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.

Circulating endothelial cell count, plasma vWF and soluble ICAM-1 levels following primary or elective percutaneous coronary intervention.

BACKGROUND: Percutaneous coronary intervention (PCI) is an important therapeutic strategy in patients with ischaemic heart disease. Our aim was to clarify the extent of endothelial injury induced by PCI in stable angina (SA) or in acute ST-elevation myocardial infarction (STEMI). METHODS: Circulating endothelial cell (CEC) count, von Willebrand factor (vWF) and soluble intercellular adhesion molecule-1 (sICAM-1) levels were determined pre-, post-, 24 and 96h after PCI in patients with SA (n=23) and with STEMI (n=28). To provide control data regarding the effect of angiography itself stable angina patients with coronarography only (n=23) were enrolled. RESULTS: PCI and coronarography in stable angina patients caused measurable, but only non-significant elevation of CEC count and plasma vWF (p=NS). In STEMI, significantly higher baseline CEC count (p=0.019) and vWF plasma levels (p=0.046) were found compared to SA with PCI/or coronarography. After PCI, explicit increase in CEC count was observed (significant peak at 24h) (p=0.036). Positive correlation was found between baseline CKMB and CEC count at 24h (r=0.51, p<0.05). CONCLUSION: Both coronary angiography and elective PCI cause only mild endothelial injury. However, in patients with STEMI, not only the procedure itself but myocardial ischemia and the ongoing atherothrombotic process might be responsible for the prolonged and more pronounced endothelial damage.

Association between the SDF1-3'A allele and high levels of CD34(+) progenitor cells mobilized into peripheral blood in humans.

Some patients unexpectedly fail to mobilize sufficient numbers of haematopoietic progenitor cells (HPCs) into the peripheral blood for autologous transplantation. Considering the important role of the chemokine stromal cell-derived factor 1 (SDF-1) in HPC homing, we investigated a possible relationship between SDF1 gene polymorphism and HPC mobilization capacity in 63 patients with malignancy. Some 67% of the good mobilizers (> or = 50 CD34(+) cells/microl) and only 36% of the intermediate/poor mobilizers were SDF1-3'A allele carriers (P = 0.032). In multivariate analysis, the presence of the SDF1-3'A allele was the only factor predictive of good CD34(+) cell mobilization (P = 0.025). This is the first report showing the involvement of genetic factors for HPC mobilization in humans and suggests a significant role for SDF-1 in this process.

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.

[Anti-pig xenogeneic response by human CD4+ T-lymphocytes]. / Reponse x¿enog¿enique anti-porc des lymphocytes T CD4+ humains.

When considering the hypothesis of xenotransplantation, and if it becomes possible to control hyperacute and delayed vascular rejection, the recognition of porcine graft by human T CD4+ lymphocytes could still constitute a very important barrier. The direct recognition of porcine MHC class II molecules (SLA-DR and SLA-DQ) by human TCR has been demonstrated in vitro. It is accompanied by a proliferative lymphocytic response, as co-stimulatory molecules are able to interact across the species barrier. In vivo, this type of recognition only applies to porcine cells with antigen-presenting functions, mainly the graft dendritic cells which emigrate into the recipient lymphoid organs. The other recognition pathway is indirect, whereby the recipient dendritic cells capture porcine xenoantigens in the graft, then process and present them to the lymphocytes in the lymphoid organs. This indirect pathway can be shown in vitro by utilizing porcine MHC class II-negative endothelial cells. In this model, human purified T CD4+ lymphocyte proliferation is tightly dependent on the presence of human antigen-presenting cells and their HLA class II molecules. As the xenogenic peptides all differ from self peptides, the indirect T-cell response will be very strong and probably difficult to control.

Trophoblast cell line resistance to NK lysis mainly involves an HLA class I-independent mechanism.

The lack of classical HLA molecules on trophoblast prevents allorecognition by maternal T lymphocytes, but poses the problem of susceptibility to NK lysis. Expression of the nonclassical class I molecule, HLA-G, on cytotrophoblast may provide the protective effect. However, the class I-negative syncytiotrophoblast escapes NK lysis by maternal PBL. In addition, while HLA-G-expressing transfectants of LCL.721.221 cells are protected from lymphokine-activated killer lysis, extravillous cytotrophoblast cells and HLA-G-expressing choriocarcinoma cells (CC) are not. The aim of this work was therefore to clarify the role of HLA class I expression on trophoblast cell resistance to NK lysis and on their susceptibility to lymphokine-activated killer lysis. Our results showed that both JAR (HLA class I-negative) and JEG-3 (HLA-G- and HLA-Cw4-positive) cells were resistant to NK lysis by PBL and were equally lysed by IL-2-stimulated PBL isolated from a given donor. In agreement, down-regulating HLA class I expression on JEG-3 cells by acid treatment, masking these molecules or the putative HLA-G (or HLA-E) receptor CD94/NKG2 and the CD158a/p58.1 NKR with mAbs, and inducing self class I molecule expression on JAR cells did not affect NK or LAK lysis of CC. These results demonstrate that the resistance of CC to NK lysis mainly involves an HLA class I-independent mechanism(s). In addition, we show that the expression of a classical class I target molecule (HLA-B7) on JAR cells is insufficient to induce lysis by allospecific polyclonal CTL.

