Stefan Margraf

Decreased neutrophil adhesion to human cytomegalovirus-infected retinal pigment epithelial cells is mediated by virus-induced up-regulation of Fas ligand independent of neutrophil apoptosis.

Human CMV (HCMV) retinitis frequently leads to blindness in iatrogenically immunosuppressed patients and in the end stage of AIDS. Despite the general proinflammatory potential of HCMV, virus infection is associated with a rather mild cellular inflammatory response in the retina. To investigate this phenomenon, the influence of HCMV (strains AD169 or Hi91) infection on C-X-C chemokine secretion, ICAM-1 expression, and neutrophil recruitment in cultured human retinal pigment epithelial (RPE) cells was studied. Supernatants from infected cultures contained enhanced levels of IL-8 and melanoma growth-stimulating activity/Gro α and induced neutrophil chemotaxis compared with supernatants from uninfected RPE cells. Despite HCMV-induced ICAM-1 expression on RPE cells, binding of activated neutrophils to HCMV-infected RPE cells and subsequent transepithelial penetration were significantly reduced. Reduced neutrophil adhesion to infected RPE cells correlated with HCMV-induced up-regulation of constitutive Fas ligand (FasL) expression. Functional blocking of FasL on RPE cells with the neutralizing mAbs NOK-1 and NOK-2 or of the Fas receptor on neutrophils with mAbB-D29 prevented the HCMV-induced impairment of neutrophil/RPE interactions. Fas-FasL-dependent impairment of neutrophil binding had occurred by 10 min after neutrophil/RPE coculture without apoptotic signs. Neutrophil apoptosis was first detected after 4 h. Treatment of neutrophils with a specific inhibitor of caspase-8 suppressed apoptosis, whereas it did not prevent impaired neutrophil binding to infected RPE. The current results suggest a novel role for FasL in the RPE regulation of neutrophil binding. This may be an important feature of virus escape mechanisms and for sustaining the immune-privileged character of the retina during HCMV ocular infection.

Human Cytomegalovirus Circumvents NF-κB Dependence in Retinal Pigment Epithelial Cells

The human CMV (HCMV) is a persistent virus that may cause severe inflammatory responses especially in immunocompromised hosts. In different cell types, HCMV infection leads to the activation of the pleiotropic transcription factor, NF-κB, which triggers virus replication but also propagates cell-mediated inflammatory mechanisms that largely depend on PG synthesis. We investigated the interactions of HCMV and the NF-κB-dependent PG synthesis pathway in cultures of retinal pigment epithelial (RPE) cells that are known to be infected in HCMV retinitis patients. Unlike in other cell types, HCMV increased neither NF-κB activity nor p65 and p105/50 mRNA levels in RPE cells. Both TNF-α and phorbol ester 12,0-tetradecanoylphorbol 13-acetate (TPA) enhanced NF-κB activity but only TPA increased HCMV replication. Cyclooxygenase-2 expression and PGE2 release was increased by TPA and TNF-α but not by HCMV infection. Stimulatory activity of TPA on HCMV replication was suppressed by protein kinase C inhibitors and inhibitors of p42/44 and p38 mitogen-activated protein kinases but not by NF-κB inhibitors. In conclusion, HCMV circumvents the NF-κB route in favor of the protein kinase C-dependent mitogen-activated protein kinase pathway in RPE cells. This virus/host cell interaction might be a mechanism that promotes HCMV persistence in immune-privileged organs such as the eye.

Diagnostic accuracy of neutrophil‐derived circulating free DNA (cf‐DNA/NETs) for septic arthritis

The release of “neutrophil extracellular traps” (NETs) has been identified as a novel immune response in innate immunity. NETs are composed of neutrophil‐derived circulating free DNA (cf‐DNA) and neutrophil cytoplasm‐derived proteins such as proteases. In this study, we analyzed the putative diagnostic value of synovial cf‐DNA/NETs for identification of septic arthritis. Forty‐two patients with a joint effusion who had undergone arthrocentesis were included. From synovial fluid, cf‐DNA/NETs (j‐cf‐DNA) levels were directly quantified. Diagnostic value of j‐cf‐DNA was compared with white blood cells (WBC), synovial white blood cells (j‐WBC), C‐reactive protein (CRP), j‐IL‐6, j‐TNF alpha, j‐IL‐1 beta, and myeloperoxidase (j‐MPO). Sensitivity, specificity, positive and negative predictive value, as well as ROC‐curves for each parameter were calculated. Synovial fluid cf‐DNA/NETs values from patients with septic arthritis (3,286 ± 386 ng/ml, n = 9) were significantly increased compared to patients with noninfectious joint inflammation (1,040 ± 208 ng/ml, n = 17) or osteoarthritis (278 ± 34 ng/ml, n = 16, p < 0.01). In conjunction with j‐cf‐DNA, j‐IL‐6 and j‐IL‐1 beta were significantly elevated (p < 0.01), but WBC, CRP, and j‐WBC were not. At a cut‐off of 300 ng/ml, j‐cf‐DNA had a sensitivity of 0.89, a specificity of 1.0, a positive predictive value of 1.0, and a negative predictive value of 0.97. Receiver operation curves revealed largest areas under the curve for cf‐DNA/NETs (0.933) and j‐IL‐6 (0.951). cf‐DNA/NETs seem to be a valuable additional marker for the diagnosis of septic arthritis or periprosthetic infections. However, this result should be confirmed in a large clinical trial.

