Combining intra-dialytic exercise and nutritional supplementation in malnourished older haemodialysis patients: Towards better quality of life and autonomy.

Protein-energy wasting (PEW), defined as a loss of body protein mass and fuel reserves, is a powerful predictor of adverse outcomes in haemodialysis (HD) patients. Robust arguments suggest that intra-dialytic exercise, combined with oral/parenteral nutrition, enhances the effect of nutritional interventions in HD patients. This pilot randomized controlled trial investigated the feasibility and the effects of a 6 month intra-dialytic cycling program combined to a nutritional support on PEW, physical functioning (gait, balance, muscle strength) and quality of life (QoL) in older HD patients (mean age 69.7 ± 14.2 years).Twenty-one patients fulfilling diagnostic criteria of PEW were randomly assigned to Nutrition-Exercise group (GN-Ex , n = 10) or Nutrition group (GN , n = 11). Both groups received nutritional supplements in order to reach recommended protein and energy intake goals. In addition GN-Ex completed a cycling program. No significant difference between groups was found in the number of patients having reached remission of PEW. Likewise, no change was observed in serum-albumin, -prealbumin, C-reactive protein, body mass index, lean- and fat-tissue index, or quadriceps force. Interestingly, we found positive effects of exercise on physical function and QoL for the GN-Ex , as evidenced by a significant improvement in the 6-min walk test (+22%), the absence of decline in balance (unlike the GN ), and a noteworthy increase in QoL (+53%). Combining intra-dialytic exercise and nutrition in HD patients is feasible, and well accepted, improves physical function and QoL but it appears not to have the potential to reverse PEW.

[Contribution of hemostatic dressings in the hemostasis of arteriovenous fistula? A quality improvement program in our center]. / Évaluation de nos pratiques professionnelles : apport des pansements hémostatiques dans l'hémostase de la fistule artério-veineuse ?

INTRODUCTION: In haemodialysis patients the length of bleeding times after fistula cannulation is an easy and fairly used method of monitoring vascular access. In the most cases, compression is performed manually by nurses and the use of haemostatic dressing is common. As data in the literature are scares, we have decided to develop a quality improvement program in our hemodialysis center to manage this issue. MATERIAL AND METHODS: After informed consent, 35 hemodialysis outpatients were selected in order to study the bleeding time using haemostatic dressing or not during two weeks in a cross over schema. The dialysis schedule was unchanged and comparative analysis of parameters such as blood flow rate or anticoagulant treatment were done between the groups. RESULTS: Compression times with and without hemostatic dressing were not different (12.6 min and 12.9 min, respectively). Patients with an anticoagulation during the dialysis session greater than 0.35 IU/kg/session had a longer bleeding time (12.75 min vs 11.75 min; P=0.008). CONCLUSION: In our evaluation, the use of haemostatic dressings is not associated with a real shorter bleeding time. Their use generate an additional cost estimated on average at 164 euros/year/patient. Patients and team realized that compression time is important for fistula monitoring and using compresses does not really increase this time.

Tuberculosis following kidney transplantation: clinical features and outcome. A French multicentre experience in the last 20 years.

BACKGROUND: Kidney transplant recipients are at high risk of opportunistic infection. The aims of this study were to describe the epidemiology, clinical features and prognosis of Tuberculosis (TB) in kidney transplant recipients. METHODS: Retrospective observational study conducted in 14 French transplant centres involving all cases of TB that occurred in kidney transplant recipients between 1986 and 2006. RESULTS: Among the 16146 kidney transplantations performed during the study period, 74 (0.45%) developed TB. The country of birth was a highly endemic area for TB in 20 (40.8%) patients. Time from kidney transplantation to TB was 10 months (4-27). Extrapulmonary and disseminated TB accounted for 33 (67.4%) cases. The most common symptoms were fever (71.7%), weight loss (41.3%) and asthenia (39.1%). Coexisting infections were diagnosed in 11 (22.4%) patients. Microbial sensitivity tests revealed no case of multidrug-resistant TB. Haemophagocytic syndrome associated with TB was diagnosed in 5 (10.2%) cases with 60% of mortality (P = 0.0005). Median length of antituberculous therapy was 12 (9.5-12) months. Immunosuppressive therapy was reduced in 22 (44.9%) patients without adverse consequence on the graft. Overall, hospital mortality was 6.1% and 1-year graft survival was 97%. CONCLUSIONS: Kidney transplantation increases the risk of TB, with a high rate of extrapulmonary disease. Symptoms of infection are often attenuated, leading to delayed diagnosis. Overall prognosis is good but haemophagocytic syndrome is associated with high mortality. Country of birth might be taken into account in the decision of post-transplantation treatment of latent TB.

