Monitoring of peripheral blood cytotoxic T-cells under fluvastatin treatment in renal transplant recipients.

Flow cytometric T-cell analysis is capable of adding valuable information for balancing immunosuppression in transplant recipients as it can take into account individual effects of immunosuppressive drugs on each patient as well as effects of other drugs which may modify the overall immunosuppression. Studies suggest that HMG-CoA-reductase-inhibitors (statins) reduce the frequency of organ rejection, although the precise mechanism of this effect is unknown. We therefore evaluated the effect of fluvastatin on size and activation of T-cell subpopulations and NK-cell activity in renal transplant recipients. At baseline, the population size of activated (HLA-DR+) T-cells was negatively correlated to serum HDL cholesterol suggesting an increased T-cell activation at low HDL levels. Fluvastatin treatment of a hypercholesterolemic group of patients for two months significantly decreased the LDL cholesterol. A longitudinal analysis revealed a relative increase in non-MHC restricted cytotoxic T-cells (CD3+/CD16+ or CD56+) over time which was significantly attenuated in fluvastatin treated patients but not in normocholesterolemic controls. Moreover, a relative decrease of activated MHC class I-restricted cytotoxic CD8+ T-cells was only observed upon fluvastatin treatment. NK-cell number and activity did not differ between groups. In summary, fluvastatin treatment of hypercholesterolemic renal transplant recipients is associated with a specific modulation of T-cells exerting cytotoxic effector functions.

Optimization of three- and four-color multiparameter DNA analysis in lymphoma specimens.

BACKGROUND: Simultaneous analysis of DNA and immunophenotype of lymphoma cells by flow cytometry allows the calculation of the proliferative activity and aneuploidy in even a small lymphoma population. Unfavorable DNA binding characteristics or spectral features of DNA dyes impair the accuracy of multiparameter DNA analysis and limit their clinical application. We describe here a reliable and reproducible application of both three- and four-color multiparameter DNA analysis. METHODS: After immunostaining of fresh samples of peripheral blood, bone marrow and single cell suspensions of lymph nodes from healthy and lymphoma patients, a methanol fixation for TO-PRO-3 and DRAQ5 staining was tested. RESULTS: The red-excitable TO-PRO-3 on a FACSCalibur is limited to two-color antigen staining including fluorescein-isothiocyanate and phycoerythrin-labeled monoclonal antibodies due to its broad excitation spectrum. Although DRAQ5 is only applicable to flow cytometers equipped with a single argon laser emitting 488-nm light, its emission spectrum can be easily separated from the FITC, PE, and PE/Texas-Red emissions. DRAQ5 showed almost identical stoichiometric DNA binding characteristics as propidium iodide. Coefficient of variation produced by DRAQ5 staining is in the range of 3.5 and is adequate for detecting aneuploid amd near-diploid cells. CONCLUSIONS: These advantageous features of DRAQ5 make it a reliable candidate for multiparameter clinical studies.

The role of plasma phospholipid transfer protein (PLTP) in HDL remodeling in acute-phase patients.

During reverse cholesterol transport plasma phospholipid transfer protein (PLTP) converts high density lipoprotein(3) (HDL(3)) into two new subpopulations, HDL(2)-like particles and pre-beta-HDL. The acute-phase response is accompanied with dramatic changes in lipid metabolism including alterations in HDL concentration, composition, and thereby its function as a substrate for HDL remodeling proteins in circulation. To evaluate how acute-phase HDL (AP-HDL) functions in PLTP-mediated HDL conversion, we collected plasma samples from patients with severe acute-phase response (n=17), and from healthy controls (n=30). Subsequently, total HDL (1.063

The influence of the duration of cardiopulmonary bypass on coagulation, fibrinolysis and platelet function.

BACKGROUND: The duration of cardiopulmonary bypass (CPB) might influence blood coagulation. This appears particularly relevant in the light of new, less invasive techniques that propose smaller incisions at the expense of a possible prolongation of time on CPB. METHODS: The time-dependent effects on coagulation, fibrinolysis and platelet function were investigated in 94 patients scheduled for elective coronary artery bypass grafting. Tests on coagulation, fibrinolysis, and platelet function (flow cytometric assay of expression densities of glycoprotein IIb/IIIa and P-selection were performed the day before surgery and after completion of surgery. RESULTS: A significant correlation was found between the duration of CPB and parameters of increased coagulation, decrease of platelet counts during CPB and platelet function. Longer duration of CPB led to an increased need for transfusion of red blood cells. CONCLUSIONS: The duration of CPB affects thrombin formation as well as platelet count and function, but not the fibrinolytic system. This may prove to be a disadvantage when employing minimally invasive techniques that prolong the duration of CPB.

