P. F. Jørgensen

Involvement of CD14 and Toll-Like Receptors in Activation of Human Monocytes by Aspergillus fumigatus Hyphae

ABSTRACT Invasive fungal infections represent an increasing problem associated with high mortality. The present study was undertaken to identify leukocyte subsets that are activated by hyphal fragments in a whole-human-blood model, as well as to examine the involvement of CD14 and Toll-like receptors (TLRs) in activation of monocytes by hyphae. Incubation of whole human blood with hyphal fragments fromAspergillus fumigatus and Scedosporium prolificans for 6 h caused induction of mRNAs for tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and IL-6 in T cells, B cells, and monocytes, but not in granulocytes, as analyzed by reverse transcription-PCR with mRNA isolated from very pure populations of these leukocyte subsets. In primary adherent human monocytes, induction of TNF-α by hyphal fragments was dependent on plasma. Heat treatment of plasma at 56°C for 30 min strongly reduced the ability of plasma to prime for activation. Pretreatment of human monocytes with different concentrations (1, 3, and 10 μg/ml) of monoclonal antibody (MAb) HTA125 (anti-TLR4) or MAb 18D11 (anti-CD14) for 30 min inhibited the release of TNF-α induced by hyphal fragments in a dose-dependent manner. Maximal inhibitions of 35 and 70% were obtained with 10 μg of HTA125 and 18D11 per ml, respectively. In contrast, pretreatment with MAb TL2.1 (anti-TLR2) did not affect signaling induced by hyphae. Pretreatment with the lipid A antagonist B975 blocked lipopolysaccharide signaling but did not inhibit TNF-α production induced by hyphal fragments. Our results suggest that T cells, B cells, and monocytes are involved in the innate immune response to invasive fungal pathogens and that serum components are relevant for activation of monocytes by hyphae. CD14 and TLR4 may be involved in signaling of Aspergillus hyphae in monocytes, but further studies to elucidate this issue are warranted.

Peptidoglycan and Lipoteichoic Acid from Staphylococcus aureus Induce Tumor Necrosis Factor Alpha, Interleukin 6 (IL-6), and IL-10 Production in Both T Cells and Monocytes in a Human Whole Blood Model

ABSTRACT We have examined the ability of peptidoglycan (PepG) and lipoteichoic acid (LTA) isolated from Staphylococcus aureusto induce the release of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-10 in whole human blood and identified the cellular origins of these cytokines. Both PepG and LTA induced transient increases in TNF-α and IL-10 in plasma, with peak values at 6 and 12 h, respectively. IL-6 values increased throughout the experimental period (24 h). The TNF-α, IL-6, and IL-10 release induced by PepG and LTA was dose dependent. Only PepG was a potent inducer of TNF-α secretion. After stimulation of whole blood with PepG or LTA, very pure populations of monocytes (CD14 positive), T cells (CD2 positive), B cells (CD19 positive), and granulocytes (CD15 positive) were isolated by immunomagnetic separation and analyzed by reverse transcription-PCR for mRNA transcripts encoding TNF-α, IL-6, and IL-10. The TNF-α mRNA results were inconclusive. In contrast, PepG induced IL-6 and IL-10 mRNA accumulation in both T cells and monocytes. LTA, as well as lipopolysaccharide, induced IL-6 and IL-10 mRNA production in monocytes and possibly in T cells. Whether granulocytes and B cells produce cytokines in response to bacterial stimuli remains obscure. Blockade of the CD14 receptors with monoclonal antibodies (18D11) had no influence on the PepG-induced release of TNF-α but attenuated the LTA-induced release of the same cytokine. In conclusion, our data indicate that circulating T cells and monocytes contribute to cytokine production in sepsis caused by gram-positive bacteria.

A randomized multicenter trial of the anti-ICAM-1 monoclonal antibody (enlimomab) for the prevention of acute rejection and delayed onset of graft function in cadaveric renal transplantation: a report of the European Anti-ICAM-1 Renal Transplant Study Group.

