Annelore Ittenson

Effects of different anaesthetic agents on immune cell function in vitro

Background and objective: Anaesthesia may affect the regulatory balance of postoperative immune response. The aim of this study was to investigate the effects of different volatile and non‐volatile anaesthetic agents and particularly of clinically used agent combinations on the proliferation capacity and cytokine production of immune cells. Methods: Peripheral blood mononuclear cells from healthy donors were PHA‐activated in the presence or absence of various concentrations of thiopental, propofol, fentanyl, sufentanil, sevoflurane, nitrous oxide and combinations of these anaesthetics. Cell proliferation was assessed by tritiated thymidine uptake. Interleukin‐2 production and release of the soluble IL‐2 receptor were determined by enzyme immunoassays and used as measures of lymphocyte activation. Results: Thiopental inhibited cell proliferation in a dose dependent manner (P < 0.001) and reduced sIL‐2R release (2090‐970 pg mL−1; P < 0.05). Propofol reduced sIL‐2R release at the high concentration of 10 μg mL−1 (2220 pg mL−1‐1780 μg mL−1; P < 0.05). Fentanyl and sufentanil did not compensate for or enhance the inhibitory effects of thiopental. Nitrous oxide, but not sevoflurane, reduced the proliferation of human peripheral blood mononuclear cells (P < 0.05). In combinations with thiopental or nitrous oxide, sevoflurane compensated the inhibitory effects of these two agents. Fentanyl, sufentanil, sevoflurane and nitrous oxide did not affect PHA‐induced IL‐2 and sIL‐2 receptor release by human peripheral blood mononuclear cells. Conclusion: Thiopental and nitrous oxide have immunosuppressive activity. In contrast, sevoflurane may have a beneficial effect by alleviating the immunosuppressive effects of both substances.

Increased expression of P-selectin in patients with chronic atrial fibrillation.

Previous studies have shown that platelets are activated during atrial fibrillation (AF). However, prophylactic therapy with aspirin is not associated with a reduction of thromboembolic complications in patients with AF. Stimulation of platelet thrombin and ADP receptors causes a release of P‐selectin, which is not affected by aspirin. The purpose of this study was to assess the influence of AF on platelet P‐selectin expression. Blood samples from 30 patients were studied ex vivo. Nineteen patients had chronic AF (> 3 months), 11 patients were in sinus rhythm (SR). P‐selectin expression was determined by flow cytometry (antibody binding capacity [BC]) at baseline and after platelet stimulation with adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP). To determine the effect of heart rate and atrial pressure (RAP), measurements were repeated after 10 minutes of ventricular pacing (120 beats/min) in patients with SR. P‐selectin expression was increased in patients with AF at baseline (AF: 1329 ±81 BC vs SR: 968 ± 108 BC; P < 0.05) and after stimulation with ADP (AF: 1445 ± 101 BC vs SR: 1061 ± 109 BC; P < 0.05) and TRAP (AF: 13783 ± 2442 BC vs SR: 5977 ± 800 BC; P < 0.05). RAP (2.0 ± 0.5 vs 6.0 ± 0.8 mmHg; P < 0.01) and atrial rate (75 ± 5 vs 114 ± 5 beats/min; P < 0.001) increased during ventricular pacing. However, P‐selectin levels remained stable. AF was accompanied by increased P‐selectin expression. In contrast, increased ventricular rate and elevated atrial pressure alone had no effect on platelet activity. Further studies are needed to determine if platelet ADP receptor inhibitors offer a therapeutic benefit in patients with AF.

High Sensitive Detection of Double-Stranded DNA Autoantibodies by a Modified Crithidia luciliae Immunofluorescence Test