ApoE-containing high density lipoproteins and phospholipid transfer protein activity increase in patients with a systemic inflammatory response.

High density lipoproteins (HDL) mediate reverse cholesterol transport as well as the clearance of oxidation products or inflammatory mediators, thereby contributing to tissue integrity. The decrease in HDL in inflammation has been attributed to decreased lecithin:cholesterol acyltransferase activity, whereas the role of phospholipid transfer protein (PLTP) and cholesteryl ester transfer protein has not been analyzed in detail. We have studied the activities of HDL-modifying proteins and the heterogeneity of HDL in healthy control subjects and three groups of postsurgery patients: no bacterial infection (group 1), bacterial focus and systemic inflammatory response (group 2), and severe sepsis (group 3). For all patients, a decrease in total HDL could be demonstrated, with a loss of mainly large, apolipoprotein A-I (apoA-I) HDL particles, an almost total loss of apoC-I, and an increase in apoE HDL (200-500 kDa), which did not contain significant amounts of apoA-I, apoA-II, or apoC-I. PLTP activity was increased in patients of groups 2 and 3, paralleled by a redistribution of PLTP into a population of small (120- to 200-kDa) particles, probably representing PLTP homodimers or lipid-complexed PLTP. In summary, the increase in apoE HDL and PLTP activity may improve the delivery of energy substrates and phospholipids to tissues that must maintain cellular membrane homeostasis under conditions of inflammatory stress.

Lipoprotein (a) up-regulates the expression of the plasminogen activator inhibitor 2 in human blood monocytes.

Elevated plasma lipoprotein (a) (Lp[a]) and cardiac events show a modest but significant association in various clinical studies. However, the influence of high Lp(a) on the gene expression in blood monocytes as a major cell involved in atherogenesis is poorly described. To identify genes influenced by elevated serum Lp(a), the gene expression was analyzed on a complementary DNA microarray comparing monocytes from a patient with isolated Lp(a) hyperlipidemia and coronary heart disease with monocytes from a healthy blood donor with low Lp(a). By using this approach, numerous genes were found differentially expressed in patient-versus-control monocytes. Verification of these candidates by Northern blot analysis or semiquantitative polymerase chain reaction in monocytes from additional patients with Lp(a) hyperlipidemia and healthy blood donors with elevated Lp(a) confirmed a significant induction of plasminogen activator inhibitor type 2 (PAI-2) messenger RNA (mRNA) in monocytes from male, but not from female, individuals with high Lp(a), indicating that this observation is gender specific. This led also to increased intracellular and secreted PAI-2 protein in monocytes from male probands with Lp(a) hyperlipidemia. Plasminogen activator inhibitor type 1 (PAI-1) mRNA was found suppressed only in the patients' monocytes and not in healthy probands with high Lp(a) levels. Purified Lp(a) induced PAI-2 mRNA and protein and reduced PAI-1 expression in monocytes isolated from various controls. The finding that PAI-2 is elevated in monocytes from male patients with isolated Lp(a) hyperlipidemia and male healthy probands with high Lp(a) and that purified Lp(a) up-regulates PAI-2 in control monocytes in vitro indicate a direct, but gender-specific, effect of Lp(a) for the induction of PAI-2 expression.

Association of primary biliary cirrhosis with vitamin D receptor BsmI genotype polymorphism in a Hungarian population.

We sought to determine the distribution of vitamin D receptor genotypes defined by the BsmI polymorphism and to investigate their association with bone mineral density in patients with primary biliary cirrhosis. Vitamin D receptor genotype and bone mineral density at the lumbar spine was determined in 31 female Hungarian patients with primary biliary cirrhosis and 51 age-matched healthy female controls. The genotype frequency (BB: 45%, Bb: 32%, bb: 22%) of the patients was significantly different from the control group (P = 0.01) due to an overrepresentation of the BB genotype. There was an apparent trend, not reaching statistical significance, for a lower bone mineral density in both the patient and control groups carrying a B allele. In conclusion, we found a strikingly high frequency of the BB genotype in patients with primary biliary cirrhosis, which raises questions about hormonal influences on the development of primary biliary cirrhosis.

Analysis of prothrombotic effects of two human monoclonal IgG antiphospholipid antibodies of apparently similar specificity.

