Volume 37, Issue 3

T-lymphocytes recognize short peptide antigens bound stably to polymorphic major histocompatibility complex (MHC)-encoded glycoproteins expressed on the surface of antigen-presenting cells (APC). Two general pathways have evolved to generate peptide-MHC complexes. The MHC class II antigen processing pathway provides a mechanism for sampling proteins present in endosomal compartments. CD4+ regulatory T-cells recognize peptides bound to MHC class II molecules, which are selectively expressed in specialized APC that have efficient mechanisms for uptake of microbial antigens, and express costimulatory molecules required for activating naive T-cells. CD8+ T-cells recognize peptides bound to MHC class I molecules. Class I molecules are widely expressed and bind peptides derived from the normal turnover of cellular proteins, providing a mechanism to display a sampling of cellular components to be monitored for abnormalities by cytotoxic T-cells. Specialized accessory proteins influence the efficiency of antigen presentation and the specificity of immune responses through their roles in generating peptides, targeting antigen and MHC glycoproteins to selected intracellular compartments, and by direct participation in the peptide-loading mechanism. It has recently been discovered that some viruses have evolved ways to inhibit or subvert discrete steps in antigen processing, providing a mechanism to evade immune recognition.

ITIM-bearing molecules represent a novel family of inhibitory receptors expressed widely through the hematopoietic compartment. These molecules share certain features such as the presence in their intracytoplasmic domain of the so-called motif ITIM (Immuno-Tyrosine-based Inhibition Motif). These molecules are able to recruit phosphatases on their phosphorylated ITIM and thus mediate a localized inhibition of the transduction pathways. The molecular basis of this inhibitory pathway is discussed below.

Severe thermal injury causes an immune dysfunction which includes a decrease of monocyte human leukocyte antigen DR (HLA-DR) expression. Interleukin-10 exerts a negative influence on this parameter in vitro . In this study we determined the prognostic value of reduced monocyte HLA-DR expression with regard to infectious complications, and the in vivo association between monocyte HLA-DR and plasma interleukin-10 concentration. Both quantities were measured serially in 19 patients with severe burns. HLA-DR expression was determined by direct immunofluorescence on a flow cytometer, and interleukin-10 was measured by ELISA. After burn trauma the percentage of HLA-DR expressing monocytes fell markedly (median: 53 % at day 2, 36 % at day 4, 31 % at day 7, 28 % at day 9, 35 % at day 12, and 42 % at day 14; compared to 93 % for healthy volunteers). Moreover, patients who became septic showed lower monocyte HLA-DR expression than non-septic patients; the differences were significant at day 2 (p < 0.01) and day 7 (p < 0.05). Plasma concentrations of interleukin-10 increased after thermal injury (median: 40 ng/l at day 2, 43 ng/l at day 4, 77 ng/l at day 7, 120 ng/l at day 9, 63 ng/l at day 12, and 82 ng/l at day 14). Individual HLA-DR expression and interleukin-10 concentration were negatively correlated, the association reaching statistical significance at day 4 (p = 0.006) and day 7 (p = 0.031). Thus, after severe burn injury monocyte HLA-DR expression has prognostic value and is negatively associated with interleukin-10 plasma concentration.

Bacterial DNA sequences containing unmethylated CpG motifs have recently been proposed to exhibit immunostimulatory effects on B-, T- and NK cells, leading to the induction of humoral and cell-mediated immune responses. In the present study we investigated the immunomodulatory effects of a CpG-containing oligodeoxynucleotide (CpG ODN) to the HIV-1 gp160 envelope (Env) protein in the BALB/c mouse model. Priming and boosting of mice with gp160 adsorbed to aluminium hydroxide (Alum) induced a typical T helper-2 (Th2)-dominated immune response with high titers of gp160-specific immunoglobulin (Ig)G1 isotypes but a weak IgG2a response. Specifically re-stimulated splenocytes from these mice predominantly secreted interleukin (IL)-5 but only minute amounts of interferon-γ (IFN-γ) upon specific re-stimulation. In contrast, a boost immunisation of gp160/Alum primed mice with a gp160/Alum/CpG combination resulted in a seven times higher production of IgG2a antibodies, without affecting the titers of IgG1 isotypes. Furthermore, approximately 10-fold increased levels of IFN-γ, but significantly reduced amounts of IL-5, were secreted from gp160-restimulated splenic cells. A further greater than 30-fold increase in the levels of specific IgG2a responses and a substantially elevated secretion of IFN-γ were observed when the mice received gp160/Alum/CpG combinations for priming and boost injections. Thus, CpG ODNs are useful as an adjuvant to induce a typical Th0/Th1 response to HIV gp160 proteins. However, despite the induction of a more Th1-like immune response, gp160/Alum/CpG combinations were not sufficient to prime an Env-specific cytotoxic T-cell (CTL) response.