Anti‐double‐stranded (ds)DNA antibodies are serological markers of systemic lupus erythematosus (SLE). Of all anti‐dsDNA antibody detection methods, the Crithidia luciliae immunofluorescence test (CLIFT) is thought to have the highest specificity for SLE. However, the clinical application is hampered by the low diagnostic sensitivity. A CLIFT with modified assay buffer (mCLIFT) was developed and compared with conventional CLIFT, using sera from 110 patients with SLE, 89 anti‐dsDNA ELISA‐positive patients with other diseases (non‐SLE group A), 157 non‐SLE patients with undetectable anti‐dsDNA antibodies by ELISA (non‐SLE group B), 77 disease controls (non‐SLE group C), and 50 healthy blood donors. Out of the 110 anti‐dsDNA antibody ELISA‐positive SLE patients, 84 (76.4%) demonstrated a positive kinetoplast staining, using the mCLIFT, compared to only 42.3%, using the conventional CLIFT. The diagnostic specificity of mCLIFT was 100% with healthy blood donors and 98.1% with the non‐SLE group C (anti‐nuclear antibodies negative; no signs or symptoms of an autoimmune disease) included. In the non‐SLE groups A and B with various other autoimmune diseases or symptoms of a possible autoimmune disease, positive mCLIFT results were obtained in 33.7% and 3.2%, respectively. In conclusion, by modification of the assay buffer, a significant increase in sensitivity of the CLIFT could be observed while retaining the high specificity for SLE. Further investigation is required to check whether the CLIFT‐positive non‐SLE patients develop SLE and whether anti‐dsDNA antibodies detected by the mCLIFT represent a pathogenetic and diagnostic subgroup of autoantibodies that may improve the early diagnosis of SLE or SLE‐overlap syndromes.

Overproduction of IL-7, IL-10 and TGF-β1 in Multiple Myeloma

Hendrik Kröning, Abteilung Onkologie und Hämatologie, Städtísches Klinikum, Krankenhaus ‘Altstadt’, PSF 1220, MaxOtten-Str. 11-15, D-39002 Magdeburg (Germany), Tel. 0391-5919-220 Multiple myeloma (MM) is characterized by bone marrow infiltration of myeloma cells, a monoclonal gam-mopathy, and osteolytic lesions. Although most patients respond to initial chemotherapy, nearly all patients relapse and are refractory to salvage regimens. It is reasonable to assume that the outcome is related to a multidrug-resistant subset of circulating myeloma cells [1]. The bone marrow infiltration by this particular cell population and the growth of myeloma cells inside osteolytic lesions are regulated by a cytokine network where interleukin-6 (IL-6) plays a key role. High serum levels of IL-6 or IL-6 receptor (IL-6R) are identified as risk factors in MM. IL-6 is suggested to act as autocrine or paracrine growth factor for myeloma cells produced by the tumour environment [2]. Additionally, B cell-regulating cytokines such as IL-7, IL-10 and IL-13 are proposed to be involved in myeloma development [3]. Furthermore, IL-4 is able to sustain the viability of activated B cells in vitro but together with TGF-ß 1 it inhibits IL-6 synthesis [4]. In a recent report it was shown that high serum level of IL-2 are associated with a smouldering course of MM. In addition, IL-7 and IL-13 have been shown to increase the level of IL-2induced activity of lymphokine-activated killer cells supporting immunologi-cal defence mechanisms against B cell malignancies, but IL-4 inhibits these effects [5, 6]. The object of this investigation was to evaluate in vivo serum levels of IL·2, IL-4, IL-6, IL-6R, IL-7, IL-10, IL-13 and TGF-ßl in different stages of MM. With respect to the hypothesis that cytokines have more localised effects, analyses of peripheral blood and bone marrow aspirates were performed in parallel. Using the nonparametric Wil-coxon-Mann-Whitney U test for statistical analysis these parameters were correlated with clinical features and different B and T cell antigens on

Rapid mitogen-induced aminopeptidase N surface expression in human T cells is dominated by mechanisms independent of de novo protein biosynthesis

Abstract The membrane bound metalloprotease aminopeptidase N (APN, CD13, EC 3.4.11.2) is a well established marker of normal and malignant cells of the myelo-monocytic lineage. It is also expressed by leukaemic blasts of a small group of patients suffering from acute or chronic lymphoid leukaemia. Recently, the expression of the APN gene in T cell lines as well as the induction of APN gene and surface expression in human peripheral T cells by mitogenic activation have been demonstrated. Here, by means of cytofluorimetric analysis evidence is provided, that the induction of APN surface expression is partially resistent to the action of the inhibitors of protein biosynthesis, puromycin and cycloheximide, and is not prevented by tunicamycin, an inhibitor of glycosylation. These data suggest that the rapid mitogen-induced surface expression of APN, detectable 20 hours after stimulation is dominated by mechanisms not dependent on de novo protein biosynthesis or glycosylation. As shown by simultaneous analyses, the inhibitors used did also differently modify the induction of surface expression of other inducible glycosylated leukocyte surface antigens, namely CD25, CD69 and CD95.