Cytomegalovirus Infection Decreases Expression of Thrombospondin-1 and -2 in Cultured Human Retinal Glial Cells: Effects of Antiviral Agents

In fibroblasts, infection with human cytomegalovirus (HCMV) inhibits expression of the extracellular matrix proteins thrombospondin-1 and -2 (TSP-1 and TSP-2). These effects may depend on expression of HCMV immediate-early (IE) genes, which are activated by cellular transcription factor NF-kappaB. The influence of HCMV infection on TSP-1 and TSP-2 expression and the ability of different antiviral drugs to prevent these cellular changes in permissive cultures of human retinal glial cells were observed. Ganciclovir inhibited only HCMV late antigen (LA) expression, whereas antisense oligonucleotide ISIS 2922 and peptide SN50, inhibitors of HCMV IE expression and NF-kappaB activity, respectively, inhibited both IE and LA expression. ISIS 2922 and SN50, but not ganciclovir, prevented down-modulation of TSP-1 and TSP-2. The results showed that HCMV-induced down-modulation of TSP-1 and TSP-2 in retinal glial cells is prevented by inhibition of HCMV IE expression. These findings may be relevant to pathogenesis and treatment of HCMV retinitis.

Inhibition of neutrophil activity improves cardiac function after cardiopulmonary bypass

BackgroundThe arterial in line application of the leukocyte inhibition module (LIM) in the cardiopulmonary bypass (CPB) limits overshooting leukocyte activity during cardiac surgery. We studied in a porcine model whether LIM may have beneficial effects on cardiac function after CPB.MethodsGerman landrace pigs underwent CPB (60 min myocardial ischemia; 30 min reperfusion) without (group I; n = 6) or with LIM (group II; n = 6). The cardiac indices (CI) and cardiac function were analyzed pre and post CPB with a Swan-Ganz catheter and the cardiac function analyzer. Neutrophil labeling with technetium, scintigraphy, and histological analyses were done to track activated neutrophils within the organs.ResultsLIM prevented CPB-associated increase of neutrophil counts in peripheral blood. In group I, the CI significantly declined post CPB (post: 3.26 ± 0.31; pre: 4.05 ± 0.45 l/min/m2; p < 0.01). In group II, the CI was only slightly reduced (post: 3.86 ± 0.49; pre 4.21 ± 1.32 l/min/m2; p = 0.23). Post CPB, the intergroup difference showed significantly higher CI values in the LIM group (p < 0.05) which was in conjunction with higher pre-load independent endsystolic pressure volume relationship (ESPVR) values (group I: 1.57 ± 0.18; group II: 1.93 ± 0.16; p < 0.001). Moreover, the systemic vascular resistance and pulmonary vascular resistance were lower in the LIM group. LIM appeared to accelerate the sequestration of hyperactivated neutrophils in the spleen and to reduce neutrophil infiltration of heart and lung.ConclusionOur data provides strong evidence that LIM improves perioperative hemodynamics and cardiac function after CPB by limiting neutrophil activity and inducing accelerated sequestration of neutrophils in the spleen.

Inhibition of neutrophil activity in cardiac surgery with cardiopulmonary bypass: a novel strategy with the leukocyte inhibition module

Recently, we showed that the arterial in-line application of the leukocyte inhibition module (LIM) within the heart-lung machine limits overshooting leukocyte activity and cardiac tissue damage. Moreover, significantly better cardiac function was found in an experimental animal model when LIM was used. In the meantime, the first promising clinical data exist. LIM has to be regarded as an essential tool in extracorporeal circulation, in the future, to improve postoperative clinical outcome and to reduce costs. This review summarizes the biological background of LIM and the current experience obtained in experimental models and clinical studies.

