Late cardiac effect of anthracycline therapy in physically active breast cancer survivors - a prospective study.

The late-onset cardiotoxic effect of anthracycline is known, however the early detection and prevention of subclinical myocardial damage has not been fully understood yet. Besides medical therapy regular physical activities may also play a role in the prevention and reduction of side effects of chemotherapy. The aim of our present study was to detect the effect of regular physical activities on the diastolic function and on the symptoms of late heart failure in case of anthracycline chemotherapy. The prospective study included 55 female patients (age 31-65 year, average 49.5 years) with breast cancer and no cardiovascular risk factors. Proper cardiologic checkup included physical examination (blood pressure, pulse, etc.), ECG, standard echocardiography parameters (EF, LV dimensions etc.) and specific tissue Doppler (TDI) measurements. Symptoms of heart failure were also recorded. After five years of follow-up, symptoms of heart failure were evaluated again. Patients were assigned into two groups depending on their physical activity: 36 patients did perform regular physical activities (mean age 49.2 years) and 19 patients did not (average age 50.1 years). There was no significant difference between the two groups in basic physiological or standard echocardiography parameters neither at the baseline nor at the later time points. Diastolic dysfunction (decreased E/A) was detected 6 months after the beginning of the treatment (T2 time point) in both groups. In the inactive group this value fell below one however there was no significant difference (1.1±0.25 vs. 0.95±0.22). One year after the beginning of the treatment (T3) a significant difference could be detected between the two groups (1.05±0.28 vs. 0.86±0.25. P=0.038). Consistent change in diastolic function (Ea/Aa) could be detected with the more sensitive TDI (Tissue Doppler Imaging) measurements after treatments in both groups, especially in the septal segment (in the non active group the Ea/Aa decreased markedly but not significantly at T2 - 1.1±0.55 vs. 0.81±0.44, and this difference became significant at T3 and 2 years after treatment (T4), p=0.007 and p=0.065). The filling pressure (E/Ea) rose above 10 (p=0.09) in the non active group at T2; and it kept rising in both groups and became significant at T3 (p=0.012). Five years after the onset of the treatment symptoms of heart failure were less frequently reported in the physically active group than in the inactive one (19.45% vs. 68.42%). The data of our study show that the diastolic dysfunction of the left ventricle related to the anthracycline therapy became evident in the physically active group later and the symptoms of heart failure were less frequent than in the non active group after five years period. Enrollment in sport activities could be a good means for partial prevention in this group of patients. Cardiologic checkup at proper intervals plays a pivotal role in detection of possible cardiotoxicity. This is a strong indication for changes in the lifestyle of the patient and the treatment protocol alike.

Developing and validating a practical decision support tool (DST) for biomass selection on marginal land.

Marginal, often contaminated, sites exist in large areas across the world as a result of historic activities such as industry, transportation and mineral extraction. Remediation, or other improvements, of these sites is typically only considered for sites with high exploitation pressure and those posing the highest risks to human health or the environment. At the same time there is increasing competition for land resources for different needs such as biofuel production. Potentially some of this land requirement could be met by production of biomass on brownfield or other marginal land, thereby improving the land while applying the crop cultivation as part of an integrated management strategy. The design and decision making for such a strategy will be site specific. A decision support framework, the Rejuvenate DST (decision support tool) has been developed with the aim of supporting such site specific decision making. This tool is presented here, and has been tested by applying it to a number of case study sites. The consequent SWOT (strength, weakness, opportunities and threats) analysis is discussed and evaluated. The DST was found to be systematic, transparent, and applicable for diverse sites in France, Romania and Sweden, in addition to the sites to which it was applied through its development. The DST is regarded as especially useful if applied as a checklist in an iterative way throughout the decision process, from identifying potential crops to identifying knowledge gaps, working/non-working management strategies and potential risks. The DST also provides a structure promoting effective stakeholder engagement.

Isolation and characterization of syncytiotrophoblast plasma membrane from human placenta.

Human full-term syncytiotrophoblast plasma membranes isolated by mechanical procedures (sieving and ultrasonic disintegration), purified by phase centrifugation, form a single band of 1.052 +/- 0.002 g/ml density in percoll gradient. The purity of the preparation was assessed by electron microscopy, enzyme analysis and beta 2-microglobulin determination.