A new dot immunoassay for simultaneous detection of celiac specific antibodies and IgA-deficiency.

Abstract Background: This study investigated whether a dot immunoassay (DIA) can provide simultaneous detection of anti-tissue transglutaminase (tTG), anti-deamidated gliadin (DG) and total IgA antibodies, as required in the work-up of celiac disease (CD) patients. Methods: Celiac disease patients (n=111) consecutively diagnosed from 2001 to 2011 at the Children’s Hospital and Institute of Immunology (Technical University Dresden) were tested for anti-tTG, anti-DG and total IgA by enzyme-linked immunosorbent assay (ELISA) and DIA retrospectively. Blood donors (n=45) and non-CD individuals with low IgA serum levels (n=8) were included as controls. Antibodies to endomysial antigens (EmA) were assessed by indirect immunofluorescence (IIF). Results: Four (3.6%) of 111 CD patients demonstrated an IgA deficiency with total IgA below 50 mg/L by ELISA. Total IgA of the 107 IgA-non-deficient CD patients varied from 70 to 6000 mg/L. All four IgA-deficient CD patients were detected by a reduced reaction control of DIA and demonstrated positive anti-tTG or anti-DG IgG by DIA or ELISA. Detection of anti-tTG and anti-DG by DIA and ELISA showed a very good agreement (IgA: κ=0.972, 0.856, respectively; IgG: 0.921, 0.895, respectively). Conclusions: Immunodot assay is a reliable and easy-to-use technique for the detection of IgA-deficient CD patients. Simultaneous assessment of anti-tTG and anti-DG IgA antibodies, and IgA deficiency by DIA can improve the efficacy of CD serology.

Pretreatment with interleukin-2 modulates peri-operative immuno-dysfunction in patients with renal cell carcinoma.

OBJECTIVE Complex peri-operative immuno-dysfunction occurs in patients with renal cell carcinoma undergoing nephrectomy. Here, the effect of pretreatment with interleukin-2 (IL-2) is addressed. METHODS Of 63 patients who underwent tumor nephrectomy, 26 patients received 4 doses of 10 Mio IE/m(2) IL-2 b.d. s.c. (i.e. a total of 40 Mio IE/m(2)) a week before operation, 37 did not. Parameters of cellular and humoral immunity (differential blood count, T-cell markers CD2, CD3, CD4, and CD8, B-cell markers CD19 and CD20, monocyte markers CD13 and CD14, NK (natural killer)-cell marker CD16, activation markers CD25, CD26, CD69, and HLA-DR, and cytokines IL-1-receptor antagonist (IL-1RA), IL-2, soluble IL-2-receptor (sIL-2R), IL-6, IL-10, and TGFbeta) were measured in venous blood. Blood was drawn before IL-2, 1 day before and immediately after the operation, and on the 1st, 3rd, 5th, and 10th postoperative day. RESULTS All patients showed postoperatively elevated leukocyte and granulocyte counts, and elevated serum levels of cytokines IL-6 and IL-10. T-cell and activation markers were decreased. However, all these alterations were less accentuated in patients who had been pretreated with IL-2. Monocyte counts and IL-2 and TGFbeta levels were decreased, but IL-1RA and sIL-2R levels were elevated in pretreated patients. IL-2 related toxicity was WHO grades I-II in all patients, grade III in one patient. The anesthetic regimen had no measurable effect. IL-6 concentrations were higher in renal venous than in venous pool blood, indicating IL-6 production in the tumor in vivo. CONCLUSIONS Pretreatment with IL-2 modulates peri-operative immuno-dysfunction in patients undergoing tumor nephrectomy. This affects in particular T-cell-mediated immunity and levels of cytokines IL-10 and IL-6. The IL-2 administration scheme used here was followed by distinct counter-regulation including monocytes, IL-2, sIL-2R, IL-1RA and TGFbeta.