Two human monoclonal antiphospholipid antibodies (APA) of the IgG type, HL-5B and RR-7F have been generated from a patient with primary antiphospholipid syndrome and recurrent cerebral microemboli (H.L.) and from a patient with SLE without evidence of recurrent thrombosis (R.R.). Both monoclonal APA have similar characteristics in ELISA tests. To further analyse the prothrombotic potential, their effect on human monocytes and platelets, and bovine aortic endothelial cells (BAEC) was investigated. Monocytes were isolated from buffy coats by standard techniques. They were incubated either with the respective monoclonal APA in different concentrations, or a control monoclonal IgG of the same subtype, or plasma of the patients, from whom the antibodies were isolated. Incubation with LPS served as positive control. BAEC were grown to confluence, and then incubated with the appropriate agonists. Procoagulant activity (PCA) was determined by a single stage clotting assay. PCA expression after incubation is given as the ratio of the coagulation times observed with media only divided by that observed with the agonist. A PCA ratio >1 indicates the induction of PCA by the agonist. At 1 microg/ml HL-5B yielded a PCA ratio of 1.63 +/- 0.16 while RR-7F induced no significant rise to 1.06 +/- 0.18. Dose response curves showed that RR-7F can induce PCA at higher concentrations. However, its effect is approx. 1/50 of HL-5B based on equimolar antibody concentration. Further analysis indicates that the majority of the PCA induced by monoclonal APA can be inhibited by a specific tissue factor antibody. Neither monoclonal antibody induced PCA in BAEC. Sera from both patients were able to induce PCA in monocytes. However, the PCA ratio of serum from H.L. was higher (1.78) than that of R.R. (1.44). Neither monoclonal APA had an effect on platelets as determined by flow cytometric analysis of CD62P, CD41, CD42b expression and fibrinogen binding with and without previous activation with 5 microM ADP or 15 microM TRAP-6. Similarly, there were no differences in platelet aggregation to different stimuli including submaximal activation. In summary, these data provide further evidence that induction of tissue factor in monocytes is one of the procoagulant effects of APA. Furthermore, the binding specificity of APA is perhaps not suited to predict the biological effects of the antibodies.

Effects of extracorporeal circulation and heparin on the phenotype of platelet surface antigens following heart surgery.

In this prospective study, the time-dependent effects of extracorporeal circulation and heparin-mediated effects on platelet surface antigens in vitro were investigated using whole blood flow cytometry. Blood samples were drawn prior to and following extracorporeal circulation in 89 patients. The response of surface antigen expression (glycoprotein IIb/IIIa, P-selectin, and glycoprotein Ib) with and without in vitro stimulation was measured. A significant correlation of the duration of extracorporeal circulation with the postoperative response of glycoprotein IIb/IIIa, glycoprotein Ib, and P-selectin to in vitro activation was found. Postoperative P-selectin and glycoprotein Ib expression stimulated with ADP correlated to blood loss. Heparin in vitro significantly reduced glycoprotein Ib expression. Heparin, as well as the duration of extracorporeal circulation, independently correlated to phenotypic changes of platelets following extracorporeal circulation. The significant correlation of these variables to postoperative blood loss demonstrates their relevance to platelet function in vivo.

The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.

Tangier disease (TD) is an autosomal recessive disorder of lipid metabolism. It is characterized by absence of plasma high-density lipoprotein (HDL) and deposition of cholesteryl esters in the reticulo-endothelial system with splenomegaly and enlargement of tonsils and lymph nodes. Although low HDL cholesterol is associated with an increased risk for coronary artery disease, this condition is not consistently found in TD pedigrees. Metabolic studies in TD patients have revealed a rapid catabolism of HDL and its precursors. In contrast to normal mononuclear phagocytes (MNP), MNP from TD individuals degrade internalized HDL in unusual lysosomes, indicating a defect in cellular lipid metabolism. HDL-mediated cholesterol efflux and intracellular lipid trafficking and turnover are abnormal in TD fibroblasts, which have a reduced in vitro growth rate. The TD locus has been mapped to chromosome 9q31. Here we present evidence that TD is caused by mutations in ABC1, encoding a member of the ATP-binding cassette (ABC) transporter family, located on chromosome 9q22-31. We have analysed five kindreds with TD and identified seven different mutations, including three that are expected to impair the function of the gene product. The identification of ABC1 as the TD locus has implications for the understanding of cellular HDL metabolism and reverse cholesterol transport, and its association with premature cardiovascular disease.