During the acute inflammatory response, there is induction of a mediator cascade which functions to activate residential macrophages and recruit blood leukocytes to the site of the inflammatory insult. Dysregulation of this process can cause an exaggerated inflammatory response and lead to tissue injury. Recent studies have focused on the transcription factor NFκB, which controls the gene expression of many pro-inflammatory mediators. Under normal conditions, NFκB is retained in the cytosol by inhibitory proteins of the IκB family. In response to an inflammatory insult, IκB proteins are degraded and the free NFκB complex translocates to the nucleus where it initiates gene transcription. An understanding of the in vivo mechanisms leading to the activation of NFκB, and the regulatory mechanisms that exist to limit this activation, may lead to the development of novel new therapeutic options for inflammatory injury. In this review we will discuss the current knowledge of the role of NFκB in the development of acute inflammation, as well as the regulatory mechanisms that exist to prevent the activation of NFκB and resolve inflammatory tissue injury.

The majority of peripheral blood monocytes strongly positive for the lipopolysaccharides (LPS)-receptor CD14 are negative for Fcγ receptor type III (CD16). However, a subset of monocytes coexpressing CD14 and CD16 accounts for about 8% of all monocytes. This population exhibits features of tissue macrophages, and is largely expanded (> 20 %) during acute and chronic inflammatory diseases including cases with pararheumatic systemic vasculitis. In addition, compared to normal controls, soluble CD14 (sCD14) is elevated (> 3 μg/ml) in serum specimens of these patients. CD14 + /CD16 + monocytes show a higher phagocytosis rate than CD14 + /CD16 negative cells, and express higher levels of interleukin-1 and major histocompatibility complex, such as histocompatibility antigens HLA-DR, -DP and -DQ antigens. Glucocorticoids downregulate expression of CD14 and rapidly deplete CD14 + /CD16 + monocytes from peripheral blood. Patients under chronic immunosuppressive therapy exhibit low CD14/+/CD16+ rates, which may rise during infectious and non-infectious inflammatory complications, however. Thus, serial analyses for sCD14 and the proinflammatory CD14 + /CD16 + subset of monocytes suggest a valuable tool monitoring patients under immunosuppressive and/or antiinflammatory therapy.

Localized juvenile periodontitis is a destructive form of periodontal inflammatory disease which has its onset at puberty. The etiopathology of the disease is still unclear but neutrophils have been suggested to play a major role both in the production and development of the disorder. About 70 % of the patients with localized juvenile periodontitis exhibit neutrophil functional abnormalities, such as decreased chemotaxis and phagocytosis. Interestingly, it has been frequently reported that the same hypoactive cells show an enhanced respiratory burst response and increased adhesion. Several possible mechanisms explaining neutrophil anomalies in localized juvenile periodontitis have been proposed. These include the presence of soluble serum factors capable of modulating neutrophil function, altered cell-surface receptor expression and/or function, and a change in the post-receptor signaling events. Recently, a growing evidence has accumulated showing that the diacylglycerol metabolism could be altered in neutrophils from patients with localized juvenile periodontitis. This change, which may be due to a defect in a major diacylglycerol metabolizing enzyme, diacylglycerol kinase, results in enhanced accumulation of diacylglycerol in activated cells. Because diacylglycerol is an endogenous activator of protein kinase C, the increased and prolonged generation of diacylglycerol could lead to abnormal pattern of protein kinase C-regulated neutrophil functions, explaining the parallel hypo- and hyperactivities.

