A. von Ruecker

Protein kinase C involvement in lipid peroxidation and cell membrane damage induced by oxygen-based radicals in hepatocytes.

We investigated the effects of oxygen-based radicals induced by t-butyl hydroperoxide or H2O2/Cu2+ on cultured hepatocytes. Radical exposure caused membrane lesions (blebs), lactate dehydrogenase release and lipid peroxidation (i.e. formation of malondialdehyde) in cells. As expected, radical scavengers (catalase, alpha-tocopherol) strongly inhibited these phenomena. A similar or even superior inhibitory effect was achieved by the protein kinase C (PKC) inhibitors H-7 and phloretin. These agents did not reveal notable radical scavenging properties as assessed by their ability to break down H2O2. The PKC stimulators 4 beta-phorbol-12-myristate-13 and 1-olyeoyl-2-acetyl-sn-glycerol intensified the detrimental actions of the radical-inducing agents. [3H]Phorbol-12,13-dibutyrate-binding studies showed that membrane association of PKC is markedly increased in hepatocytes after exposure to H2O2/Cu2+ or t-butyl hydroperoxide. These results suggest that PKC membrane translocation and activation may be important for mediating membrane damage and lipid peroxidation after cells are exposed to oxygen-based radicals.

Bladder cancer cells acquire competent mechanisms to escape Fas-mediated apoptosis and immune surveillance in the course of malignant transformation

Mechanisms of resistance against Fas-mediated cell killing have been reported in different malignancies. However, the biological response of immune escape mechanisms might depend on malignant transformation of cancer cells. In this study we investigated different mechanisms of immune escape in 2 well-differentiated low-grade (RT4 and RT112) and 2 poorly differentiated high-grade (T24 and TCCSUP) bladder cancer cell lines. Fas, the receptor of Fas-ligand, is expressed and shedded by human transitional bladder carcinoma cell lines RT4, RT112, T24 and TCCSUP. Cytotoxicity and apoptosis assays demonstrate that in spite of the Fas expression, poorly differentiated T24 and TCCSUP cells are insensitive towards either recombinant Fas-ligand or agonistic apoptosis-inducing monoclonal antibody against Fas. In poorly differentiated T24 and TCCSUP cell lines we were able to detect marked Fas-ligand protein by flow cytometry and Western blot analysis. In grade 1 RT4 and RT112 cells only minor expression of Fas-ligand possibly because of proteinase action. Fas-ligand mRNA translation or post-translational processing seems to be regulated differentially in the cancer cell lines depending on malignant transformation. In co-culture experiments we show that poorly differentiated cells can induce apoptosis and cell death in Jurkat cells and activated peripheral blood mononuclear cells. This in vitro study suggests that bladder cancer cells can take advantage of different mechanisms of immune evasion and become more competent in avoiding immune surveillance during transformation to higher-grade malignant disease.

Increased susceptibility to apoptosis in circulating lymphocytes of critically ill patients.

Abstract. Background and aims: Lymphocyte apoptosis may influence immune responsiveness in systemic inflammation. Therefore, we investigated whether early signs of apoptosis (i.e., annexin-V binding and cell shrinkage) in peripheral lymphocytes were different among patients with severe sepsis, critically ill, nonseptic patients after major surgery, and healthy individuals. Patients/methods: Ten patients with severe sepsis and ten critically ill, nonseptic patients after major surgery admitted to a surgical intensive care unit in a university hospital were included in the study. In addition, ten healthy blood donors were included for comparison. We investigated early signs of apoptosis using flow cytometric measurement of annexin-V binding to the cell surface and cell shrinkage of peripheral lymphocytes. Results: The percentage of apoptotic lymphocytes determined as annexin-V positive and propidium iodide negative cells was increased in freshly prepared cells of patients with severe sepsis (11.4±0.5%) and critically ill, nonseptic patients after major surgery (18.5±2.0%) relative to healthy blood donors (4.4±0.5%) (P<0.05). No significant difference between patients with severe sepsis and patients after major surgery were found. Annexin-V binding increased significantly after OKT-3 stimulation of lymphocytes in patients with severe sepsis (34.4±1.6%), patients after major surgery (33.8±3.4%), and healthy blood donors (21.1±2.8%). No significant difference among groups was detected following OKT-3 stimulation. Furthermore, freshly isolated peripheral lymphocytes of patients with severe sepsis and critically ill, nonseptic patients after major surgery revealed a significantly higher proportion of cell shrinkage than in healthy blood donors (55.0±2.2%, 21.5±2.4% vs 3.6±0.7%; P<0.05). Conclusion: Circulating lymphocytes of critically ill patients show a high degree of early signs of cellular apoptosis. This may contribute to hyporesponsiveness of immune cells in systemic inflammation.

Endoscopic vs conventional hernia repair from an immunologic point of view.