Flow cytometric immunophenotyping of mature lymphatic neoplasias using knowledge guided cluster analysis.

Flow cytometry is widely used for the immunological characterization of hematopoietic malignancies. Discrimination of normal and malignant cellular immunophenotypes is the most critical step in data analysis, especially if multi-color analysis is performed on highly heterogenous cell suspensions. We therefore investigated, whether adaptive, simultaneous multiparameter gating allowed automated, operator independent analysis of data obtained from the immunophenotyping of blood or bone marrow samples with regard to the presence of non-Hodgkin lymphoma cells. The identification of physiological and malignant cells was achieved by predefining population boundaries, based on the expectations of the population's location in two-dimensional dot plots. The prospective application of these predefined region boundaries in 52 blood and bone marrow samples enabled identification of lymphoma cells with regard to their presence and immunophenotype, based on the correlation of markers as defined in multiple tubes. Our data confirm that highly standardized data analysis methods can reduce the variability of analysis and support the expert in establishing a rapid classification of the sample.

Predictors of blood loss after coronary artery bypass grafting.

OBJECTIVE: To assess the predictive value of variables possibly associated with blood loss after coronary artery bypass grafting (CABG). DESIGN: A prospective study. SETTING: A university hospital. PARTICIPANTS: Eighty-nine patients scheduled for elective CABG. INTERVENTIONS: Blood samples were drawn before and after surgery. Chest tube drainage was measured hourly until removal of drains. MEASUREMENTS AND MAIN RESULTS: Activation of coagulation and fibrinolysis, routine clotting tests, and expression of platelet surface antigens were analyzed using flow cytometry. A significant correlation was found among blood loss and activated partial thromboplastin time, fibrinogen, prothrombin fragment 1 + 2, D-dimers, platelet count, GPIb and P-selectin expression on platelets, use of internal thoracic artery, cross-clamp time, and thrombin-antithrombin III complex. In a multiple regression model, glycoprotein (GP) Ib expression on platelets, platelet count, use of internal thoracic artery, and D-dimers were significantly associated with blood loss. Logistic regression analysis showed that GPIb and D-dimers predicted an increased blood loss with a positive predictive value of 73% and a negative predictive value of 91%. CONCLUSIONS: Postoperative D-dimers and GPIb expression may be useful to exclude nonsurgical causes in bleeding patients after CABG.

[Effect of processed blood volume, leukocyte count and concentration of CD34-positive cells in peripheral blood on efficiency of stem cell apheresis]. / Einfluss von prozessiertem Blutvolumen, Leukozytenzahlen und Konzentration CD34-positiver Zellen im peripheren Blut auf die Effizienz von Stammzellapheresen.

Despite many published studies no parameter could be identified yet to acceptably and individually predict collection results in stem cell apheresis. We analyzed leukocyte counts and processed blood volume, absolute and relative CD34+ cell counts, and overall collection efficiency in 120 patients with hematological and solid malignancies (354 leukaphereses using the Cobe Spectra cell separator, a median of 3 per patient, span 1-9). Stem cells were mobilized into peripheral blood by conventional chemotherapy followed by daily doses of G-CSF. CD34+ progenitor cell counts were monitored through multiparametric flow cytometry. Blood and collection flows varied in the range of 45-90 ml/min and 0.7-1.5 ml/min, respectively. CD34+ progenitor cells were enriched 38-fold in the apheresis product as compared to peripheral blood at a processed blood volume lower than one total blood volume. Efficiency continuously declined, on to a 25-fold concentration at a processed blood volume above the 3-fold total blood volume. Total collection efficiency, calculated from the absolute content of CD34+ progenitor cells in peripheral blood and apheresis concentrate (a parameter for progenitor cell mobilization during the apheresis), reached a plateau at a processed blood volume above the 3-fold total blood volume. However, variation among individual patients was high. The concentration rate of CD34+ cells at a leukocyte count below 5,000/microliter averaged 50 and declined continuously to 8 at leukocyte counts between 45,000 and 50,000/microliter. To summarize, in 70% of patients with leukocyte counts below 5,000/microliter and CD34+ progenitor cell counts above 10,000/ml, more than 1.5 x 10(6) progenitors per kg body weight could be collected in a single leukapheresis. According to the presented data, the variation in overall collection efficiency is mainly due to: 1) varying mobilization of progenitors during the apheresis procedures itself and 2) dependence on peripheral leukocyte counts.