H. pylori -associated gastric mucosal inflammation is characterized by the presence of polymorphonuclear (PMN) leukocyte infiltrate, which is more severe when the infecting strain is cagA positive. After appropriate stimuli, such as bacterial products, PMN release large amounts of oxygen derived free radicals and proteases, to kill the bacterium. H. pylori seems to be particularly resistant to the oxidative machinery of PMN, which can in turn damage the host gastric mucosa. We evaluated peripheral PMN oxidative burst response after stimulation with water extracts from cagA positive (WE cagA +) or negative (WE cagA −) H. pylori strains in infected (n=31) and non-infected patients (n=32) in comparison with healthy controls (n=16); the influence of gastric mucosal inflammatory infiltrate and activity grade on PMN oxidative burst were also assessed. PMN oxidative burst was measured by FACS analysis. H. pylori water extracts were obtained from bacterial culture. H. pylori genotype was determined by means of the polymerase chain reaction. The PMN oxidative burst in H. pylori infected patients was significantly higher than that in H. pylori negative or healthy controls, no differences being found when the results following WE cagA + and WE cagA -stimulation were compared. The difference in PMN oxidative burst obtained after WE cagA − and E. coli (standard stimulus for PMN oxidative burst) stimulation discriminated H. pylori infected from non-infected patients with a sensitivity of 90 % and a specificity of 97 %. The grade of PMN oxidative burst correlated with PMN infiltration grade of the gastric mucosa. Our findings allow to conclude that PMN oxidative burst activation by H. pylori WE is species-but not strain-correlated. PMN priming, probably consequent to the action of soluble mediators released by mononuclear cells, makes PMN hyper-responsive to H. pylori products, thus favoring the release in the gastric mucosa of infected patients of large amounts of oxygen-derived free radicals, which are not enough to eliminate the infection, but may contribute to damaging the gastric mucosa itself. Peripheral PMN oxidative burst response to H. pylori WE might furthermore be of help in diagnosing H. pylori infection.

Flow cytometry was adapted to measure lymphocytes in human cerebrospinal fluid (CSF). The method was sufficiently precise, reproducible and accurate despite low cell counts. In lumbar CSF of controls with 500 to 3500 (10 3 /l) leukocytes, lymphocyte counts correlated with those in corresponding venous blood: blood/CSF ratios of ≈ 2000 : 1 were found for total T cells (CD3 + ) and CD3 + HLA-DR − , CD3 + 4 + , CD3 + 8 + subsets, ratios were increased for the lymphocyte subsets CD3 + HLA-DR + ≤ CD3 + 16 + 56 + < CD16 + 56 + 3 − < CD8 + 3 − << CD19 + ; CD8 + 4 + ratio was half of CD3 + ratio. Data indicate selective barriers (blood-brain and blood-CSF barriers) to blood lymphocyte subsets which favor the transfer of T subsets. Correlation of the subset ratios to the CD3 + ratio indicates distinct barrier properties which changed differently with acute and subacute inflammations and neuroimmunological diseases of central nervous system (CNS) in lumbar or ventricular CSF, but not with simple protein barrier disturbance. HLA DR + T ratios were higher than HLA DR − T ratios only with controls and some neuroimmunological diseases. Lymphocyte barrier characteristics were related to protein leakage situated at the same barriers, indicating for the lymphocyte subsets selective transfer routes in control subjects and non-selective routes in patients with CNS inflammation where altered ratios revealed a mixture of routes.

The integrin heterodimer CD11b/CD18 (αMβ2, Mac-1, CR3) expressed on monocytes or polymorphonuclear leukocytes (PMN) is a receptor for iC3b, fibrinogen, heparin, and for intercellular adhesion molecule (ICAM)-1 on endothelium, crucially contributing to vascular cell interactions in inflammation and atherosclerosis. In this report, we summarize our findings on the effects of lipid mediators and lipid-lowering drugs. Exposure of endothelial cells to oxidized low density lipoprotein (oxLDL) induces upregulation of ICAM-1 and increases adhesion of monocytic cells expressing Mac-1. Inhibition experiments show that monocytes use distinct ligands, i.e. ICAM-1 and heparan sulfate proteoglycans for adhesion to oxLDL-treated endothelium. An albumin-transferable oxLDL activity is inhibited by the antioxidant pyrrolidine dithiocarbamate (PDTC), while 8-epi-prostaglandin F2α (8-epi-PGF2α) or lysophosphatidylcholine had no effect, implicating yet unidentified radicals. Sequential adhesive and signaling events lead to the firm adhesion of rolling PMN on activated and adherent platelets, which may occupy areas of endothelial denudation. Shear-resistant arrest of PMN on thrombin-stimulated platelets in flow conditions requires distinct regions of Mac-1, involving its interactions with fibrinogen bound to platelet αllbβ3, and with other platelet ligands. Both arrest and adhesion strengthening under flow are stimulated by platelet-activating factor and leukotriene B4, but not by the chemokine receptor CXCR2. We tested whether Mac-1-dependent monocyte adhesiveness is affected by inhibitors of hydroxy-methylglutaryl-Coenzyme A reductase (statins) which improve morbidity and survival of patients with coronary heart disease. As compared to controls, adhesion of isolated monocytes to endothelium ex vivo was increased in patients with hypercholesterolemia. Treatment with statins decreased total and low density lipoprotein (LDL) cholesterol plasma levels, surface expression of Mac-1, and resulted in a dramatic reduction of Mac-1-mediated monocyte adhesion to endothelium. The inhibition of monocyte adhesion was reversed by mevalonate but not LDL in vitro , indicating that isoprenoid precursors are crucial for adhesiveness of Mac-1. Such effects may crucially contribute to the clinical benefit of statins, independent of cholesterol-lowering, and may represent a paradigm for novel, anti-inflammatory mechanisms of action by this class of drugs.

