Perioperative immunomodulation with interleukin-2 in patients with renal cell carcinoma: results of a controlled phase II trial.

We conducted a non-randomised controlled phase II trial to investigate the role of preoperative administration of interleukin-2 (IL-2) in patients with renal cell carcinoma undergoing tumour nephrectomy. A total of 120 consecutive patients were allocated alternately to the two study groups: perioperative immunomodulation with IL-2 (IL-2 group; n=60) and perioperative immunomonitoring without immunomodulation (control group; n=60). Patients from the IL-2 group received four doses of 10 x 10(6) IU m(-2) twice daily subcutaneously a week before operation followed by a daily maintenance dose of 3 x 10(6) IU m(-2) subcutaneously until a day before the operation. Parameters of cellular and humoral immunity (leucocytes, T-cell markers CD3, CD4, and CD8, B-cell marker CD19, monocyte marker CD14, natural killer (NK) cell markers CD16, CD56, and CD57, activation markers CD6, CD25, CD28, and CD69, progenitor cell marker CD34, as well as IL-2, IL-6, IL-10, soluble IL-2 receptor, IL-1 receptor antagonist, transforming growth factor-beta1, and vascular endothelial growth factor) were measured in peripheral venous blood at various intervals. Interleukin-2-related toxicity was WHO grade 1 (24%), 2 (67%), and 3 (9%). In the postoperative period, T-cell markers, activation markers, and NK cell markers decreased, and IL-6 and IL-10 increased. However, all these alterations were significantly less accentuated in patients who had been pretreated with IL-2. Median follow-up was 40 months. Tumour-specific survival in the IL-2 group and the control group was 98 vs 81% after 1 year and 86 vs 73% after 5 years (P=0.04). A similar effect was found for progression-free survival. We conclude that IL-2 can be safely administered in the perioperative period and modulates immunological parameters. However, to validate the survival data, a larger randomised phase III trial is needed.

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 microg mL(-1); P < 0.05). Propofol reduced sIL-2R release at the high concentration of 10 microg mL(-1) (2220 pg mL(-1) 1780 microg 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.

Expression of cell surface antigens in diabetic patients and healthy controls after injury.

AIMS: Due to the systemic character of Type 2 diabetes, cellular disturbances paralleled by an altered expression of various growth factors constitute the basis for impaired wound healing. Cell-surface antigens are altered in chronic wounds and may also have an effect on the persistance of diabetic foot lesions. METHODS: We investigated blood samples of diabetic patients with diabetic foot ulcers (n=21) in comparison with those from healthy control patients subsequent to an injury (n=9). A blood sample (EDTA) was taken from each participant (in the trauma control group on the third day after injury) and examined by flow cytometry [fluorescence-activated cell sorter (FACS)]. Typical cell surface antigens involved in wound healing were studied [cluster of differentiation (CD)2, CD3, CD4, CD25 and human leukocyte antigen (HLA)-diabetic retinopathy (DR)]. RESULTS: known to adversely affect wound healing were elevated in diabetic patients (CD2 p<0.001; CD3 p=0.016, CD4 p=0.22, CD25 p<0.001). HLA-DR expression was also decreased in diabetic foot patients (p=0.023). CONCLUSIONS: Cell-surface antigens appear to be altered in diabetic patients when compared to healthy controls. Thus, due to the systemic character of Type 2 diabetes, cellular disturbances may well constitute the basis for impaired wound healing in diabetes.

Pretreatment with interleukin-2 modulates perioperative immunodysfunction in patients with renal cell carcinoma.