Complete saturation of protamine sulphate by dsDNA is necessary in order to obtain a highly sensitive and specific anti-dsDNA ELISA.

A protamine sulphate (PS) pretreated solid phase coated with different amounts of dsDNA has been used to develop a sensitive, specific and reproducible anti-dsDNA ELISA. Using low concentrations of a dsDNA coat 50% of SLE sera were found to be positive and false positive reactivity due to anti-PS reactivity was found in 3/40 patients with other auto-immune diseases (OAID). In contrast, when PS was saturated with higher concentrations of dsDNA 80% of SLE sera were detected, the reproducibility of the results was better and anti-PS reactivity of OAID patients with an anti-PS reactivity disappeared. The sera of three other OAID patients contained low avidity anti-dsDNA, measured after a salt elution step in the ELISA procedure, and 2/60 patients with non-auto-immune disease exhibited a false positive anti-dsDNA reactivity since they reacted with the solid phase even in the absence of PS and dsDNA. Thus an ELISA procedure using a PS pretreated solid phase permits the sensitive, specific and reproducible measurement of anti-dsDNA antibodies only if a high concentration of dsDNA is coated on the PS and appropriate controls are performed.

ELISA for the detection of anticardiolipin antibodies. High specificity based on the use of adult bovine serum as buffer and systematic subtraction of non-specific binding.

We have used an ELISA procedure to compare the reactivity of samples with or without IgG anticardiolipin antibodies (ACA) on cardiolipin-coated wells (target wells) and cardiolipin-free wells (control wells), using 10% fetal calf serum (10% FCS), 10% adult bovine serum (10% ABS) or 1% bovine serum albumin (1% BSA) as buffer. With 1% BSA, ACA reactivity was very low which suggests that this buffer would be inappropriate for use in ELISA procedures for ACA. 10% FCS induced non-specific binding of normal IgG only on target wells, particularly in the case of IgG hypergammaglobulinemia. With 10% ABS, this non-specific binding occurred on the solid phase with and without cardiolipin and could be accurately evaluated by subtracting the absorbance of control wells. Results of assays on 35 systemic lupus erythematosus sera using 10% ABS as buffer showed that highly specific results-could be obtained only if non-specific binding was systematically subtracted.

Docosahexaenoic and eicosapentaenoic acids inhibit in vitro human lymphocyte-endothelial cell adhesion.

Dietary supplementation with fish oil, which contains high amounts of long chain omega 3 ((n-3)) polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), has recently been shown to have protective and ameliorative effects on diseases characterized by chronic inflammatory reactions. Interactions between vascular endothelium, mononuclear cells, and cytokines are crucial steps in the course of inflammatory processes such as chronic graft rejection. We therefore studied the effects of DHA and EPA on both the adhesion of peripheral blood lymphocytes (PBL) to human endothelial cells (EC) in culture and the expression of EC-adhesion molecules and their counterreceptors on PBL. The addition of DHA or EPA to the adhesion assay significantly decreased the adhesion of PBL to untreated EC and tumor necrosis factor-alpha (TNF alpha)-, interleukin (IL) 4-, and lipopolysaccharide-stimulated EC. When EC were pretreated with (n-3) PUFAs for 18 hr, washed, and then stimulated by TNF alpha, IL-4, or lipopolysaccharide, PBL adhesion was also significantly reduced compared with controls. We also showed that PBL preincubated with DHA or EPA, and then washed and chromium radiolabeled, still exhibited an adhesion inhibition to TNF alpha- and IL-4-treated EC as well as untreated EC. Cytofluorometry and immunoenzymatic analyses indicated that pretreatment of EC with (n-3) PUFAs before their activation significantly reduced the EC-induced expression of vascular cell adhesion molecule 1, whereas the level of expression of intercellular adhesion molecule 1 and E-selectin was not modified. Furthermore, we showed that incubation of PBL with DHA or EPA moderately reduced the level of cell surface expression of L-selectin and leukocyte function-associated antigen 1, but not of very late antigen 4. In all cases, the inhibitory effect of (n-3) PUFAs was specific and dose dependent. In addition, DHA seems to be a more potent inhibitor than EPA, but the two compounds in association had an additive effect. Regardless of the mode of action, this inhibitory effect may explain the protective and ameliorative effects of (n-3) PUFAs on diseases involving chronic inflammatory reaction.

