug-resistant bacteria. Peptidoglycan (PepG) is a cell wall component of gram-positive bacteria that stimulates inflammatory responses both ex vivo and in vivo. The systemic effects of PepG on inflammation have not been studied in a large animal model. METHODS: Anesthetized pigs were subjected to 8-h continuous intravenous infusions of lipopolysaccharide (LPS) (4 mcg/kg/h), PepG (40 mcg/kg/h), LPS plus PepG, or saline. The concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8, and IL-10 were measured in the plasma prior to infusion (time 0) and thereafter every second hour until the end of the experiments. Heparinized whole blood samples drawn at time 0 and after a 6-h infusion of LPS or PepG were incubated ex vivo with PepG (10 mcg/mL), LPS (10 ng/mL), or a combination of PepG and LPS to study the immunologic consequences of systemic inflammation. Concentrations of TNF-alpha, IL-8, and IL-1beta were measured in the supernatant liquids. RESULTS: In vivo, there was transient upregulation of TNF-alpha after infusion of LPS, PepG, or the combination. Interleukin-6 and IL-8 were upregulated by LPS but not by PepG. In vitro studies of whole blood obtained at time 0 revealed a synergistic effect of LPS and PepG on the release of TNF-alpha. Incubation of whole blood obtained after 6 h of infusion of LPS or PepG revealed tolerance and cross-tolerance between the two bacterial components in the induction of TNF-alpha, IL-8, and IL-1beta. CONCLUSIONS: Peptidoglycan is a potent inducer of TNF-alpha in this large animal model. Peptidoglycan and LPS synergized to increase the formation of the proinflammatory cytokine TNF-alpha. The study demonstrates for the first time the development of tolerance and cross-tolerance between LPS and PepG in a large animal model. These phenomena could be of importance for the signs and symptoms of sepsis.     

An association between inflammatory state and left ventricular hypertrophy in hemodialysis patients

This study was performed to investigate the potential relationship between left ventricular hypertrophy (LVH) and proinflammatory cytokines in hemodialysis (HD) patients and the effect of HD on cytokine production. Serum interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) measurements and echocardiographic studies were performed in 35 stable HD patients. A variety of probable risk factors for LVH including age, HD duration, blood pressure (BP), body mass index, lipid profile, hemoglobin, albumin, parathormone and homocysteine levels were also investigated. Additionally, the effect of HD procedure on cytokine levels was evaluated. Predialysis serum levels of IL-1beta, IL-6, TNF-alpha, and homocysteine in HD patients were compared with 12 healthy subjects. Left ventricular hypertrophy was demonstrated in 20 (57%) of HD patients by echocardiography. Left ventricular mass index (LVMI) was correlated positively with systolic BP (r=0.556, p=0.001), diastolic BP (r=0.474, p=0.004), and serum levels of TNF-alpha (r=0.446, p=0.009). Multiple regression analysis showed that systolic BP and TNF-alpha levels were significant independent predictors of LVH. No relationship was observed between LVH and other parameters. The mean predialysis serum level of IL-6 was significantly higher in HD patients compared to healthy controls (15.7 +/- 8.7 vs. 7.3 +/- 0.7 pg/ mL, p=0.001). Predialysis serum levels of TNF-alpha in HD patients were higher when compared to healthy subjects, but the difference was not statistically significant (8.3 +/- 3 vs. 7 +/- 1.45 pg/mL, respectively, p>0.05). However, serum levels of IL-6 and TNF-alpha significantly elevated after HD, when compared to predialysis levels (from 15.7 +/- 8.7 to 17.8 +/- 9.5 pg/mL, p=0.001 and from 8.3 +/- 3.0 to 9.9 +/- 3.5 pg/mL p=0.004, respectively). As a conclusion, in addition to BP, proinflammatory cytokines, TNF-alpha in particular, seem to be associated with LVH in ESRD patients.     

Evidence for interleukin-1 beta production by cultured normal human osteoblast-like cells.