Pretreatment with Interferon-α2a Modulates Perioperative Immunodysfunction in Patients with Renal Cell Carcinoma

Introduction: Complex perioperative immunodysfunction occurs in patients with renal cell carcinoma undergoing surgery. Here, we report on the effect of preoperative treatment with interferon-α2a (IFN-α2a). Materials and Methods: 30 patients with a renal tumour received preoperative IFN-α2a for 6 days beginning 1 week before nephrectomy, 30 did not. Parameters of cellular and humoral immunity were measured in venous blood at various intervals using flow cytometry and ELISA. Endpoints included effects on immune parameters, toxicity, and survival. Results: Toxicity was grade 1 in 52%, 2 in 30%, and 3 in 4%. During IFN-α2a administration, leukocytes, monocytes, granulocytes, B-cell marker CD19, activation markers, CD4+CD25+ regulatory T-cells, and vascular endothelial growth factor (VEGF) dropped significantly, but no difference was observed in T-cell and natural killer (NK)-cell markers, and IL-10. Postoperatively, T-cell and activation markers decreased in both groups, but CD4, CD28, IL-6, IL-10, and HLA-DR alterations were significantly less accentuated in patients who had been treated with IFN-α2a. After a median follow-up of 23 months, survival did not differ between the groups (p = 0.54). Conclusions: Perioperative immunodysfunction can be modulated by preoperative administration of IFN- α2a. IFN-α2a decreased the level of VEGF and CD4+CD25+ regulatory T-cells implicating a potential combination with tyrosine kinase inhibitors and vaccines.

Dipeptidylpeptidase IV (DPIV) and Alanyl-Aminopeptidases (AAPs) as a New Target Complex for Treatment of Autoimmune and Inflammatory Diseases—Proof of Concept in a Mouse Model of Colitis

In summary these results strongly support the idea that AAPs and DPIV represent a promising target complex for the pharmacological therapy of T cell-mediated diseases by preserving and enhancing endogenous immunosuppressive mechanisms. Whereas inhibitors of AAPs appear to preferentially act on CD4+CD25+ regulatory T cells by preserving their immunosuppressive activity via enhanced expression of immunosuppressive cytokines and FOXP3, inhibition of DPIV leads to increased production/release of TGF-β1 and inhibition of cellular proliferation of predominantly activated effector T cells. Thus, specific inhibition of DPIV and AAPs via small molecular compounds provides a new approach for the pharmacological treatment of autoimmune and inflammatory diseases that simultaneously interferes with two major axis of T cell function.

COMPLEX PERIOPERATIVE IMMUNO-DYSFUNCTION IN PATIENTS WITH RENAL CELL CARCINOMA

PURPOSE Patients with renal cell carcinoma have an impaired function of the immune system, which is the basis for different approaches of immunotherapy. We address perioperative changes of several parameters of the immune system in these patients. MATERIALS AND METHODS Parameters of cellular and humoral immunity, including differential blood count, T cell markers CD2, 3, 4 and 8, B cell markers CD19 and 20, monocyte markers CD13 and 14, natural killer cell marker CD16, activation markers CD25, CD26 and HLA-DR, and cytokines interleukin-1 (IL-1) receptor antagonist, IL-2, soluble IL-2 receptor, IL-6, IL-10 and transforming growth factor-beta, were measured in the venous blood of patients who underwent renal surgery extracorporeal shock wave lithotripsy (ESWL, Dornier Medical Systems, Inc., Marietta, Georgia). Patients were grouped and age matched, and 37 underwent tumor nephrectomy, 20 open renal surgery for nonmalignant reasons and 24 ESWL. A group consisting of 39 controls received no treatment. RESULTS Little change was detected in controls and those patients who received ESWL. Patients who underwent open renal surgery had increased leukocyte and granulocyte counts until postoperative day 3 but had low T cell counts. The postoperative decrease in CD25 expressing cells corresponded to an increase in the soluble IL-2-receptor. Cytokines IL-6 and 10, which also have immunosuppressive properties, were markedly increased postoperatively. These changes were more noted (p <0.01) in those patients who underwent tumor nephrectomy than open renal surgery for nonmalignant reasons and remained detectable when paired patients with similar surgical trauma were compared. In tumor nephrectomy cases renal venous IL-6 was higher than peripheral venous levels. CONCLUSIONS Patients with renal cell carcinoma suffer from selective immuno-dysfunction, indicating a rationale for perioperative immunomodulation.