Evidence of noninvolvement of swine MHC class II in the in vitro proliferative response of human lymphocytes to porcine endothelial cells.

Successful pig-to-human xenotransplantation may expose swine endothelium to the human immune system. Since endothelial MHC class II expression is crucial in the genesis of an allogeneic lymphocyte response, the involvement of porcine MHC (SLA) class II molecules in the induction of human lymphocyte proliferation was studied. When cocultured with a confluent monolayer of irradiated porcine aortic endothelial cells (PAEC), human peripheral blood mononuclear cells (PBMC) incorporated tritiated thymidine. Monocyte depletion strongly reduced the magnitude of the lymphocyte proliferative response. Resting cultured PAEC were SLA class II-negative and an induction of these molecules during the xenogeneic mixed lymphocyte endothelial cell culture (XMLEC) was not observed. Moreover, the addition of an antibody directed against the SLA-DR molecule was without effect. Lymphocyte proliferation was also studied in response to SLA class II-positive stimulating cells--either human TNF-alpha-stimulated PAEC or porcine splenocytes. Induction of SLA class II molecules on PAEC had no effect on the human PBMC proliferative response. Moreover, human PBMC did not proliferate in response to porcine splenocytes. These results suggest (1) that SLA class II molecules are not involved in the induction of the human lymphocyte proliferative response and (2) that the endothelial nature of the stimulating cells plays a key role in this proliferation.

Removal of terminal alpha-galactosyl residues from xenogeneic porcine endothelial cells. Decrease in complement-mediated cytotoxicity but persistence of IgG1-mediated antibody-dependent cell-mediated cytotoxicity.

To determine the role of the terminal alpha-galactosyl residue in the endothelial damage mediated by human xenoreactive natural antibodies (IgM and IgG), we treated porcine endothelial cells in culture with green coffee bean alpha-galactosidase. A practically complete removal of terminal alpha-Gal residues (as evaluated by flow cytometry with Bandeiraea simplicifolia isolectin B4) and concomitant exposure of N-acetyllactosamine were obtained without altering cell viability. A dramatic decrease in IgM and IgG binding (from a pool of human sera) was observed, confirming the key role of the alpha-galactosyl residues. The enzyme treatment did not induce any nonspecific immunoglobulin binding sites, but led to the exposure of new epitopes for a minor fraction of IgM. The main residual IgM and IgG binding could be due to xenoantigens other than the alpha-galactosyl residues. When alpha-galactosidase-treated endothelial cells were used as targets in cytotoxicity experiments, they were less susceptible than untreated cells to complement-mediated cytotoxicity induced by fresh human serum. In contrast, they did not acquire resistance to human IgG-dependent cellular cytotoxicity, despite the decrease in IgG binding. Because it is known that antibody-dependent cytotoxicity mediated by CD16+ NK cells is dependent on IgG1 and IgG3, and not on IgG2 or IgG4, which was confirmed by blocking experiments, we studied the binding of all four subclasses to intact and alpha-galactosidase-treated endothelial cells. Two major subclasses, IgG1 and IgG2, bound to untreated endothelial cells, whereas IgG3 binding was low and IgG4 binding was negligible. A decrease in IgG1, IgG2, and IgG3 binding was observed upon alpha-galactosidase treatment, indicating that antibodies belonging to these three subclasses recognize alpha-galactosyl residues. The decrease in IgG2 binding was more pronounced than the decrease in IgG1 binding. Collectively, these data indicate that IgG1 xenoreactive natural antibodies, including those which are not directed at the alpha-galactosyl residues, could play a major role in the early delayed vascular rejection of pig xenografts.

Tissue factor activity of syncytiotrophoblast plasma membranes and tumoral trophoblast cells in culture.