To determine if bone cells produce interleukin-1 beta (IL-1 beta), a potent bone resorption-stimulating agent, we studied well-characterized, nearly homogeneous cultures of normal human osteoblast-like (hOB) cells. With four strains of such cells, vehicle-treated cultures produced minimal IL-1 beta (mean +/- SEM, 1.3 +/- 0.3 pg/ml per 10(6) cells per 24 h) and showed dose-dependent (r = 0.99) increases to 2.2 +/- 0.7, 5.0 +/- 0.9, or 17.8 +/- 6.7 pg/ml, respectively, after treatment with lipopolysaccharide (LPS) at 3, 10, or 30 micrograms/ml (for increases after 10 and 30 micrograms/ml treatments, P less than 0.05). After treatment with tumor necrosis factor alpha (TNF-alpha) at 10 U/ml, IL-1 beta increased to 16.2 +/- 3.7 pg/ml (P less than 0.05). Neither 17 beta-estradiol nor bovine parathyroid hormone(1-34) (each at 10 nM), alone or in combination with LPS or TNF-alpha, affected IL-1 beta release. Northern blot analysis of total cellular RNA preparation revealed a single hybridization band at 1.9 kb when probed with a partially deleted cDNA for human IL-1 beta. The steady-state IL-1 beta mRNA levels showed a significant increase with LPS treatment and a lesser increase with TNF-alpha treatment in hOB cells. Moreover, TNF-alpha produced an even greater increase in IL-1 mRNA in HOBIT cells, a well-differentiated clonal cell line derived from normal hOB cells transfected with the SV40 large T antigen. We conclude that human cells of the osteoblast lineage produce IL-1 beta in response to well-recognized stimuli for IL-1 release from responsive tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

TNF Alpha−308 Genotype and Renin–Angiotensin System in Hemodialysis Patients: An Effect on Inflammatory Cytokine Levels?

BACKGROUND: Renin-angiotensin system (RAS) was suggested to modulate inflammatory cytokine production. Angiotensin II was consistently shown to increase production of tumor necrosis factor alpha (TNF-alpha). However, inflammatory cytokines and RAS were modulated by genetic polymorphisms such as TNF-alpha-308 G > A and angiotensin-converting enzyme (ACE) I/D gene polymorphisms. The aim of this study was to investigate the effects of ACE and TNF-alpha genotypes on inflammatory cytokines in hemodialysis (HD) patients. METHODS: ACE I/D and TNF-alpha-308 G > A genotypes, pre- and postdialysis plasma renin activity (PRA), serum ACE, interleukin-1 beta (IL-1beta), and TNF-alpha levels were determined in 22 HD patients. RESULTS: Predialysis serum ACE activity is correlated with TNF-alpha (r = 0.63; P = 0.01), and PRA was correlated with IL-1beta levels (r = 0.49; P = 0.02). Pre/postdialysis IL-1beta and TNF-alpha were similar in DD and II/ID ACE genotypes. Predialysis TNF-alpha and IL-1beta (32.4 +/- 5; 35.1 +/- 4.2 vs. 28.1 +/- 3.7; 26.5 +/- 6.2 pg/mL; P < 0.05) and postdialysis TNF-alpha levels (30.4 +/- 1.4 vs. 28.4 +/- 0.82 pg/mL; P < 0.05) were significantly higher in TNF1/2 than TNF1/1 patients. CONCLUSION: ACE and TNF-alpha-308 G > A (1/2) gene polymorphisms may contribute to modulation of proinflammatory cytokine production and hence chronic inflammation in HD patients.     

Heat shock protein 27 inhibits apoptosis in human neutrophils.

BACKGROUND: Prolonged neutrophil(PMN) survival has been implicated in tissue injury following sepsis. A variety of bacterial products have been identified which inhibit PMN apoptosis including lipopolysaccharide(LPS). Extracellular heat shock proteins(Hsp) have recently been identified as potent regulatory signals for the innate immune system during the inflammatory response. We hypothesized that Hsp 27 can affect PMN phenotype with respect to apoptosis and cytokine profile. MATERIALS AND METHODS: PMN were isolated from the peripheral blood of healthy human volunteers by red blood cell sedimentation and gradient centrifugation. Cells were placed in media and cultured for 18 h with and without recombinant human Hsp 27 at various concentrations. In parallel experiments, PMN were stimulated with LPS, a known inhibitor of PMN apoptosis, for comparison. Apoptosis was quantified using annexin V and propidium iodide staining with flow cytometric analysis. Culture supernatants were assayed for secretion of TNF-alpha, IL-10, and IL-12. RESULTS: Hsp 27 significantly inhibits PMN apoptosis [control; 81.8 +/- 3.6%, vs Hsp 27, 60.4 +/- 4.1% p < 0.05]. The reduction is similar to that signaled by LPS, alone. Together their effect is not synergistic. The Hsp 27 response is dose-dependent. Hsp 27 does not induce secretion of TNF-alpha, IL-10, or IL-12, whereas LPS does signal IL-12 and TNF-alpha secretion. CONCLUSION: These data demonstrate that exogenous Hsp 27 may play a role in neutrophil-mediated tissue injury during trauma and sepsis via its ability to inhibit neutrophil apoptosis. However, Hsp 27 does not significantly alter neutrophil phenotype with respect to cytokine production profile.     