A possible synergistic effect of mycophenolic acid (MPA) and the immunosuppressive cytokines IL-4, IL-10 and IL-13 was investigated in human umbilical vein endothelial cells (HUVEC). These cytokines, which are produced by Th2-lymphocytes, were shown to induce the expression of vascular cell adhesion molecule-1 (VCAM-1) and production of IL-6 in endothelial cells. In this study we found IL-4 to induce both intercellular cell adhesion molecule-1 (ICAM-1) and VCAM-1 expression, whereas IL-13 induced VCAM-1 only. The surface expression of E-selectin was not influenced by any of the cytokines tested. MPA on its own led to statistically significant ICAM-1 expression on HUVEC. The combination of MPA with IL-4 led to a significant ICAM-1 expression in an additive manner compared to the cytokine alone. In contrast, MPA neither induced VCAM-1 nor did it influence the effects of IL-4 and IL-13 on VCAM-1 expression. A clinically relevant concentration of mycophenolic acid (10 μmol/l) decreased intracellular guanosine-5′-triphosphate (GTP) levels significantly. Since intracellular nucleotides are responsible for the glycosylation of proteins, a disturbance of the endothelial nucleotide balance could be responsible for the effects of MPA on ICAM-1. Guanine and guanosine prevented and partially reversed the actions of MPA, on both intracellular GTP and ICAM-1 expression, which strongly implies that MPA by interfering with nucleotide metabolism, affects the adhesive properties of endothelial cells, and by acting synergistically with IL-4 probably influences Th2 cytokine effects.

The effect of the inhibitor of inosine-5′-monophosphate dehydrogenase (IMPDH), mycophenolic acid, on intracellular nucleotides and the synthesis of cellular glycoproteins was evaluated in human umbilical vein endothelial cells. A clinically attainable concentration (10 μmol/l) of mycophenolic acid decreased guanosine-5′-triphosphate (GTP) levels significantly and led to a strong elevation of uridine-5′-triphosphate (UTP), whereas intracellular adenosine-5′-triphosphate (ATP) pools remained unaffected. The staining of the endothelial cell membranes with lectins specific for fucose and mannose ( Ulex europaeus- and Galanthus nivalis agglutinin, respectively) was reduced, reflecting an inhibition of fucose and mannose incorporation into endothelial glycoproteins. The surface expression of E-selectin, an important determinant for leuko-endothelial interactions decreased significantly. Guanine and guanosine prevented the actions of mycophenolic acid and reversed the drug-induced decrease in GTP and its associated effects. The findings that mycophenolic acid produces alterations in the formation of glycoproteins and in the membrane architecture are indicative of metabolic lesions induced by an agent that depresses guanine nucleotide synthesis through inhibtion of IMPDH. The pronounced reduction of E-selectin surface expression on endothelial cells accompanied by changes of endothelial cell fucosylation, a prerequisite for the contact with lymphocytic L-selectin, indicates an inhibitory effect of mycophenolic acid in the rolling phase of leukocyte recruitment and strongly implies a new and additional immunosuppressive mechanism of this agent.

Human C-reactive protein is a Ca 2+ -binding acute phase-protein with binding specificity for phosphocholine. Recent crystallographic and mutagenesis studies have provided a solid understanding of the structural biology of the protein, while experiments using transgenic mice have confirmed its host-defense function. The protein consists of five identical protomers in cyclic symmetry. On one face of each protomer there is a binding site for phosphocholine consisting of two Ca 2+ ions that ligate the phosphate group and a hydrophobic pocket that accommodates the methyl groups of phosphocholine. On the opposite face is a deep cleft formed by parts of the N and C termini and bordered by an α-helix. Mutational studies indicate that the C1q-binding site of the molecule is located at the open end of this cleft with Asp 112 and Tyr 175 representing contact residues. Using human C-reactive protein transgenic mice, we investigated the host defense functions of the protein. Transgenic mice infected with Streptococcus pneumoniae had increased lifespan and lowered mortality compared to wild-type mice. This was attributable to an up to 400-fold reduction in bacteremia mediated mainly by the interaction of C-reactive protein with complement. A complement-independent host protective effect was also demonstrated.