AbstractBackground: In this study we tried to estimate the local surgical trauma in patients undergoing endoscopic or conventional hernia repair via the changes in peripheral blood T cell subpopulations (i.e., T-helper 1 (TH1) and TH2 cells), recently shown to be recruited differentially to inflammatory sites. Methods: Cells were identified flow-cytometrically by intracellular cytokine staining on a single cell level in 30 patients undergoing conventional (Shouldice) or total extraperitoneal patch (TEPP) hernia repair. Results: The TH1 cells decreased postoperatively in Shouldice patients on an average of 20.8–31.4%, whereas in TEPP patients only a minor decline (mean, 7.8–9.2%) was observed. The TH2 cells did not change significantly in TEPP patients, and a small increase (mean, 7.7%) was detected in Shouldice patients. Conclusions: Our results suggest that the postoperative reduction in TH1 cells reflects local surgical trauma and can be helpful in evaluating different surgical procedures. When conventional and endoscopic hernia repair were compared, the latter proved less traumatizing.

Qualitative and quantitative changes in platelets after coronary-artery bypass surgery may help identify thrombotic complications and infections

SummaryWe studied the effect of coronary-artery bypass surgery on blood cells and platelets. Hematological parameters of eighty-three patients were measured by an automated cell counting and sizing analyzer. Sampling time was from 24 h prior to 10 days after surgery. During this time leukocytes and platelets showed characteristic changes in numbers and size, whereas red blood cells revealed no typical modifications. Even though it seems clear that changes of hematological parameters occur after bypass surgery, it is important to be aware of the actual extent of such changes. Therefore the data of 50 patients who had had no post-operative clinical complications were combined to generate diagrams of those parameters that had changed in a characteristic fashion. The diagrams showing average changes, and 99% confidence intervals in mean platelet volume and platelet count were able to identify seven (out of 7) cases with complications up to 48 h before clinical signs were apparent. Complications ranged from mild (3 cases with infections) to severe (4 cases with thrombosis, embolic thrombosis and/or reinfarction). Diagrams showing changes in leukocyte parameters were able to identify only two cases with infections. Even though the number of cases is yet small, the results suggest that surveillance of platelet parameters may be useful in postoperative care. Furthermore, this study was able to confirm the recent findings of Trowbridge and Martin [18] that an abnormal increase in platelet volume distribution width and low platelet counts found in patients with coronary heart disease may serve as good indicators for the prethrombotic state and the risk of myocardial infarction.

Mechanismen der regenerativen Wundheilung nach Hauttransplantation: der SMAD-Signalweg

Scars arise in the late phase of wound healing as a response to tissue injury and are characterised by fibroplasia. For the patient, the function deficiency is not only a cosmetic problem, in some cases it can present a life-threatening complication and lead to severe loss in health related quality of life. Current studies with the MRL mice could demonstrate an accelerated wound healing without the generation of scars as well as regenerative wound healing. The aim of the present study was to elucidate the mechanism of regenerative wound healing in a skin transplantation model. Material and Methods: Full skin grafts from MRL/+ and B10 mice were cross transplanted. 3 d and 10 d after transplantation we measured tissue pO2 (Licox-pO2), microcirculation (Laser Doppler), graft size and graft vascularization. In tissue slides, we evaluated the collagen thickness and cell apoptosis by the TUNEL-assay. Quantitative mRNA Analysis was performed with the Taqman method. Results: (Mean+/– SD; n = 5; *p < 0.05; T = 10 d; MRL vs. 0B10) Tissue pO2 was significantly higher in MRL mice (148 +/– 15.3* vs. 76 +/– 28.7 mm Hg), as was the microcirculation (233 +/– 65* vs. 77+/–33.2 aU) and the size of the full thickness skin graft (100+/–32 vs. 46+/– 17.6 mm2) which at 10 d was significantly larger than in the B10-group. The thickness of the collagen layer (298+/–32.82 vs. 545.6+/–39.2 µm) and the TUNEL reaction were significantly higher in the B10-group. The infiltration of the wound area with inflammatory cells (leucocytes) was markedly reduced in the MRL-group. Discussion: As possible mechanism for accelerated wound healing the SMAD pathway has been implicated. Our results showed that in MRL-mice, in contrast to B10 mice, the signal molecule SMAD7 expression is down regulated; therefore the signals from transforming growth factor beta are enhanced. Conclusions: The enhanced regenerative wound healing in MRL/+ mice is associated with improved vascularization and microcirculation. The inflammatory reaction is blunted. Causative for these results are most likely changes in the SMAD signal pathway.

Atrial Natriuretic Peptide (ANP) Protects Cells against Injury by Hypoxia and Hypochlorous Acid

Recent reports show that ANP protects against post-ischemic renal failure in vivo and in the isolated perfused kidney [4, 8]. Since ANP has been shown to be a potent renal vasodilatator, this protective effect may be mediated exclusively by improving hemodynamics. To determine if ANP also has a protective effect at the cellular level, studies were performed in hepatocyte cell cultures. Hepatocytes showed a considerable increase in cellular cGMP content after ANP stimulation, suggesting the presence of ANP receptors. Cell cultures were exposed to hypoxia and reactive oxygen (hypochlorous acid, HClO), which cause cell damage similar to postischemic injury in vivo [3]. The following report demonstrates that ANP can protect cell cultures.

Atrial natriuretic peptide involvement in human platelet aggregability in vitro

SummaryAtrial natriuretic peptide (ANP) counteracts the destabilization and aggregation of platelets which is induced by vasopressin, angiotensin, and adrenaline.