Endotoxin desensitization of human mononuclear cells after cardiopulmonary bypass: role of humoral factors

BACKGROUND: The ability of leukocytes to release proinflammatory cytokines on lipopolysaccharide stimulation in vitro is impaired after cardiopulmonary bypass (CPB). This study tested contribution and interaction of humoral factors in altered leukocyte responsiveness to lipopolysaccharide. METHODS: Whole blood and isolated peripheral-blood mononuclear cells (PBMCs) from 10 patients obtained after induction of anesthesia (T1) and 20 min (T2) and 24 h (T3) after CPB were cultured in the absence or presence of lipopolysaccharide and assessed for release of tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-1beta and their functional antagonists, IL-1 receptor antagonist (IL-1ra) and IL-10. In addition, dose-response characteristics and interaction of IL-10 and norepinephrine as modulators of TNF-alpha release were studied. RESULTS: Cardiopulmonary bypass induced release of antiinflammatory (T2: IL-10: median 25 pg/ml, 25th-75th percentile 9-42; IL-1ra: median 1,528 pg/ml, 25th-75th percentile 1,075-17,047; P < 0.05 compared with T1) but failed to induce proinflammatory cytokines (T2: TNF-alpha: median 0 pg/ml, 25th-75th percentile 0-6; IL-1beta: median 1 pg/ml, 25th-75th percentile 0-81; nonsignificant). Removal of plasma at T2 increased TNF-alpha response to lipopolysaccharide (+83.8%; P < 0.05), whereas it suppressed IL-10 (-36.8%; P < 0.05). Similarly, incubation of PBMCs (T1) with plasma obtained after CPB (T2) as well as addition of IL-10 or norepinephrine in concentrations present in plasma after CPB led to a reduced lipopolysaccharide-stimulated TNF-alpha and an increased IL-10 response. Coadministration of norepinephrine and IL-10 had synergistic effects. Although pretreatment with an anti-IL-10 antibody and labetalol before addition of plasma obtained at T2 largely restored the TNF-alpha response in vitro, their addition post-treatment failed to restore the monocytic TNF-alpha response. CONCLUSIONS: Plasma contains interacting factors that inhibit the release of TNF-alpha and increase the release of IL-10, presumably attenuating the inflammatory response to CPB. Although norepinephrine fails to induce a cytokine response in the absence of other stimuli, its administration seems to augment the antiinflammatory IL-10 response while attenuating the TNF-alpha response.     

Inosine exerts a broad range of antiinflammatory effects in a murine model of acute lung injury

OBJECTIVE: To investigate the effects of inosine on the acute lung inflammation induced by lipopolysaccharide (LPS) in vivo and on the activation and cytotoxicity elicited by proinflammatory cytokines on human lung epithelial (A549) cells in vitro. SUMMARY BACKGROUND DATA: Inosine is an endogenous purine recently shown to exert immunomodulatory and antiinflammatory effects. METHODS: Mice challenged with intratracheal LPS (50 microg) were treated after 1, 6, and 12 hours with inosine (200 mg/kg intraperitoneal) or vehicle. After 24 hours, bronchoalveolar lavage fluid was obtained to measure proinflammatory (tumor necrosis factor-alpha [TNF-alpha], interleukin [IL]-1beta, IL-6), and antiinflammatory (IL-10, IL-4) cytokines, chemokines (MIP-1alpha and MIP-2), myeloperoxidase activity and total cell counts, nitric oxide production, and proteins. Lung histology and immunohistochemical detection of 3-nitrotyrosine, a marker of nitrosative stress, were performed in inflated-fixed lungs. In vitro, cell viability and production of the chemokine IL-8 were evaluated in A549 cells stimulated with a mixture of cytokines in the presence or absence of inosine. RESULTS: Inosine downregulated the LPS-induced expression of TNF-alpha, IL-1beta, IL-6 and MIP-2 and tended to reduce MIP-1alpha, whereas it enhanced the production of IL-4. Total leukocyte counts, myeloperoxidase, nitric oxide production, and proteins were all significantly decreased by inosine. The purine also improved lung morphology and suppressed 3-nitrotyrosine staining in the lungs after LPS. Inosine attenuated the cytotoxicity and the expression of IL-8 induced by proinflammatory cytokines in A549 cells. CONCLUSIONS: Inosine largely suppressed LPS-induced lung inflammation in vivo and reduced the toxicity of cytokines in lung cells in vitro. These data support the proposal that inosine might represent a useful adjunct in the therapy of acute respiratory distress syndrome.     