BACKGROUND T-cell activation through T-cell receptor engagement requires co-stimulatory molecules and also adhesion molecules such as ICAM-1. Moreover ICAM-1 mediates leukocyte invasion from the blood into tissue during inflammatory processes. In animal studies using mouse monoclonal antibodies against ICAM-1 (enlimomab), renal allograft survival has been improved and reperfusion damage from ischemia reduced. The European Anti-ICAM-1 Renal Transplant Study (EARTS) was a randomized, double-blind, parallel-group, placebo-controlled study lastingl year and performed in 10 transplant centers in Europe. METHODS A total of 262 recipients of cadaveric kidneys were given either enlimomab or a placebo for 6 days and were given triple immunosuppressive therapy of cyclosporine, azathioprine, and prednisolone. The primary efficacy endpoint was the incidence of the first acute rejection within 3 months, and each event was assessed by a committee including investigators and independent pathologists. RESULTS There was no significant difference in the incidences of first acute rejection at 3 months between the placebo and enlimomab groups (39% vs. 45%), and enlimomab did not reduce the risk of delayed onset of graft function (DGF) (26% vs. 31%). Neither was there a difference in patient survival (95% vs. 91%) or graft survival (89% vs. 84%) at 1 year. Fatal events occurred in 19 (7%) patients (7 placebo, 12 enlimomab). Clinically, the most important non-fatal adverse events were infections; however, there was no statistically significant difference between the incidences in the two groups (70% vs. 79%). CONCLUSION Short term enlimomab induction therapy after renal transplantation did not reduce the rate of acute rejection or DGF.

Liver transplantation for nonresectable liver metastases from colorectal cancer.

Objective:The objective of this pilot study was to investigate the potential for long-term overall survival (OS) after liver transplantation for colorectal liver metastases (CLMs). Background:Patients with nonresectable CLMs have poor prognosis, and few survive beyond 5 years. CLMs are currently considered an absolute contraindication for liver transplantation, although liver transplantation for primary and some secondary liver malignancies shows excellent outcome in selected patients. Before 1995, several liver transplantations for CLMs were performed, but outcome was poor (5-year survival rate: 18%) and liver transplantation for CLMs was abandoned. Since then, the survival rate after liver transplantation in general has improved by almost 30%. On the basis of this, a 5-year survival rate of about 50% after liver transplantation for CLMs could be anticipated. Methods:In a prospective pilot study, liver transplantation for nonresectable CLMs was performed (n = 21). Main inclusion criteria were liver-only CLMs, excised primary tumors, and at least 6 weeks of chemotherapy. Results:Kaplan-Meier estimates of the OS rate at 1, 3, and 5 years were 95%, 68%, and 60%, respectively. Metastatic recurrence of disease was common (mainly pulmonary). However, a significant proportion of the recurrences were accessible for surgery, and at follow-up (after median of 27 months; range, 8–60), 33% had no evidence of disease. Hepatic tumor load before liver transplantation, time from primary surgery to liver transplantation, and progressive disease on chemotherapy were identified as significant prognostic factors. Conclusions:OS exceeds by far reported outcome for chemotherapy, which is the only treatment option available for this patient group. Furthermore, OS is comparable with liver resection for resectable CLMs and survival after repeat liver transplantation for nonmalignant diseases. Selection strategies based on prognostic factors may further improve the outcome (ClinicalTrials.gov: NCT01311453).

Peptidoglycan primes for LPS-induced release of proinflammatory cytokines in whole human blood

The pathophysiological mechanisms involved in mixed bacterial infections caused by gram-positive and gram-negative bacteria are largely unknown. The present study examines the potential interaction between lipopolysaccharide (LPS) and peptidoglycan (PepG) in the induction of the sepsis-associated cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-10 in whole human blood. Plasma values of these cytokines were measured by enzyme immunoassays and a TNF bioassay. Co-administration of PepG (10 microg/mL) or muramyl dipeptide (MDP, 1 microg/mL) with LPS (10 ng/mL) caused significantly elevated values of TNF-alpha and IL-6 in the blood that could not be obtained by the sum of the values obtained by each stimulant alone, or by 3-fold higher doses of either bacterial component alone. This phenomenon was observed 1 h after stimulation, throughout the experimental period (24 h), and with different doses of LPS and PepG. In contrast, the release of IL-10 was not influenced by the co-administration of PepG or MDP with LPS. The TNF-alpha release induced by co-administration of LPS and PepG was abrogated after pretreatment with a monoclonal antibody against CD14 (18D11). Addition of PepG or MDP to whole blood caused a 2-fold increase in the surface expression of CD14 on monocytes, as measured by flow cytometry. In contrast, LPS caused decreased expression of this receptor. Our data suggest that PepG and MDP primes human whole blood leukocytes for LPS-induced release of proinflammatory cytokines. We speculate that synergy between PepG and LPS may contribute to the pathogenesis in sepsis caused by mixed bacterial infections.