During pregnancy, important modifications of hemostasis occur resulting in mothers in hypercoagulability and the role of placental cells such as trophoblast cells has been hypothesized. In this study, we first showed that syncytiotrophoblast plasma membranes, isolated from normal human placenta, expressed a strong tissue factor (TF) activity. We then studied TF activity of two continuous trophoblast cell lines (JEG-3 and BeWo) in comparison to human umbilical vein endothelial cells (HUVEC) and transformed human endothelial cells (ECV-304). TF assays were performed on intact detached confluent cells. Unstimulated JEG-3 and BeWo cells exhibited a very high TF activity which slightly increased after 2 to 4 h TNF-alpha stimulation. In contrast, HUVEC and ECV-304 had a lower basal TF activity which was mainly inducible by TNF-alpha, with a maximum effect after 4 to 6 h stimulation. For both cell types, TF activity was decreased to basal value after 16-hour TNF-alpha stimulation. These results support that trophoblast cells are able to express TF but the involvement of this property in the hemostatic physiological changes observed during pregnancy, remains to be demonstrated.

Lipid and protein components of the syncytiotrophoblast plasma membrane inhibit lymphocyte proliferation by two distinct pathways.

The ability of syncytiotrophoblast plasma membrane lipid and protein fractions (STPM lipids, STPM proteins), tested under a reconstituted form, to inhibit lymphocyte proliferation induced by PHA was investigated. The cytostatic activity of STPM proteins appeared greater than that of the STPM lipids. Furthermore, IL-2 production and IL-2 receptor expression by activated lymphocytes were markedly decreased in the presence of STPM proteins compared to the native membrane but remained unaffected in the presence of STPM lipids. Finally, the inhibition of lymphoproliferation could be maintained after removal of the protein fraction from lymphocytes prior to stimulation by PHA. The biological and immunological significance of these results is discussed.

Correlation of plasma hormone levels and peripheral circulating lymphocyte subpopulations during human pregnancy.

Using monoclonal antibodies (OKT3, OKT4, OKT8) peripheral blood lymphocyte subsets were determined in 40 normal primiparous pregnant women and compared with those of 31 nonpregnant controls. In pregnant women plasma concentrations of estradiol, progesterone, human placental lactogen (HPL), beta subunit of human chorionic gonadotropin (beta HCG), and alpha-fetoprotein were measured by means of radioimmunoassay. We studied if correlations between peripheral lymphocyte subsets and plasma hormone levels might exist. We observed in pregnant women from 10 to 40 wk of amenorrhea a decrease in the percentage of OKT3 and OKT8 cells, and during the course of pregnancy an increase in the percentage of OKT4 cells. This increase inversely correlated with plasma beta HCG levels and directly correlated with plasma HPL levels.

Two-dimensional echocardiographic diagnosis of papillary muscle rupture.

A 69-year-old man developed a rupture of the papillary muscle on the fifth day of acute posterior myocardial infarction. The two-dimensional echocardiographic features of ruptured papillary muscle included (1) mobile mass of echos attached to normal chordae tendineae in the left ventricle, (2) absent tip of papillary muscle, and (3) mitral valve prolapse. Noninvasive, two-dimensional echocardiography can reveal correct diagnosis of ruptured papillary muscle suspected clinically.

Syncytiotrophoblast plasma membrane inhibits membrane expression of activation markers on PHA-stimulated human lymphocytes.

Studies have been carried out on the effects of full-term human syncytiotrophoblast plasma membrane (STPM) preparations on the membrane expression of the lymphocyte activation markers HLA-DR, IL-2R, TfR and CD69 during PHA-induced lymphoproliferation. STPM considerably decreases the expression of both late (HLA-DR) and early (IL-2R, TfR, CD69) activation markers at doses which inhibit PHA-induced lymphoproliferation. These results favour the hypothesis that STPM inhibits a very early phase of PHA-induced lymphocyte activation.

Germ cell-Sertoli cell interactions and production of testosterone by purified Leydig cells from mature rat.