Induction of higher expression of IL-beta and TNF-alpha,lower expression of IL-10 and cyclic guanosine monophosphate by pulmonary arterial hypertension following cardiopulmonary bypass

OBJECTIVE: Pulmonary arterial hypertension [PAH] and cardiopulmonary bypass [CPB] induce systemic inflammatory cytokines that are critical factors related to postoperative mortality of open heart surgery. We studied the expression of proinflammatory cytokines and cyclic guanosine monophosphate [cGMP] in patients suffering from PAH after CPB. METHODS: Seventy-six patients who underwent valve replacement surgery were recruited and divided into two groups according to their pulmonary arterial pressure [< 50 mmHg for Group A and > or = 50 mmHg for Group B]. Blood samples were taken to measure the concentrations of interleukin-1 beta [IL-1 beta], tumour necrosis factor alpha [TNF-alpha], interleukin-10 [IL-10] and cGMP.RESULTS: IL-1 beta and TNF-alpha were significantly higher in Group B [28.6 +/- 9.1 mmHg] than in Group A [65.8 +/- 10.2 mmHg] at baseline. After CPB, IL-1 beta of both groups rose significantly, while only TNF-alpha of Group B rose significantly higher. There were significant differences between the two groups after CPB. IL-10 and cGMP in Group B were lower than in Group A at baseline. They all decreased significantly after CPB. Significant differences were seen between the groups after CPB. CONCLUSION: Patients suffering from PAH had different levels of proinflammatory and anti-inflammatory cytokines compared to normal patients. PAH aggravates the production of IL-1 beta and TNF-alpha, while it decreases the production of IL-10 and cGMP after CPB.     

Effects of anaesthesia based on high versus low doses of opioids on the cytokine and acute-phase protein responses in patients undergoing cardiac surgery

Background : Cardiac surgery with cardiopulmonary bypass (CPB) evokes a systemic inflammatory response involving the proinflammatory cytokines tumor necrosis factor-α (TNFα), interleukin (iL)-1, IL-6, IL-8 and anti-inflammatory cytokines such as IL-10. Like IL-10, opioids downregulate the immune responses in vivo and in vitro, including the activity of the cytokine-producing monocytes and granulocytes. The proinflammatory cytokines are potent inducers of the hepatic acute-phase protein synthesis. The aim of the present study was to investigate if choice of anaesthesia, based on high-dose opioids (fentanyl) versus low-dose opioids influenced the release of IL-6, IL-8, and IL-10. Secondly, it was investigated whether serum amyloid P-component (SAP) is an acute-phase protein in man such as C-reactive protein (CRP), with which it is physically and structurally related. Methods : Sixteen patients submitted to elective coronary artery bypass grafting (CABG) surgery were randomized to either low-dose opioid anaesthesia consisting of thoracic epidural analgesia combined with inhalational anaesthesia (group I) or high-dose fentanyl anaesthesia (group II). From each patient 18 blood samples were taken perioperatively. Cytokine analyses were performed with ELISA, CRP and SAP mere measured with rocket immunoelectrophoresis (REI). Results : Surgery and CPB elicited a marked, transient and almost simultaneous proinflammatory and anti-inflammatory cytokine response with no differences between the groups. The cytokine levels returned to preoperative levels 1–3 d after operation. Anaesthesia and surgery did not affect SAP plasma levels while patients showed a major increase in CRP concentrations preceding the cytokine responses. Conclusion : CABG performed during two different anaesthetic techniques, high-dose fentanyl versus low-dose opioid anaesthesia, elicited a well-defined cytokine response with minor variation in the time course of each cytokine. The cytokine production was not modified by type of anaesthesia. Finally, SAP is not an acute-phase protein in men.     