Complex perioperative immunodysfunction occurs in patients with renal cell carcinoma undergoing nephrectomy. Here, the effect of pretreatment with IL-2 is addressed. Of 63 patients who underwent tumour nephrectomy, 26 patients received four doses of 10 Mio IE/m2 IL-2 b.d. s.c. (i.e. a total of 40 Mio IE/m2) 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-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 peripheral venous blood. Blood was drawn before IL-2, one day before and immediately after the operation, and on the 1st, 3rd, 5th, and 10th postoperative day. 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 grade I-II in all patients, grade III in one patient. The anaesthetic regimen had no measurable effect. IL-6 concentrations were higher in renal venous than in venous pool blood, indicating IL-6 production in the tumour in vivo. Tumour-specific survival was better in pretreated patients with tumours extending beyond the kidney. Pretreatment with IL-2 modulates perioperative immunodysfunction in patients undergoing tumour nephrectomy. This affects in particular T-cell-mediated immunity and levels of cytokines IL-10 and IL-6. The IL-2 application scheme used here was followed by distinct counter regulation including monocytes, IL-2, sIL-2R, IL-1RA and TGFbeta. Taken together, pretreatment with IL-2 may complement surgery in the treatment of patients with renal cell carcinoma, and may help close the therapeutic gap between neo-adjuvant and adjuvant immunotherapy.

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.

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.

MRP8/MRP14, CD11b and HLA-DR expression of alveolar macrophages in pneumonia.

Activation of alveolar macrophages is characterised by specific alterations to the expression pattern of surface markers under certain pathological conditions. MRP8/MRP14 and CD11b are involved in the regulation of macrophage migration and adhesion. HLA-DR regulates the antigen presentation by alveolar macrophages. The aim of this study was to investigate the phenotype of alveolar macrophages in pneumonia particularly in relationship to the changes in concentrations of TGF-beta1 and IL-8. Using cytofluorimetry, we analysed the surface expression of MRP8/MRP14, CD11b, and HLA-DR on alveolar macrophages of 42 pneumonia (PN) patients, 14 patients with interstitial lung diseases (ILD), five patients with chronic obstructive lung disease (COPD), and 58 patients without lung disease. Phenotypic characteristics were correlated to the concentration of TGF-beta1 and IL-8 in the bronchoalveolar lavage fluid (BALF) of the same patients. The direct influence of TGF-beta1 and IL-8 on expression of MRP8/MRP14, CD11b and HLA-DR of cultured monocytes and MonoMac cells was analysed. Significantly more MRP8/MRP14 and CD11b positive macrophages and less HLA-DR-positive macrophages were found in PN but not in ILD or COPD. The percentage of CD11b-positive macrophages correlated with the TGF-beta1 as well as the IL-8 concentrations. The amount of HLA-DR-positive macrophages correlated negatively to the concentration of TGF-beta1 and IL-8. These findings document a significant activation of alveolar macrophages during pneumonia. TGF-beta1 led to a modulation of HLA-DR and MRP8/MRP14-antigen expression in vitro. In conclusion, it was shown that in pneumonia but not in ILD or COPD alveolar macrophages were characterised by an increased MRP8/MRP14 and CD11b expression and a diminished HLA-DR expression. The characterisation of subpopulations within the alveolar macrophages may be a useful tool for the monitoring of disease progression.

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: 13,783 +/- 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.

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

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.

The main neutral aminopeptidase activity of human lymphoid tumour cell lines does not originate from the aminopeptidase N-(APN; CD13) gene.

Lymphocytes and related cell lines are predominantly CD13-negative, however, there are reports describing neutral aminopeptidase activity in or on these cells. The aim of this study was to answer the question, whether this activity originates from APN-gene expression. The total cellular activities (Ala-pNA hydrolysis) of lymphoid cell lines are up to 15 times higher than that of normal lymphocytes. Despite weak or lacking CD13 surface expression all lymphoid cell lines tested contain APNmRNA as quantified by competitive RT-PCR as well as low enzymatic activity in their particulate fractions. By isoelectric focusing two enzyme species with isoelectric points of 5.4 or between 3.5 to 4.8, respectively, were detected. To investigate whether these activities result from APN-gene we established transfectants lacking cellular APN expression of the CD13-positive histiocytic cell line U937 and the CD13-negative T-cell line H9. Studies on these transfectants proved (I) that the main neutral aminopeptidase activity expressed in lymphoid cells is definitively not related to APN and (II) that APN is also expressed in lymphoid cells, although on a low level only.

gamma-Glutamyl transpeptidase-cellular expression in populations of normal human mononuclear cells and patients suffering from leukemias.