Acute phase proteins are extremely helpful markers for indicating a disturbance of the homeostasis within the organism and for monitoring the course of a disease. Despite the availability of several serum acute phase markers, a better and more specific prediction of sepsis and related disorders, such as systemic inflammatory response syndrome (SIRS) is still needed, as these diseases still have a high mortality rate and have to be detected early and with high specificity. Here a novel acute-phase protein is introduced, that has certain biological functions in host defense and that may be a useful addition for the diagnosis and monitoring of sepsis. Lipopolysaccharide (LPS or endotoxin), binding protein (LBP) is a class 1 acute-phase protein with the ability to bind and transfer bacterial LPS. Changes in serum levels of LBP have profound effects on the host's ability to react to endotoxin stimulation and to defend itself against sepsis. Results obtained from in vitro studies and from an animal model are reviewed here and a perspective on ongoing clinical studies is given. There is evidence that LBP, along with other LPS-recognizing molecules, is an important parameter for monitoring the acute phase and the ability of the host to react to LPS-challenge.

Objective: Established parameters, e.g. C-reactive protein (CRP), do not differentiate specifically enough between patients developing an infection and those exhibiting an acute phase response following cardiac surgery. The objective of this prospecitive study was to investigate if procalcitonin (PCT) ist more helpful than CRP. Methods: During a 1-year period, seven out of 563 patients (1.2 %) developed systemic infections (group A) after cardiac operations with cardiopulmonary bypass (CPB), and additional eight patients (1.4 %) had local wound infections requiring surgical therapy (group B). Blood samples for PCT and CRP measurements were taken preoperatively, at the onset of infection (d1), as well as on the third day (d3), fifth day (d5), and seventh day (d7) following diagnosis of infection. Forty-four randomly selected patients undergoing cardiac surgery with CPB without clinical signs of infection, additional intensive care unit (ICU) management or additional antibiotic treatment served as control (group C) to assess the PCT and CRP contribution to acute phase response. PCT and CRP levels were measured preoperatively, on the first (d1), third (d3) and fifth day (d5) after operation. Results: At the onset of infection, PCT levels (median interquartile range 25 %–75 %) increased significantly in group A as compared to baseline values (10.86 (3.28–15.13) ng/ml vs. 0.12 (0.08–0.21) ng/ml), and decreased during treatment to still significantly elevated values on d5 (0.56 (0.51–0.85) ng/ml). CRP levels were significantly elevated on all days investigated with no trend towards normalisation (d1: 164.5 (137–223) mg/l) vs. 1.95 (1.1–2.8) mg/l preoperatively, d5: 181.1 (134–189.6) mg/l. In group B, no increase in PCT levels, but a significant increase of CRP from d1 (165.9 (96.6–181.6) mg/l) vs. 3.7 (2–4.3) mg/l preopratively) until d5 98 (92.8–226.2) mg/l was detected. In group C, postoperative PCT levels increased slightly but significantly in the absence of infection on d1 (0.46 (0.26–0.77) ng/ml vs. 0.13 (0.08–0.19) ng/ml preoperatively), and d3 (0.37 (0.2–0.65) ng/ml and reached baseline on d5 (0.24 (0.11–0.51) ng/ml)). CRP levels were significantly elevated as compared to baseline on all postoperative days investigated (baseline: 1.75 (0.6–2.9) mg/l, d1: 97.5 (74.5–120) mg/l), d3: 114 (83.05–168.5) mg/l, d5: 51.4 (27.4–99.8) mg/l)). PCT showed a significant correlation to CRP in group A (r = 0.48, p < 0.001), a weak correlation in group C (r = 0.27, p = 0.002) and no correlation in group B. Intergroup comparison revealed a significant difference for PCT between all groups (A > C >B) and significantly higher CRP levels in group A vs. C and in group B vs. C. Thus, the pattern high PCT/high CRP appears to indicate a systemic infection, while low PCT/high CRP indicates either acute phase response or local wound problems, but no systemic infection. The cost for PCT measurements was 5.6-fold higher as compared to CRP. Conclusion: Due to the significant differences in the degree of increase, PCT appears to be useful in discriminating between acute phase response following cardiac surgery with CPB or local problems and systemic infections, with additional CRP-measurement increasing the specifity.