Fast detection of tissue factor and tissue factor pathway inhibitor messenger RNA in endothelial cells and monocytes by sensitive reverse transcription-polymerase chain reaction.

We developed fast and sensitive reverse transcription-polymerase chain reaction (RT-PCR) procedures to study the expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI-1) mRNA in human endothelial cells and monocytes. The sensitivity of the technique was checked by performing RT-PCR with limited numbers of cells. Cells were stimulated either with tumor necrosis factor (TNF-alpha) or endotoxin to induce TF mRNA expression or with phorbol ester to increase TFPI-1 mRNA expression. Thus, RT-PCR specific for TF mRNA provided detection from as few as 10(3) TNF-alpha stimulated endothelial cells and 5 x 10(2) monocytes stimulated by endotoxin. TF mRNA expression was increased by TNF-alpha in endothelial cells and in monocytes stimulated by endotoxin. Elevated expression of TF mRNA in monocytes without stimulation by endotoxin was mainly related to cell adhesion. TFPI-1 mRNA was constitutively expressed in endothelial cells and was detected in only 5 x 10(2) unstimulated cells and 10(2) phorbol ester-stimulated cells. Expression was increased upon stimulation with phorbol ester. With this technique, TFPI-1 mRNA in monocytes was rather low even when cells were stimulated with phorbol ester or after adhesion.

Human allogeneic lymphocytes trigger endothelial cell tissue factor expression by a tumor necrosis factor-dependent pathway.

Lymphocyte adhesion to endothelial cells and the extravascular deposition of fibrin are 2 important processes during pathologic situations such as allograft rejection. Tissue factor (TF) expression was therefore measured on human umbilical vein endothelial cells (HUVECs) after coculture with allogeneic lymphocytes (PBLs) by a factor Xa generation assay. When cocultured with PBLs, HUVECs expressed strong procoagulant activity related to the TF/factor VII-dependent pathway, which was enhanced when endothelial cells were treated with interferon-gamma (IFN-gamma). The highest TF activity was measured when 10(5) lymphocytes were incubated with 10(4) HUVECs (ratio 10: 1) for 4 hours, a time-dependent course similar to that obtained with tumor necrosis factor-alpha (TNF-alpha), and direct contact between the 2 cell types was necessary. PBL-induced TF activity was inhibited by cycloheximide or actinomycin D, indicating active protein synthesis that was confirmed by the increase in TF mRNA detected by reverse transcription-polymerase chain reaction. It was then demonstrated that 1 of the primary signaling pathways leading to endothelial cell TF expression was a rapid initial interaction between membrane TNF expressed on PBLs and the 75-kd TNF receptor, with subsequent involvement of platelet-activating factor and P-selectin. Finally, we showed that the transduction of external signals involving the activation of protein kinase C and protein tyrosine kinases also contributed to the regulation of TF expression.

Human T lymphocyte proliferative response to resting porcine endothelial cells results from an HLA-restricted, IL-10-sensitive, indirect presentation pathway but also depends on endothelial-specific costimulatory factors.

To investigate the mechanisms of cellular rejection in pig-to-human xenotransplantation, the proliferation of different human purified lymphocyte subpopulations in response to swine leukocyte Ag class II-negative porcine aortic endothelial cells (PAEC) was measured in the presence or absence of human autologous adherent cells (huAPC). CD8+ lymphocytes proliferated moderately in the absence of huAPC, and the immune response was slightly increased when huAPC were added. CD56+ lymphocytes failed to proliferate in response to PAEC whether huAPC were present or not. CD4+ lymphocytes alone did not proliferate in response to PAEC, but a strong proliferative response was observed in the presence of metabolically active huAPC. This response was totally abolished by mAbs directed against HLA class II molecules or by pretreatment of huAPC by human IL-10. Even in the presence of huAPC, CD4+ lymphocytes failed to respond to fixed PAEC or to PAEC-lysates, suggesting that PAEC must be viable to support lymphocyte proliferation. Finally, none of the nonendothelial porcine adherent cells tested was able to induce human lymphocyte proliferation, despite the fact that they also provided a large set of xenogeneic peptides. Our results show that the indirect presentation pathway of xenoantigens by huAPC to CD4+ lymphocytes is crucial in the response to porcine endothelial cells, and that IL-10 could be of therapeutic interest to prevent human lymphocyte activation by this pathway. Furthermore, we demonstrated that stimulatory signals specifically provided by endothelial cells are also necessary for this huAPC-restricted proliferative response.