The expression of the ectoenzyme gamma-glutamyl transpeptidase (EC2.3.2.2., gamma GT) was investigated by flow cytometry on populations of peripheral blood mononuclear cells (PBMC) from healthy subjects and patients suffering from several types of leukemia before and under chemotherapy. In unstimulated PBMC, 28% of these cells were found to be gamma GT positive. The highest expression was measured on monocytes (CD14/gamma GT+ cells: 60%). Within the subsets of T lymphocytes (CD3/gamma GT+ cells: 18%) we saw no clear differences between CD4+ and CD8+ cells. B lymphocytes, NK cells, and activated cells showed low expressions (up to 10%). Treatment of PBMC with mitogens, alpha-IFN, IL-2, and GM-CSF did not affect the enzyme expression on normal mononuclear cells (MNC). However, a rapid increase of gamma GT+ cells was found in the presence of glutathione (GSH) and n-acetyl cysteine (nAC), particularly on monocytes, B cells, and NK cells. Comparing 40 healthy subjects and untreated patients suffering from leukemias, a significantly higher expression of gamma GT+ cells in the total MNC populations (B-CLL: 57%, CML: 62% gamma GT+ cells) was observed in B-chronic lymphocytic leukemia (B-CLL) and chronic myelogenous leukemia (CML), whereas other leukemias did not show clear differences. Most interestingly, the gamma GT expression was diminished in all populations of CML cells after 5 h of incubation in the presence of 10 units/ml IFN-alpha. These data suggest a possible protective role of gamma GT in MNC and a regulatory function of this enzyme in the development of CML.

Thiol-proteindisulfide-oxidoreductase (proteindisulfide isomerase): a new plasma membrane constituent of mature human B lymphocytes.

The thiol-proteindisulfide-oxidoreductase (TPO, EC 1.8.4.2., proteindisulfide isomerase, EC 5.3.4.1.) is known as an cytoplasmatic enzyme, and is thought to be involved in the post-translational folding of disulfide containing proteins. Using monoclonal and polyclonal antibodies the authors were able to prove that this enzyme or an unknown homologous protein is localized also to the plasma membrane of B lymphocytes. In peripheral blood from healthy donors 11% of the mononuclear cells (PBMNC) expressed this surface antigen whereas in PBMNC of patients with B-cell chronic lymphocytic leukaemia 76% of the MNC were positive. This value correlates well with the known B-cell markers CD19 and CD20. However, this antigen is different from all known clustered B-cell markers. Immunoprecipitation analysis of PHA-stimulated PBMNC and of cells from patients suffering from chronic lymphocytic leukaemia revealed a membrane protein with the same molecular weight (61 kDa) as the TPO. These data suggest that this enzyme is present not only in the cytoplasm but also on the surface of B cells and that it is possibly involved in the regulation of the SH-SS status of the cell membrane proteins of B lymphocytes.

[Biofreezer BF 80--a universally employed apparatus in preservation at very low temperatures]. / Biofreezer BF 80--eine universell einsetzbare Tieftemperaturkonservierungsapparatur.

A computer-controlled freezing apparatus is described which uses liquid nitrogen (LN2) as coolant. The freezing equipment BF 80 consists of a big cryochamber (volume 24 l), a LN2 tank and a computer for the control of the freezing procedure. The biofreezer functions in the range from 40 to -180 degrees C. A complete cooling curve can be composed of up 15 linear segments with freezing rates between 0 and 45 K.min-1. In the computer up to 80 different freezing curves can be stored and activated by simple operations. The reliability of the biofreezer BF 80 was successfully tested for the cryopreservation of lymphocytes.

[Determination of cellular and humoral parameters of fatty liver]. / Untersuchungen von zellulären und humoralen Parametern bei Patienten mit Fettleber.

Cellular and humoral immunological parameters were examined in mononuclear cells from peripheral blood of patients with alcoholic and nonalcoholic steatosis hepatis. The ratio of T4 and T8 positive lymphocytes and the number of monocytes of these patients were in the normal range. The percentage of NK-cells, B-lymphocytes and DR-antigen-positive cells was increased.