The addition of seminiferous tubule (ST) culture medium (STM) prepared from testes of either busulfan-treated (Bus) or cryptorchid (Cryp) or genetically sterile (hd) rats, to Percoll purified Leydig cells leads to a further increase of LH-stimulated testosterone (T) output (26, 43 and 14%, respectively). Taking into account that the Sertoli cell number per cm of ST is 2.6, 1.8 and 1.4-fold greater in Bus, Cryp and hd rats than in controls, the above STM effects on T output, expressed per 10(6) Sertoli cells are in fact lower (63, 44 and 43%, respectively) that those of control STM. Similar results have been obtained for the STM transferrin levels which are decreased, 74, 67 and 45%, respectively in Bus, Cryp and hd animals. So, it is likely that the Sertoli cell secretion of both the paracrine factor involved on Leydig cell T production and the transferrin is influenced mainly by spermatids and to a lesser extent by spermatocytes of mature rat testis.

Human lymphocyte adhesion to xenogeneic porcine endothelial cells: modulation by human TNF-alpha and involvement of VLA-4 and LFA-1.

Considering that in the allogeneic situation the adhesion of recipient lymphocytes to donor endothelial cells initiates the cellular rejection, we questioned the possible occurrence of a similar process in the xenogeneic situation. The adhesion of human peripheral blood lymphocytes (PBL) to porcine aortic endothelial cells (PAEC) was thus studied in an in vitro porcine-to-human xenogeneic model. It was found that 25.9% of human PBL adhered to resting PAEC. Furthermore, this adhesion increased significantly when the PAEC were stimulated by the human cytokine TNF-alpha (tumor necrosis factor-alpha). The effect of human TNF-alpha was concentration- and time-dependent and was maximal (from 25.9% to 35.6%) with 100 U/ml during 6 h. Moreover, blocking experiments with monoclonal antibody (mAb) demonstrated the role of the PBL adhesion molecules LFA-1 and especially VLA-4. Indeed, an anti-CD11a mAb decreased PBL adhesion to resting PAEC by 17.1% and to TNF-alpha stimulated PAEC by 16.9%, whereas an anti-CD49d mAb decreased dramatically PBL adhesion to resting PAEC by 53.1% and to TNF-alpha stimulated PAEC by 41.0%. Finally, phenotypic analysis of the adherent PBL showed that 50.5% of adherent cells to resting PAEC were NK (natural killer) cells, whereas 50.7% of adherent cells to TNF-alpha stimulated PAEC were T lymphocytes, showing the preferential adhesion of NK cells to resting PAEC, and that the stimulation of the PAEC with human TNF-alpha affects predominantly T lymphocyte adhesion. These results indicate that human PBL could bind to xenogeneic PAEC and that this interaction could be a first step of a xenogeneic cellular rejection.

Human NK cell-mediated direct and IgG-dependent cytotoxicity against xenogeneic porcine endothelial cells.

Once hyperacute rejection has been prevented, the pig-to-human xenograft might be exposed to vascular cell-mediated rejection directed against vascular structures. In order to evaluate the relative importance of direct and antibody-dependent anti-endothelial cell-mediated cytotoxicity in different individuals, freshly isolated human blood leukocytes were incubated with confluent porcine aortic endothelial cells (PAEC) in a 4 h Cr-release cytotoxicity assay. Peripheral blood mononuclear cells (PBMC) and lymphocytes (PBL) of all subjects tested (but not monocytes or neutrophils) directly killed PAEC, with wide interindividual variations (from 2.8% to 32%). The addition of heat-inactivated autologous serum to PBMC and PBL (but not to myeloid cells) always enhanced cytotoxicity. This antibody-dependent cell-mediated cytotoxicity (ADCC) was also observed in the presence of adult pooled serum and cord blood pooled serum and was eliminated after adsorption of adult pooled serum to insoluble protein A, demonstrating that IgG is the only class of immunoglobulin involved in this phenomenon. Moreover, blocking Fc gamma RIII with an anti-CD16 mAb eliminated ADCC without affecting direct cytotoxicity. When the ADCC exerted by the PBL of all subjects was assessed with the same preparation of purified IgG, wide interindividual variations were again observed. Surprisingly, there was no correlation between direct cytotoxicity and ADCC although, as depletion experiments demonstrated, both were due to CD16+ natural killer (NK) cells. These results argue that CD16+ NK cells could play an important role in early vascular rejection of porcine discordant xenografts, by both a direct and an IgG xenoreactive natural antibody-dependent cell-mediated cytotoxicity.