[Fluvastatin affects HLA class I expression on endothelial cells]. / Effet de la fluvastatine sur l'expression du CMH de classe I par les cellules endothéliales humaines.

Originally designed to target elevated lipids, the "traditional" cause of atherosclerosis, statins might also confer vascular benefit by directly or indirectly modulating both the inflammatory and immune responses. Statins have been shown to downregulate MHC class II and CD40 expression on activated endothelial cells (EC). In this study, we investigate the potential effect of statins on MHC class I expression and regulation in response to IFNgamma. Primary cultures of human ECs have been treated with increasing doses of fluvastatin (0.01; 0.1 and 1 microM) with or without IFNgamma for 48 hours. Surface expression of MHC class I and class II has been analyzed by flow cytometry. Our data indicate that fluvastatin increases MHC class I expression on quiescent ECs by a dose-dependent effect. Furthermore, fluvastatin potentiates the MHC class I upregulation but prevents MHC class II induction triggered by IFNgamma. These effects are reversed by mevalonate. In conclusion, our results suggest that while decreasing MHC class II expression, fluvastatin (at 0.1 and 1 microM) upregulates MHC class I expression on ECs. Functional consequences of statin-mediated modulation of MHC on ECs have still to be elucidated in vitro and in vivo.

Vascular endothelial cells evade apoptosis triggered by human leukocyte antigen-DR ligation mediated by allospecific antibodies.

BACKGROUND: Human leukocyte antigen (HLA)-DR ligation mediates cell death of antigen-presenting cells (APC), including mature B cells, macrophages, and dendritic cells. This study investigates the apoptotic effects of HLA class II ligation mediated by anti-HLA antibodies on activated human vascular graft endothelial cells (ECs). METHODS: HLA class II expression was examined by flow cytometry using a panel of HLA-typed vascular ECs isolated from transplant donors and compared with that of B lymphocytes. The apoptotic effects of anti-HLA-DR monoclonal antibodies (mAbs) were investigated using viability assays, DNA content analysis, and annexin-V labeling. Intracellular signaling pathways mediated by HLA-DR ligation on ECs were examined by Western blotting. RESULTS: Even with optimal stimulation, the expression of HLA-DR on interferon (IFN)-gamma-treated ECs was quantitatively lower (3-5-fold) than that on B cells. Whereas anti-HLA-DR monomorphic mAbs induced apoptosis of B cells (approximately 22%), no significant apoptosis of IFN-gamma-activated (DR-positive) ECs ( < 5%), collected from the same donor, was observed under the same conditions. Similarly, specific polymorphic anti-HLA-DR11 or -DR16 antibodies were unable to induce EC apoptosis. Nevertheless, antibody-binding to HLA-DR on ECs is sufficient to induce intracellular signaling, as evident in the modulation of tyrosine phosphorylation and protein kinase (PK)C-alpha/beta and PKB/Akt activation. Our results suggest that HLA-DR ligation induces both common and divergent signaling events in ECs and B cells. CONCLUSION: Collectively, our data suggest that, in contrast with professional APC, graft ECs evade apoptosis mediated by HLA-DR ligation, not as a result of moderate HLA-DR expression but rather as a result of a specific signaling pathway.

Exogenous tissue inhibitor of metalloproteinase-1 promotes endothelial cell survival through activation of the phosphatidylinositol 3-kinase/Akt pathway.

Control of molecular targets and signaling pathways which improve endothelial cell survival may be an attractive concept for interfering with dysregulated vascular injury and remodeling, a key mechanism for transplant arteriosclerosis and chronic allograft rejection. In addition to inhibiting matrix metalloproteinase activity, it has been suggested by recent studies that the tissue inhibitor of metalloproteinase (TIMP)-1 may inhibit apoptosis in various cell types. The present work examines the possibility that TIMP-1 belongs to a protective pathway via antiapoptotic properties and investigates the signaling pathway mediated by TIMP-1 in human ECs. We demonstrate that exogenous, recombinant, TIMP-1 efficiently prevents apoptosis induced by TNFalpha in cycloheximide-sensitized ECs. The antiapoptotic effect of TIMP-1 was dose-dependent and a maximal effect of TIMP-1 (30% protection) was reached using 250 ng/mL of recombinant TIMP-1. We present evidence that TIMP-1 induces activation of PI3-kinase but not NFkappaB pathway in ECs. Our findings further indicate that TIMP-1-induced EC survival is mediated through activation of PI3-kinase pathway and the downstream phosphorylation of Akt kinase. Blocking the PI3-kinase pathway with wortmannin or LY294002 restores TNFalpha-mediated EC death. In conclusion, our findings suggest that TIMP-1, generated upon inflammation, acts as an antiapoptotic molecule that can prevent EC apoptosis through activation of the PI3-kinase and phosphorylation of the Akt kinase.