[Fc-IgG and Fc-IgM receptor-carrying lymphocytes in human blood. Optimization of determining T(M) lymphocytes]. / Fc-IgG- und Fc-IgM-Rezeptor tragende Lymphozyten im menschlichen Blut. Optimierung der Bestimmung der TM-Lymphozyten.

Several methods for enumeration of Fc receptor bearing T lymphocytes and mononuclear cells from human peripheral blood were compared. The detection of Fc receptors is based on the formation of EA rosettes by using bovine erythrocytes and purified rabbit IgG or IgM antibodies. As alternative method the mixed rosette assay (EA rosettes plus sheep erythrocyte rosettes) (3) was applied for determining TG lymphocytes without the need of T cell separation. Independent of the method used for T cell separation (preparative rosetting with sheep erythrocytes stabilized by AET or HSA) the number of TG and TM lymphocytes was found to be identical. TG values obtained by use of the mixed rosette assay were significant lower (10 +/- 2%) than those obtained with the classical test (18 +/- 5%) (EA rosettes after T cell separation). Obviously this difference is due to a contamination of T lymphocyte preparations by non-T cells. On freshly isolated T lymphocytes without overnight culture we obtained 29% and 35%, respectively TM lymphocytes after separation of T cells using sheep erythrocyte rosettes stabilized with AET or HSA. The expression of FcIgM receptors was found to be strongly dependent on the composition and pH value of the culture medium. In the presence of human AB serum the maximum of FcIgM receptor expression on isolated T cells was obtained at pH 8.5. Under optimum conditions we found 63% and 66% respectively TM lymphocytes after T cell separation using AET or HSA stabilized sheep erythrocytes.

[Human T-lymphocytes--clinical and methodological studies on the detection of Fc-IgM- and Fc-IgG-receptor bearing T-lymphocytes (Tm and Tg)]. / Humane T-Lymphozyten--Klinische und methodische Studien zum Nachweis von Fc-IgM- und Fc-IgG-Rezeptor-tragenden T-Lymphozyten (TM und TG).

Several methodical aspects for determination of T lymphocytes with Fc receptors for IgM (TM) and IgG (TG) were studied including separation technique of T cells with E-rosetting, culture conditions of T cells for determination of TM and the rosetting of TM and TG with EA complexes. The bests results were obtained by stabilization of E-rosettes with human serum albumin, after separation of E-rosette forming cells lysis of sheep erythrocytes with save hypotonic shock, culturing of T cells in medium containing 20% AB Serum. Furthermore it was shown the possibility using EA complexes produced with not purifieded IgG or IgM anti-ox-red-blood cells antisera without lost of specifity for TM and TG. The percentage of TM and TG in peripheral blood of thirty healthy persons as well as monitoring TM and TG in three cases was investigated.

Dipeptidyl peptidase IV and alpha-naphthylacetate esterase in human T lymphocytes: cytochemical and biochemical investigations.

Dipeptidyl peptidase IV (DP IV) activity of human lymphocytes was measured using biochemical assays of cell suspensions and enzyme cytochemical staining of smears from capillary blood and mononuclear cells (MNC). The hydrolysis rate of Gly-Pro-pNA in suspensions of MNC correlates well with the number of DP IV reactive cells as determined by cytochemical staining. MNC from healthy volunteers were shown to contain 44 +/- 10% lymphocytes reactive for DP IV. Using preparations of T lymphocytes and adherent MNC it was shown that DP IV is specific for T lymphocytes. About 60% of T lymphocytes contain DP IV and 94% of DP IV reactive cells form rosettes with sheep erythrocytes. Parallel staining for DP IV and unspecific acid alpha-naphthylacetate esterase (ANAE) yielded nearly the same figure of cells stained for ANAE in a dot-like pattern (50 +/- 10% MNC) as was observed for DP IV. Correlation of both markers indicates that DP IV expressing lymphocytes presumably represent that subpopulation of TM cells which is characterized by a dot-like reaction pattern of ANAE.