Kidneys From Deceased Donors More Than 75 Years Perform Acceptably After Transplantation

Background. Organ shortage has resulted in an increased use of expanded criteria donors for transplantation, in particular kidneys from older donors. There is limited data on the impact of donor age more than 75 years on kidney transplant outcome. Methods. A retrospective single-center analysis on deceased donors more than 75 years and kidney transplant outcome in an old for old setting was performed. Histologic findings (global kidney score) in graft biopsies and deceased-donor scores were evaluated to observe if this information could be helpful in predicting outcome. Results. Evaluation of data from 54 single kidney transplantations from 29 donors more than 75 years (median 77.5, range 75.2–86.1) were assessed. Ninety-three percent of the donors died of intracranial bleeding, and 69% had a history of hypertension or cardiovascular event(s). Median recipient age was 70.1 (range 50.6–82.4). Fifty-two grafts (96%) had posttransplant function. Death censored graft survival at 1, 3, and 5 years were 87%, 83%, and 83%, respectively. Patient survival was 81%, 75%, and 59% at the same time points. At follow-up at median 23 months (range 6–144 months), thirty-five recipients were alive with a median serum creatinine of 163 &mgr;mol/ L (range 103–348). Global kidney score and deceased donor score did not predict graft outcome. Conclusion. Kidney transplants from deceased donors more than 75 years perform acceptable as single transplants and should be considered for use in older recipients.

Sirolimus Interferes with the Innate Response to Bacterial Products in Human Whole Blood by Attenuation of IL‐10 Production

Current immunosuppressive strategies are aimed at abrogating the allospecific T‐cell response against donor tissues or organs. However, little information is yet available on the potential influences of these drugs on innate immune responses. In order to address this, we have employed a whole blood model. Human whole blood was pretreated with sirolimus, cyclosporine A or tacrolimus in therapeutic as well as supra therapeutic doses, and subsequently stimulated with lipopolysaccharide (LPS), peptidoglycan (PepG) or lipoteichoic acid (LTA). Plasma cytokine analyses revealed a potent inhibitory effect of sirolimus on interleukin(IL)‐10 production induced by all bacterial products tested. In contrast, cyclosporine A and tacrolimus inhibited the tumour necrosis factor (TNF)‐α production in response to LPS, but not to PepG and LTA. Using a quantitative mRNA analyses, we also observed that sirolimus significantly decreased the IL‐10 mRNA accumulation to sub‐basal levels in peripheral blood mononuclear cells (PBMC). This suggests that the sirolimus inhibits IL‐10 production by interfering with the IL‐10 gene transcription. However, the molecular mechanism of this inhibition remains unclear. Based on the present study and observations by others, we postulate that the clinical use of the sirolimus may be associated with a dysregulated innate immune response to bacterial infection and thus an increased risk of hyperinflammation and sepsis.