Lactoferrin is an iron-binding glycoprotein found in exocrine secretions of mammals and released from neutrophilic granules during inflammation. This review describes the biological roles of lactoferrin in host defence. Secreted lactoferrin exerts antimicrobial action either by chelation of iron or by destabilization of bacterial membranes. Furthermore, lactoferrin modulates the inflammatory process, mainly by preventing the release of cytokines from monocytes and by regulating the proliferation and differentiation of immune cells. Some of these activities are related to the ability of lactoferrin to bind lipopolysaccharides (LPS) with high affinity. Indeed, recent in vitro studies indicate that lactoferrin is able to compete with the LPS-binding protein for LPS binding and therefore to prevent the transfer of LPS to CD14 present at the surface of monocytes. Moreover, the prophylactic properties of lactoferrin against septicemia in vivo have been demonstrated. Taken as a whole, these observations strongly suggest that lactoferrin is one of the key molecules which modulate the inflammatory response.

Annexins, protein kinases C and cytosolic phospholipase A 2 belong to three families of ubiquitous cytoplasmic proteins involved in signal transduction. All annexins share the property of binding to phospholipids in the presence of calcium. Most annexins are substrates for protein kinases C except annexin V, the most ubiquitous and abundant annexin. Protein kinases C (PKC) belong to three distinct groups of kinases, conventional PKCs (cPKCs) that depend on calcium, diacylglycerol and negatively charged phospholipids for their activity, novel PKCs (nPKCs) and atypical PKCs (aPKCs), that do not require calcium for their activity, although they both require negatively charged phospholipids. Cytosolic phospholipase A 2 (cPLA2) depends on calcium for its catalytic activity as well as on serine phosphorylation by MAP kinases. We report that annexin V modulates the activity of cPKCs as well as of cPLA2 by interfering with their ability to bind to negatively charged phospholipids and calcium. We propose that annexin V could interfere with the calcium and phospholipid signalling pathway.

Lipoxins are lipox ygenase in teraction products formed by transcellular metabolism during host defence and inflammation. In model systems, lipoxins modulate polymorphonuclear leukocytes (PMN) chemotaxis, adhesion molecule expression, inhibit PMN-endothelial cell adhesion, and attenuate cytokine release from epithelial cells. These observations raise the possibility that lipoxins are ‘stop signals’ for PMN-mediated tissue injury and promote the resolution of acute inflammation.

Eicosanoids are known to play important roles in inflammation. Recent findings have given rise to several new concepts regulating the generation of eicosanoids, illustrated in Figure 1. Lipoxins (LX) are trihydroxytetraene-containing eicosanoids that are generated within vascular lumen by platelet-leukocyte interactions and at mucosal surfaces by leukocyte-epithelial cell interactions. During these cell-cell interactions, transcellular biosynthetic pathways are used as major routes, and thus, in humans, LX are formed in vivo during multicellular responses such as inflammation, atherosclerosis, and thrombosis. This branch of the eicosanoid cascade generates specific tetraene-containing products that appear to function as stop signals, since they inhibit key steps in leukocyte-mediated inflammation. Of special interest, it appears that aspirin also functions in part via production of novel epimers of lipoxins or 15-epi-lipoxins (Figure 1). Here, we review recent developments on the cellular interactions of these novel anti-inflammatory mediators.

Three documented cell death pathways, apoptosis, necrosis, and oncosis will be discussed. The end result of each pathway is cell death; however, the path by which death is achieved and the morphological and physiological traits of each may be strikingly distinct. Now that well characterized models have been established for particularly apoptosis, the induction pathway(s) has received much attention and the pathway pathology is beginning to be understood. Three model systems were investigated: APO-1/Fas, hypoxia, and oncosis. Cell death was induced, and during a time course sampling, a variety of methodologies, including DNA fragmentation by flow cytometry and gel electrophoresis, DNA staining, flow cytometric light scatter, transmission electron microscopy, anti-tubulin, Trypan blue, annexin V, and anti-APO2.7 were employed to monitor the cell death progress. The apoptotic pathway in the CD95-induced Jurkat cell model was further investigated using caspase inhibitor peptides and analyzed for APO2.7 antigen expression and DNA fragmentation by flow cytometry. Time course sampling characterized the cell death pathway and helped to differentiate the capabilities of the methods. The time to response and duration of the response were dependent upon cell type and method of induction. The CD95-induced Jurkat cell model showed a classical apoptotic response; however the MDA-MB-175-VII hypoxia model and the anti-5A9 induced oncosis model were not as clear. Each methodology shows advantages and disadvantages that allow the investigator to select several methods to identify, monitor, and enumerate cells with respect to cell death progression using time course studies.