Ganciclovir pharmacokinetics and cytokine dynamics in renal transplant recipients with cytomegalovirus infection

Ganciclovir is considered to be the first-line treatment for cytomegalovirus (CMV) in renal transplant recipients. This infection is also associated with elevations of specific plasma cytokines post-transplantation. To investigate daily cytokine response to therapy and ganciclovir pharmacokinetics, 4 transplant recipients (3 males, 1 female) with stable renal allograft function diagnosed with CMV infection were enrolled less than 4 months post-transplant. A creatinine clearance (ClCr) was generated by the Cockroft-Gault (C-G) equation (range: 42.3-68.5 mL/min) to determine ganciclovir dosing. Blood samples were collected for ganciclovir and cytokine [including interleukin (IL)-1beta, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, TNF-alpha, GM-CSF, and interferon (IFN)-gamma analyses after 7 d of intravenous (i.v.) ganciclovir (dosage range: 165-400 mg daily) therapy and again after 7 d of oral (p.o.) ganciclovir (dosage range: 1000 mg, 2-3 times daily) therapy. Pharmacokinetic ganciclovir was described with a two-compartment model. Total clearance of ganciclovir was consistently greater than ClCr, suggesting tubular secretion. Peak concentrations for i.v. ganciclovir averaged 8.39+/-1.87 microg/mL with minimum concentrations of 0.48+/-0.35 microg/mL. Plasma concentrations were lower but more sustained during a p.o. dosing interval (max=2.12+/-0.58 microg/mL, min=1.15+/-0.34 microg/mL). IL-6, IL-8, IL-10, and TNF-alpha were detectable at multiple times during the study periods while the remainder of the cytokines were only intermittently detectable. Average concentrations (i.v. versus p.o. study period) for TNF-alpha were 40.1+/-17.5 versus 22.1+/-11.2 pg/mL, for IL-8 were 17.1+/-15.6 versus 4.12+/-2.59 pg/mL, and for IL-10 were 7.39+/-5.54 versus 2.64+/-1.06 pg/mL. Concentrations were similar for IL-6 during both studies (9.39+/-5.42 versus 14.7+/-14.8 pg/mL). TNF-alpha, IL-8, and IFN-gamma appeared to correlate with CMV antigenemia. Further investigation of ganciclovir disposition and changes in plasma cytokines in renal transplant recipients during CMV infection may provide insight into variable antiviral responses in renal transplant recipients.     

Different effects of raloxifene and estrogen on interleukin-1beta and interleukin-1 receptor antagonist production using in vitro and ex vivo studies

The increase in lumbar spine BMD in response to Raloxifene (RLX), a selective estrogen receptor modulator, is smaller in magnitude compared to the response to treatment with estradiol (E2). The reasons for this observation are unclear. Estrogen has a potent effect on the production of proinflammatory cytokines which support osteoclastogenic and bone resorption. Therefore the different response to RLX may relate, at least in part, to a difference in the ability of RLX to modulate the production of proinflammatory cytokines which are abundant in the red marrow of the vertebrae. The aim of this study was to determine the effect of RLX and E2 both in vitro and ex vivo on the production of the pro-resorptive cytokine interleukin-1beta (IL-1beta) and its antagonist, interleukin-1 receptor antagonist (IL-1ra). We obtained samples of peripheral blood from (a) 10 untreated postmenopausal women with osteopenia (ages 53 to 72 years, mean 61 years), (b) 15 postmenopausal women (ages 52 to 72 years, mean 63 years) at baseline and after 6 months of RLX therapy (60 mg/day) and (c) 10 postmenopausal women (ages 60 to 75 years, mean 64 years) at baseline and 6 months after a single E2 implant (25 mg). Cultures of whole blood from the untreated women were incubated with RLX or 17beta-E2 at 1 pM, 100 pM, 10 nM and 1 microM concentrations. LPS-stimulated whole blood cultures from the raloxifene- and estradiol-treated women were prepared at baseline and at 6 months. IL-1beta and IL-1ra were measured by ELISA in the conditioned media. In vitro there was a significant dose-dependent decrease in IL-1beta and IL-1ra in response to 17beta-E2 (both P<0.0001) which was not apparent in response to RLX (both P>0.05). In ex vivo cultures from women receiving 6 months treatment with E2 implants, there was a significant decrease in IL-1beta (-36+/-8%, P=0.01) but no significant change in IL-1ra (+29+/-20%, P=0.3). There was no significant change in either IL-1beta or IL-1ra after 6 months RLX therapy (+20+/-14% and +12+/-10%, both P>0.05). We conclude that treatment with RLX, unlike estradiol does not modulate the production of the proinflammatory cytokines IL-1beta and IL-1ra using in vitro or ex vivo whole blood culture methods. This may account, at least in part for the reduced efficacy of RLX therapy compared to estrogen which has been observed in vivo on bone mineral density, bone turnover and reduction in fracture risk.     