[Clinical and flow cytometry outcome of improved photopheresis methods in dermatologic patients]. / Klinische und durchflusszytometrische Ergebnisse eines verbesserten Photophereseverfahrens bei dermatologischen Patienten.

In photopheresis, 8-methoxypsoralen (8-MOP) is added to a mononuclear cell concentrate and then activated by UVA light, thus forming covalent bonds between DNA strands. Infusion of these modified cells that are not able to replicate any more seems to lead to the elimination of pathogenic T-cell clones and clinical improvement in patients suffering from cutaneous T-cell lymphoma, graft versus host reactions, and various autoimmune diseases. The original method described by Edelson et al. (1987) was improved by the following modifications proposed by Andreu et al. (1994): i) by using a cell separator (COBE Spectra) that produced purer concentrate with less red blood cells absorbing UVA light. ii) By applying 8-MOP directly into the collection bag, the drug side effects due to the oral application of the thousandfold dose and varying serum levels were avoided. iii) By irradiating the concentrate after collection, all cells received the same irradiation dosage. We treated 2 men with cutaneous T-cell lymphoma, 2 women with atopic eczema and 1 man with severe pustular psoriasis with overall 123 extracorporeal photochemotherapy (ECPC) sessions. In addition to routine laboratory analysis, detailed characterization of lymphocyte subpopulations was carried out by flow cytometry to differentiate T-helper and T-suppressor cells and their activation (HLA-DR, CD 25), B, NK cells, and monocyte subpopulations. The following mediators soluble were analyzed as well: Il-2-receptor and as indicators of acute phase reaction Il-8, neopterin and Il-1 beta. We observed a good clinical improvement independent of no significant trend in the immunological phenotype of circulating blood leukocytes. Our results suggest that ECPC effects are not mediated by a systemic immune response or alternatively are not measured in the blood compartment.

Fluorescence flow cytometry of human leukocytes in the detection of LDL receptor defects in the differential diagnosis of hypercholesterolemia.

A flow-cytometric method with fluorescence-labeled monoclonal antibodies (MABs) against the low density lipoprotein (LDL) receptor (C7A MAB) or 3,3'-dioctadecylindocarbocyanin-iodide (DiI) LDL has been developed that allows the quantification of LDL receptors on leukocytes and the identification of patients with familial hypercholesterolemia (FH) within 48 hours. Leukocytes were isolated from 10 mL anticoagulated blood by density gradient centrifugation. To induce maximal expression of LDL receptors, mononuclear cells were preincubated with either phytohemagglutinine (PHA) or lipoprotein-deficient serum (LPDS). LPDS-treated monocytes provided a more homogeneous cell population with regard to LDL receptor activity than did the PHA-treated lymphocytes; they also provided a greater discrimination between the fluorescence of the receptor probes and cellular autofluorescence. The C7A MAB was able to compete for DiI LDL binding by about 40%. In competition with unlabeled LDL, DiI LDL revealed linear binding, indicating an affinity similar to native LDL. The binding characteristics of DiI LDL were also similar to 125I-LDL binding. LDL isolated from familial defective apolipoprotein B-100 was not able to compete for DiI LDL binding on monocytes, whereas native LDL reduced it by about 80%. In monocytes from FH heterozygous patients, the cellular mean fluorescence using either C7A MAB or DiI LDL at 4 degrees C was 30% to 70%; in FH homozygotes, cellular mean fluorescence was less than 20% of that in monocytes from normal individuals. In patients with familial defective apolipoprotein B-100 antibody binding was normal, but one patient's own LDL failed to compete with normal DiI LDL for 4 degrees C binding on U937 test monocytes. Patient monocytes having internalization defects showed normal 4 degrees C DiI LDL binding, but at 20 degrees C cell-associated fluorescence was reduced by about 40%. In our study 384 hypercholesterolemic patients (preselected according to serum cholesterol levels, clinical symptoms, and family history) were analyzed for LDL receptor expression using the C7A MAB-based assay. In 71.8% of the patients with cholesterol levels higher than 300 mg/dL, an LDL receptor deficiency was observed. Apolipoprotein E isoforms and lipoprotein[a] were found to be independent from the LDL receptor status. In some patients with high cholesterol levels but normal LDL receptor expression with the C7A MAB assay, LDL receptor defects could be diagnosed when either reduced binding or internalization of DiI LDL or familial defective apolipoprotein B-100 was detected.(ABSTRACT TRUNCATED AT 400 WORDS)