Various lines of evidence indicate that oxidative stress resulting in lipid peroxidation and protein modification is involved in the pathogenesis of atherosclerosis and coronary heart disease. We have investigated the effect of modified (oxidized) low-density lipoproteins (oxLDL) on collagen and fibronectin synthesis in cultured human coronary artery smooth muscle cells (HCA-SMC). As shown by immunofluorescence microscopy and time-resolved fluorescence immunoassay, oxLDL dose-dependently stimulated collagen type I and fibronectin synthesis in cultured HCA-SMC. The effect on matrix synthesis was biphasic, with a maximum effect at concentrations between 1 and 10 μg/ml oxLDL. Higher oxLDL concentrations (>25 μg/ml) were cytotoxic. Beside oxLDL, malondialdehyde-modified LDL also stimulated extracellular matrix synthesis. In the presence of 100 μg/ml ascorbic acid, 25, 50 and 100 μg/ml oxLDL induced apoptosis within 6–8 hours (demonstrated by TUNEL-reaction, annexin-V binding and APO-2.7-expression). Apoptosis was not induced by normal (unmodified) LDL and malondialdehyde-modified LDL. The radical scavengers and antioxidants TROLOX and probucol and the hydrogen peroxide eliminator catalase significantly reduced oxLDL-induced apoptosis. Our results demonstrate that low concentrations of oxLDL are profibrogenic by stimulating extracellular matrix synthesis, whereas higher oxLDL concentrations induce oxidative stress and apoptosis in coronary artery smooth muscle cells. The profibrogenic effect might be relevant in the formation of atherosclerotic plaques, and the proapoptotic effect might contribute to an increased plaque vulnerability.

During sepsis there is an increase in the plasma content of several compounds, e.g., bacterial toxins, cytokines, cell debris, free hemoglobin and myoglobin. In blood, these compounds activate various cascade systems, which in large amounts or in more vulnerable patients lead to a disseminated intra-vascular coagulopathy (DIC) with multiorgan dysfunction syndrome (MODS) and death, despite conventional intensive care unit therapy. Therapeutic attempts to reverse these conditions have so far been of limited benefit. These effects have mainly been focused on lowering the blood concentration of single substances such as tumor necrosis factor. By the use of low-and high-flux hemodialysis filters, usually only small amounts of these substances are removed. By the use of plasmapheresis or plasma exchange, the extent of removal is considerably increased. The efficacy varies between the techniques (centrifugation vs. filtration or adsorption) and has also different influences on e.g. the complement system. This report describes these techniques and the therapeutical possibilities given by them. In small trials, blood or plasma exchange has been used as rescue therapy in critically ill patients with a progressive MODS and DIC. A survival of about 80 % of the patients has been reported in these studies and the use of combined therapy will be discussed. Controlled trials are required in this field.

Adequate volume loading may be the most important step in the treatment of patients with septic shock. Techniques allowing us to achieve and tightly control volume loading and regional perfusion are considered to be helpful. An elevated oxygen delivery may be beneficial in some patients but the increase of oxygen delivery should be guided by the measurement of parameters assessing global and regional oxygenation. Forcing an increase in oxygen delivery by the use of very high dosages of catecholamines can be harmful. Vasopressors should be used for achieving an adequate perfusion pressure. For norepinephrine, no negative effects on regional perfusion have been demonstrated. Epinephrine and dopamine should be avoided because they seem to redistribute blood flow away from the splanchnic region. There are no convincing data yet to support the routine use of low dose dopamine or dopexamine in patients with sepsis. Neither low dose dopamine nor dopexamine has been proven to prevent renal failure in septic patients. Furthermore, there is evidence that low dose dopamine may reduce mucosal perfusion in the gut in some patients. There is some suggestion that dopexamine can improve splanchnic perfusion but since these effects remain somewhat controversial, there is no reason for a general recommendation for dopexamine in septic patients.

Intravenous immune globulins are widely used as supplemental treatment of sepsis, septic shock and systemic inflammation in the critically ill, although this indication has at best been validated in part. Likely beneficial mechanisms of action may include the improvement of serum bactericidal activity due to neutralizing and opsonizing immunoglobulin (Ig)G- and IgM-antibodies, as well as stimulation of phagocytosis and neutralization of bacterial endo- and exotoxins; another attractive mode of action may represent immune globulin-mediated modification and specific suppression of proinflammatory cytokine release from endotoxin- and superantigen-activated blood cells. For the “entire group of patients with sepsis and septic shock” a reduction in mortality by intravenous immune globulin could not be documented; however, in the score-based immunoglobulin in sepsis (SBITS)-study with 653 patients included, a moderate improvement in sepsis morbidity and multiple organ dysfunction syndrome was demonstrated. In defined sepsis subgroups, a reduction in mortality by intravenous immune globulin has been seen in individual small, not yet confirmed trials. Finally, the incidence of some severe infections in well characterized “patients at risk” and “operations at risk” is reduced by intravenous immune globulin prophylaxis. Thus, intravenous immune globulin is not a “magic bullet” of sepsis treatment, but it may reduce morbidity and thereby represent a useful piece of stone in the therapeutic mosaic of sepsis treatment.