Release of (1-->3)-beta-D-glucan from depth-type membrane filters and their in vitro effects on proinflammatory cytokine production

To clarify the origin of (1-->3)-beta-D-glucan in blood products and assess the biological activity of filter extracts, we evaluated (1-->3)-beta-D-glucan extraction from depth filters used to process blood products and their in vitro effects on proinflammatory cytokine production from macrophages. Cellulose or nylon filters were analyzed for (1-->3)-beta-D-glucan using the Fungitec G test. To evaluate the biological activity of the filter extracts, Mono Mac 6 cells (a human macrophage cell line) were cultured with filter extracts with or without lipopolysaccharide, and tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) concentrations in the culture media were measured. (1-->3)-beta-D-Glucan was released from seven cellulose filters but the nylon filter level was undetectable. Proinflammatory cytokine production ranged from 74.3% to 119.0% of the control for TNF-alpha and 81.2% to 115.9% for IL-1beta. TNF-alpha and IL-1beta levels were low without lipopolysaccharide. The data indicate that (1-->3)-beta-D-glucan in blood products is contaminated with the depth filters and that these filter extracts modulate proinflammatory cytokine production from macrophages.     

Inflammatory response during hyperglycemia and hyperinsulinemia in a porcine endotoxemic model: the contribution of essential organs

BACKGROUND: During euglycemia acute hyperinsulinemia diminishes the cytokine response to endotoxin [Lipopolysaccharide (LPS)] exposure. In this study we elucidated whether acute hyperglycemia and hyperinsulinemia modify the cytokine content in several organs during LPS challenge in a porcine model. METHODS: Pigs (35-40 kg) were randomized to either normoglycemia (group 1, n = 8) or hyperglycemia and hyperinsulinemia (group 2, n = 8), anesthetized and mechanically ventilated. Both groups received a 180-min intravenous infusion of LPS (total 10 microg kg(-1)). Groups 1 and 2 were clamped at plasma glucose concentrations of 5 mM and 15 mM, respectively. Group 1 maintained a baseline insulin level while the hyperglycemic group exhibited increased insulin levels. RESULTS: Circulating cytokines, cytokine mRNA and cytokine protein content were examined in the heart, liver, kidneys, lungs, spleen, adipose and muscle tissue. After LPS exposure, in both groups vast and equal plasma cytokines were elicited by approximately 70-5000-fold. A 10-fold higher level of IL-10, IL-6 and TNF-alpha protein was found in kidney tissue compared to the other organs together with a 3-10-fold increase of TNF-alpha in adipose tissue. However, cytokine mRNAs as well as organ function were without statistical difference between the groups. CONCLUSION: Endotoxemia elicited a pronounced cytokine response in both plasma and at organ level. The kidneys and adipose tissue showed the highest cytokine protein content. Acute hyperglycemia apparently counteracts the well-established anti-inflammatory effects of insulin on the inflammatory response in a LPS challenged porcine model. Whether the observation can be extrapolated to more long-term stress-exposure remains to be clarified.     

Proinflammatory cytokines are not released in the circulation following acute pulmonary thromboembolism in pigs.

Histological examination of acute lung injury associated with sepsis often revealed thromboembolic lesions in the pulmonary microcirculation. Several inflammatory mediators such as platelet activating factor, thromboxane, and endothelins have also been implicated in the pathogenesis of acute pulmonary thromboembolism (APTE). In the present study we examined the roles of three proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and IL-8, in the early phase of APTE. APTE was induced in 13 anesthetized piglets (22+/-4 kg) by injecting thrombin-induced blood clots directly into the left lower lobar pulmonary artery. Five animals that received only warm sterile saline served as controls. Arterial plasma samples were collected regularly over 8 h so that cytokine levels could be measured later by enzyme-linked immunosorbent assay (ELISA). Administration of clots doubled the mean pulmonary arterial pressure (from 13+/-5 to 26+/-7 mm Hg) and caused significant decrease in arterial oxygen tension (PaO2 from 390+/-85 to 256+/-89 mm Hg while the FiO2 was maintained at 1.0). Mean arterial blood pressure and cardiac output remained comparable throughout the experiments after initial fluid resuscitation. Plasma levels of TNF-alpha, IL-1beta, and IL-8 were not significantly increased in the APTE group when compared with their baseline values or the control group. Our results thus show that APTE is associated with pulmonary hypertension and deterioration of gas exchange but not with the systemic release of TNF-alpha, IL-1beta, or IL-8. We conclude that these cytokines have minimal impact on the systemic circulation during APTE.