End stage coronary disease treated with the transmyocardial CO2 laser revascularization: a chance for the 'inoperable' patient.

OBJECTIVE: The aim of this study is to evaluate the short and mid-term efficacy of the Transmyocardial High Power CO2 Laser Revascularisation (TMLR) as a last resource method for end-stage coronary disease patients. METHOD AND PATIENTS: The High Power CO2 Laser 800 W Heart Laser (PLC Medical Systems) was used since February 1994 to treat 268 patients. In 52% of the cases (140) the indication for TMLR treatment was virtual inoperability by the classical bypass revascularisation. In the other 128 patients (48%), where only an incomplete revascularisation was expected, the TMLR was combined with a feasible bypass revascularisation (CABG). Of all patients, 71% were operated on 1-5 times before and or treated by several percutaneous transluminal coronary angioplasty (PTCA). All patients were sufferers of angina pectoris and most were classified Canadian Cardiac Society (CCS) 3-4, despite the maximal medical treatment. The ejection fraction was normal in 13% of patients only, and in 47% of them it was below 40% (10-68%). RESULTS: The operation itself was generally well tolerated. We lost only one patient at the table. The hospital survival was 89.2%; 88.2% in the combined group and 90.3% in the TMLR only group. After the routine follow up screening 3, 6 and 12 months postoperatively (262 patients--131 TMLR and 131 TMRL/CABG), 40% of the TMLR patients upgraded into the functional class CCS 0-1; the combined group of patients scored up even in 84%. All considering their quality of life to be 'better than years ago'. The ergometry stress test, impossible for most of them before, became feasible and better in 80% of the patients. In the follow up period of the combined group, another 6 (4.7%), and in the TMLR only group, 12 (9.4%) patients died. CONCLUSION: The short and middle term results of this--until now the largest single institution series of TMLR treated patients--were that patients almost without exception were refused for any kind of surgery by several other centres; this shows an acceptable survival rate and a surprising level of pain relief, increased activity and better quality of life then ever expected. In our experience, TMLR is a suitable method for treatment of end stage coronary disease, if all standard measures, medical therapy, PTCA and redo coronary revascularisation possibilities are exhausted. The favourable results imply the question as to whether this method will become an alternative for a second bypass operation in the future. The TMLR as an alternative for heart transplant is already a reality for some of our patients.

Docosahexaenoic and eicosapentaenoic acids inhibit in vitro human endothelial cell production of interleukin-6.

The interaction between lymphocytes, cytokines, and endothelial cells (EC) is a key step in the inflammatory process. Interleukin-6 (IL-6) a pleiotropic cytokine in its effects, seems to be an early indicator of acute systemic inflammation. In this study, we have examined the effects of polyunsaturated fatty acids (PUFAs) on the production of IL-6 by human unstimulated EC or EC stimulated with TNF-alpha (100 U/ml); IL-4 (100 U/ml); LPS (1 ug/ml); or allogeneic peripheral blood lymphocytes (PBL). Twenty-four hour culture supernatants of immunoreactive IL-6 were measured by Sandwich ELISA. We have shown that the production of IL-6 was potentiated when EC were stimulated with TNF-alpha; IL-4; LPS; or monocyte-depleted PBL in comparison to unstimulated EC. The addition of n-3 PUFAs in culture medium (100 ug/ml DHA or EPA) significantly reduces the production of IL-6 by unstimulated EC; or stimulated with TNF-alpha; IL-4 pg/ml); LPS or depleted PBL respectively for DHA and EPA, whereas the n-6 PUFAs (Arachidonic acid), even used at the highest concentration, was ineffective. This inhibitory effect is PUFA dose dependent but is more potent with EPA than DHA. Regardless of the mode of action, since IL-6 is known to be involved in hematopoiesis, in the regulation of the immune response and in the inflammatory reaction, these results suggest that n-3 PUFAs may play a role in suppressing inflammation. Further studies are needed to elucidate the mechanism involved and the choice between the two fatty acids for clinical and therapeutic purposes.

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.

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.