Hemodialyis with cellulose-based membranes is associated with an array of adverse reactions, including leukopenia, pulmonary sequestration and dysfunction of leukocytes. Activation of the alternative pathway of complement due to direct contact of plasma with dialysis membrane is considered to be responsible for the induction of these side effects. In recent years, evidence has accumulated that other neutrophil effector functions such as reactive oxygen intermediate production play an important role as well. Here the importance of burst formation in cooperation with other inflammatory effector functions in the mechanisms of hemodialysis-related adverse effects will be discussed.

Sepsis with multiple organ failure is frequently associated with a substantial decrease of cholesterol levels. This decrease of cholesterol is strongly associated with mortality suggesting a direct relation between inflammatory conditions and altered cholesterol homeostasis. The host response during sepsis is mediated by cytokines and growth factors, which are capable of influencing lipid metabolism. Conversely lipoproteins are also capable of modulating cytokine production during the inflammatory response. Therefore the decrease in circulating cholesterol levels seems to play a crucial role in the pathophysiology of sepsis. In this review the interaction between cytokines and lipid metabolism and its clinical consequences will be discussed.

Patients (n = 242) admitted to intensive care unit for longer than 48 hours were categorised for sepsis according to American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) Consensus Conference criteria. Body temperature, leukocyte count, C-reactive protein (CRP) and procalcitonin (PCT) as well as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, IL-10 and HLA-DR expression on monocytes were determined. Data of one randomly choosen day per patient entered analysis. Immunologic mediators contributing significantly to outcome were determined by logistic regression analysis. Area under the curves (AUC) of receiver operating characteristic curves of clinical markers of inflammation predicting prognosis were compared with AUC of relevant immunologic mediators. TNF-α, IL-6 and HLA-DR expression on monocytes were significantly associated with outcome; the AUC's were 0.835, 0.844 and 0.761 respectively. AUC's for clinical markers were 0.878, 0.811, 0.620 and 0.614 for PCT, CRP, leukocyte count and body temperature respectively. PCT had the highest AUC compared to other clinical markers. These data indicate that PCT might be a better marker than the classic criteria of inflammation, CRP, leukocyte count, and body temperature to identify patients endangered by severe infection or sepsis.

A detailed study of the immune dysregulation involved in systemic inflammation requires the analysis of the main inflammatory cells in the circulation – the monocytes. Blood samples available from patients are necessarily restricted so that a rapid and efficient method is required to isolate these cells. Here we present a protocol to isolate blood monocytes in high yield from small samples of heparinised blood. The method yields monocyte preparations with a purity greater than 96 %.

Extracorporeal apheresis of endotoxins and pro-inflammatory cytokines is still a therapeutic option in the early hyper-inflammatory phase of gram-negative sepsis. There is therefore ongoing interest in adsorber materials suitable for that kind of clinical application. Here we describe lipopolysaccharide (LPS) and cytokine adsorption characteristics of a new adsorbent based on purified human serum albumin (HSA) covalently linked to macroporous polymer beads (iHSA). Multipoint attachment of HSA to acrylic beads via carboxyl groups of the protein resulted in an increased affinity to LPS. In adsorption experiments (adsorbent/plasma ratio 1:3) a 70–80 % reduction of limulus amoebocyte lysate (LAL) activity from 8.59 ± 2.07 EU/ml (mean ± SD) to 1.82 ± 0.77 EU/ml ( S. abortus equi; n = 40) (p < 0.001) and from 115.13 ± 53.76 EU/ml to 17.70 ± 11.68 EU/ml ( E. coli F583 ; n = 6) (p < 0.01) was achieved. iHSA-purified plasma samples showed a decreased capability of inducing cytokine release from peripheral monocytes. Direct haemoperfusion of LPS pre-stimulated whole blood over iHSA resulted in decreased tumour necrosis factor α (TNFα) concentrations (30–40 % reduction) whereas induced levels of interleukin (IL)-β and IL-6 were not affected. Depending on the means of immobilization, iHSA shows higher affinity for LPS than native albumin present in plasma. We demonstrated an efficient removal of LPS from plasma in vitro . Adsorption over immobilized HSA appears to be a simple and effective means of removing LPS and perhaps pro-inflammatory cytokines from the circulation.