Development of an ex vivo model of pig kidney perfused with human lymphocytes. Analysis of xenogeneic cellular reactions.

BACKGROUND: Because of the explosive nature and the extremely rapid process of hyperacute rejection (HAR), significant infiltration of the xenograft by immunocompetent cells is not observed, and the role and the mechanism of action of cell-mediated rejection in discordant xenografts are therefore still under discussion. METHOD: We developed an experimental approach using pig kidneys perfused with human peripheral blood lymphocytes (PBL) in which the immunologic barrier of hyperacute rejection was excluded and which mimics the in vivo situation. RESULTS: PBL retention in the kidney was evaluated at 20-minute intervals for 3 hours. Retention increased from 30% to 80% with the time of perfusion and was specific because significantly fewer syngeneic lymphocytes were retained. Phenotype analysis of recovered PBL showed a significant decrease in natural killer (NK) cells. Immunohistochemical studies revealed the presence of NK cells and T lymphocytes in the glomerular and interstitial tubular structures of the kidney. Functional studies showed a progressive cessation of diuresis and augmentation of renal vascular resistance when the kidney was perfused with PBL. Electron microscopy examinations of kidney sections perfused with PBL showed swollen endothelial zones, suggesting alterations to and damage of the endothelium. CONCLUSIONS: This system provides a valuable model for the study of early discordant xenogeneic cellular rejection and demonstrates the predominance of xenograft infiltration by NK cells.

New developments in medical-surgical treatment of acute myocardial infarction.

Selective intracoronary thrombolysis with streptokinase was successful in 72 of 84 (86%) patients admitted to the hospital with definitive signs of acute transmural myocardial infarction due to complete occlusion of either the left anterior descending coronary artery, the right coronary artery, or the circumflex artery. The average time between onset of acute symptoms and medically induced reperfusion was 241 +/- 90 minutes (SD). Reperfusion resulted in prompt relief of pain, regression of cardiogenic shock, and normalization of electrocardiograms. Follow-up treatment was either medical or surgical. The 32 medically treated patients had a high reocclusion rate, with 6 fatal (19%) and 9 nonfatal (28%) reinfarctions. In order to the reduce the risk of reinfarction, additional simultaneous transluminal balloon angioplasty was done in a recent series of patients with stenoses accessible to this technique. The best early and long-term results were achieved in 17 patients who underwent coronary artery bypass grafting within three days after successful thrombolysis. There was no operative mortality, and subsequent bleeding has not been a problem. It is concluded that early operation is the treatment of choice in all patients suitable for such intervention who have undergone successful intracoronary thrombolysis within 4 hours after onset of acute myocardial infarction. Late coronary bypass operation should be reserved for symptomatic patients who have definitive signs of infarction in spite of successful thrombolysis.

Carotid endarterectomy: factors influencing perioperative complications.

Early carotid surgery has been suggested to be an important cause of perioperative deterioration due to secondary haemorrhage into a recent brain infarction. It has also been suggested that the existence of preoperative neurological deficits may worsen the prognosis of surgical treatment in carotid disease. Neither of these observations could be confirmed; severe perioperative complications (5%) in this study of carotid endarterectomy were strongly related to the degree of carotid stenosis. This aspect of carotid surgery has not been previously studied. Even though technical difficulties may play an exaggerated role in a training hospital system, it is nevertheless necessary to assess the impact of surgical procedures of different extent when the "acceptable risk" for carotid surgery is calculated.

Preferential use of dilutions of single sera than mixture of sera to standardize the quantitation of anticardiolipin antibodies.

The aim of this study was to compare four house standards coming from two University Hospital laboratories with the standards provided by the Antiphospholipid Standardization Laboratory (ASL) in order to quantify anticardiolipin antibodies. Using two different plates and two different buffered protein solutions, slopes from the serial dilutions of each of the four house standards were found comparable. In contrast different slopes were obtained when using the ASL standards which consist of a mixture of sera. Our results indicate that dilutions of single sera are more suitable than mixture of sera when quantification of anticardiolipin antibodies is required.