Cytokine Modulation in Experimental Endotoxemia: Characterization of an ex vivo Whole Blood Model

A new model was developed to study cytokine regulation and modulation in whole blood ex vivo. The model is characterized by stable leukocyte counts and high leukocyte viability throughout the experimental period. Oxygen consumption per time decreased slowly, whereas carbon dioxide partial pressure increased accordingly throughout the experiment. In this model, the anti-inflammatory effects of recombinant human (rh) interleukin (IL)-4, rhIL-10 and rhIL-13 on lipopolysaccharide (LPS) stimulated (10 ng/ml) leukocytes were examined and compared by measuring their ability to inhibit the release and mRNA levels of tumor necrosis factor (TNF)α, IL-6 and IL-1β. rhIL-10 potently inhibited the release of TNF-α, IL-6 and IL-1β in a potent and dose-dependent manner, but did not influence the mRNA levels of these cytokines in CD14-positive cells. Also, rhIL-4 and rhIL-13 inhibited the release of IL-6 and IL-1β in a potent and dose-dependent manner, however, stronger maximal inhibition of IL-1β (85%) than of IL-6 (60%) was obtained. In contrast, rhIL-4 and rhIL-13 seemed to have both stimulatory and inhibitory effects on plasma values of TNF-α. The effects of 10 ng/ml LPS showed to be signalling through the CD14 receptor, since blood treated with a monoclonal anti-CD14 antibody did not produce any TNF-α. The whole blood model described in this study is in our opinion a useful tool for investigating immunomodulating effects on a mixed white blood cell population.

Peptidoglycan and lipoteichoic acid modify monocyte phenotype in human whole blood.

ABSTRACT We examined the influence of the gram-positive cell wall products peptidoglycan (PepG) and lipoteichoic acid (LTA), compared to lipopolysaccharide (LPS), on the monocyte expression of receptors involved in antigen presentation (HLA-DR, B7.1, and B7.2), cell adhesion (intercellular adhesion molecule-1 [ICAM-1] and lymphocyte function associated antigen-3 [LFA-3]), phagocytosis (FcγRI), and cell activation (CD14). We also evaluated possible influences of the immunosuppressive drugs cyclosporine A, tacrolimus, and sirolimus on the expression of these receptors. Pretreatment of whole blood for 4 h with the immunosuppressive drugs did not influence the expression of the surface receptors in normal or stimulated blood. Stimulation with both PepG and LTA caused significant up-regulation of the surface expression of ICAM-1 and HLA-DR on whole blood monocytes, similar to that obtained with LPS, whereas B7.1, B7.2, LFA-3, and FcγRI were not modulated. PepG and LTA also caused increased expression of CD14, whereas LPS down-regulated this molecule. In contrast, we did not detect any significant influence of any of the bacterial products on the plasma concentration of soluble CD14. We hypothesized that the increased expression of surface CD14 in blood stimulated with PepG would prime for cellular activation by LPS. Indeed, we show that PepG and the partial PepG structure muramyl dipeptide acted in synergy with LPS to cause the release of tumor necrosis factor-α. The results suggest that PepG and LPS provoke partly different responses on monocyte phenotype and that CD14 may play different roles in the innate response to gram-positive and gram-negative bacteria.

Nitric Oxide Administration Restores the Hepatic Artery Buffer Response During Porcine Endotoxemia

The hepatic artery buffer response, which is lost during endotoxemia, plays a central role in the autoregulation of liver perfusion. A temporarily decreased synthesis of nitric oxide during early endotoxemia might be responsible for this dysfunction; hence exogenous administration of nitric oxide could reestablish the autoregulation of hepatic blood flow and help prevent hepatic damage later in septic shock. Fifteen pigs were treated with lipopolysaccharide +/− the nitric oxide donor nitroprusside-sodium via the portal vein. Hemodynamics were measured, and serum chemistry and liver biopsies for nitric oxide synthase expression were obtained. Lipopolysaccharide decreased arterial liver perfusion after 5 hours by 38% (p =. 012), which was reversed by addition of nitroprusside (8%). Administration of nitroprusside preserved an increase of 28% in hepatic arterial upon portal vein flow reduction (p <. 001). Nitroprusside maintained mRNA levels of constitutive nitric oxide synthase in liver tissue which were decreased by lipopolysaccharide (p =. 026 vs. p =. 114) and tempered the burst in inducible nitric oxide synthase expression at t = 3 hours. The early administration of the nitric oxide donor sodium nitroprusside during endotoxemia is able to reestablish the autoregulatory response of the hepatic artery following reduction of hepatic blood flow. This beneficial effect might help to prevent subsequent hepatic damage in the course of abdominal sepsis.