ANTISENSE OLIGONUCLEOTIDE ISIS 2922 TARGETS IE-EXPRESSION AND PREVENTS HCMV-IE-INDUCED SUPPRESSION OF TSP-1 AND TSP-2 EXPRESSION

ISIS 2922, but not ganciclovir (GCV), inhibits HCMV immediate early protein (IE) expression in different infected cell lines and prevents down-modulation of extracellular matrix proteins thrombospondin-1 and -2 induced by IE proteins. While action of ISIS 2922 is mainly due to specific inhibition of IE 2 mRNA, there is also evidence for unspecific effects in terms of inhibition of virus adhesion and penetration.

Cardiac Surgery With Extracorporeal Circulation: Neutrophil Transendothelial Migration Is Mediated by β1 Integrin (CD29) in the Presence of TNF-Alpha

Cardiac surgery with extracorporeal circulation is associated with neutrophil activation, inflammation, and edema. Endothelial hyperpermeability elicited by the interaction of activated neutrophils and/or cytokines with endothelial cells may be critical in this regard. However, the immune and cellular mechanisms involved are not fully understood. Cocultures with human endothelial cells and neutrophils from cardiac surgery patients were used to evaluate the role of β1 integrin activity and the proinflammatory cytokine tumor necrosis factor (TNF)-α in neutrophil transendothelial migration and in impairment of the integrity of endothelial cell-to-cell contacts. Blocking of CD29 (heavy chain of β1 integrins) totally prevented neutrophil adhesion and transendothelial migration. Pretreatment of neutrophils with either a CD29-stimulating monoclonal antibody or the addition of TNF-α (0.1–10 U/ml) to the coculture failed to induce transendothelial migration. However, coculture of endothelial cells with CD29-stimulated neutrophils in the presence of 0.1–10 U/ml TNF-α strongly induced neutrophil transmigration. CD29/TNF-α-mediated transmigration was associated with intracellular redistribution of endothelial β-catenin. We further showed that CD29/TNF-α-mediated effects involved PI3K and tyrosine kinase-dependent signaling via MAPK but were independent of nuclear transcription factor (NF)-κB activity. Inhibition of CD29/TNF-α might be a therapeutic option to limit endothelial dysfunction following cardiac surgery with extracorporeal circulation.

Impaired Cell Viability and Functionality of Hepatocytes After Incubation With Septic Plasma—Results of a Second Prospective Biosensor Study

Liver dysfunction (LD) and liver failure are associated with poor outcome in critically ill patients. In patients with severe sepsis or septic shock, LD occurred in nearly 19% of patients. An early diagnosis of LD at time of initial damage of the liver can lead to a better prognosis of these patients because an early start of therapy is possible. We performed a second prospective study with septic patients to test a new cell-based cytotoxicity device (biosensor) to evaluate clinical relevance for early diagnosis of LD and prognostic capacity. In the clinical study, 99 intensive care unit patients were included in two groups. From the patients of the septic group (n = 51, SG), and the control (non-septic) group [n = 49, control group (CG)] were drawn 20 ml blood at inclusion, after 3, and 7 days for testing with the biosensor. Patients’ data were recorded for hospital survival, organ function, and demographic data, illness severity [acute physiology and chronic health evaluation (APACHE) II-, sepsis-related organ failure assessment (SOFA) scores], cytokines, circulating-free deoxyribonucleic acid/neutrophil-derived extracellular traps (cf-DNA/NETs), microbiological results, and pre-morbidity. For the developed cytotoxicity test, the human liver cell line HepG2/C3A was used. Patients’ plasma was incubated in a microtiter plate assay with the test cells and after 6 days incubation the viability (trypan blue staining, XTT-test) and functionality (synthesis of albumin, cytochrome 1A2 activity) was analyzed. An impairment of viability and functionality of test cells was only seen in the SG compared with the CG. The plasma of non-survivors in the SG led to a more pronounced impairment of test cells than the plasma of survivors at inclusion. In addition, the levels of cf-DNA/NETs were significantly higher in the SG at inclusion, after 3, and after 7 days compared with the CG. The SG showed an in-hospital mortality of 24% and the values of bilirubin, APACHE II-, and SOFA scores were markedly higher at inclusion than in the CG. Hepatotoxicity of septic plasma was already detected with the liver cell-based biosensor at inclusion and also in the course of disease. The biosensor may be a tool for early diagnosis of LD in septic patients and may have prognostic relevance.