Endothelial cell activation and proliferation modulate NKG2D activity by regulating MICA expression and shedding.

MICA are major histocompatibility complex class I-related molecules, expressed by endothelial cells (ECs), that may be targets for alloantibodies and NKG2D-expressing natural killer (NK) and T effector cells in organ allografts. This study shows that basal levels of MICA expressed on vascular ECs is sufficient to functionally modulate the expression and activity of the immunoreceptor NKG2D in allogeneic NK cells. We found that MICA expression is differentially regulated at the EC surface in response to cytokines. TNFα upregulates MICA while IFNγ significantly decreases MICA at the EC surface. Both cytokines induce the release of soluble MICA by ECs. Modulation of NKG2D correlates with the MICA level on the EC surface. Glycosylation and metalloproteinase activities account for major post-transcriptional mechanisms controlling MICA level and the function in ECs. Our results indicate that, in addition to the NFκB pathway, the mitogen-activated protein kinase pathways JNK, ERK1/2 and p38 are key signaling pathways in the control of MICA by the cytokines. Finally, we show that EC proliferation mediated by FGF-2 or wound healing increases the MICA level. Together, our data suggest that inflammation and proliferation regulate endothelial MICA expression and shedding, enabling ECs to modulate NKG2D activity on effector NK and T cells, and provide further evidence of a role for ECs in immunoregulation.

Profiling posttransplant circulating antibodies in kidney transplantation using donor endothelial cells.

BACKGROUND: Pathogenesis of antibody (Ab) responses to transplant are yet not well defined. This study aimed to detect and to analyze posttransplant circulating allo-Abs reacting toward graft endothelial cells (ECs) using primary EC cultures prospectively isolated from the transplant donor at the time of transplantation. METHODS: This study shows a retrospective analysis performed using a dedicated EC crossmatch (ECXM) assay that we developed for the experimental assessment of donor-specific EC-reactive Abs. Donor-specific ECXM was performed by flow cytometry on posttransplant sera (n=256) from an historical cohort of 22 kidney allograft recipients. RESULTS: In this study, we show that 27.3% (6/22) of recipients have a positive ECXM that strictly correlates (100%, 6/6) with the presence of anti-human leukocyte antigen (HLA) Abs posttransplantation. ECXM identifies both donor-specific Abs (DSA; 50%) and non-DSA (50%) reactive to EC. DSA and non-DSA are mostly IgG1 and exhibit peak titers ranging from 1/8 to 1/1024. ECXM indicates that DSA correspond to anti-HLA class II Abs; this immunization is late (M3-M60) but persistent (still detected at M60). In contrast, non-DSA are non-HLA-type Abs reacting with third-party EC and reflecting an early but transient immunization (ended at M3-M12). Our findings demonstrate selective regulatory pathways initiated by anti-HLA class II and non-DSA in graft EC reflected by CCR4 and interleukin 1ß up-regulation, respectively. CONCLUSIONS: We provide evidence that circulating Abs in HLA-sensitized transplant recipients include both DSA and non-HLA/non-DSA able to bind to graft EC and induce specific gene transcription.

Inflammation dysregulates Notch signaling in endothelial cells: implication of Notch2 and Notch4 to endothelial dysfunction.

Although the involvement of the Notch pathway in several areas of vascular biology is now clearly established, its role in vascular inflammation at the endothelial level remains to be elucidated. In this study, we demonstrated that pro-inflammatory cytokines drive a specific regulation of the Notch pathway in vascular endothelial cells (ECs). In arterial ECs, TNFα strongly modulates the pattern of Notch expression by decreasing Notch4 expression while increasing Notch2 expression. Changes in Notch expression were associated with a reduction in hes1 and hey2 expression and in CBF1 reporter gene activity, suggesting that TNFα regulates both Notch expression and activity. Notch2 and Notch4 regulations occurred independently and were found to be mostly mediated by the NFκB signaling pathways and PI3-kinase signaling pathways, respectively. Functionally, TNF-mediated Notch regulation promotes caspase-dependent EC apoptosis. Finally, our findings confirmed that dysregulated Notch signaling also occurs upon inflammation in vivo and correlates with caspase activation and apoptosis. In conclusion, inflammatory cytokines elicit a switch in Notch expression characterized by Notch2 predominance over Notch4 leading to a reduced Notch activity and promoting apoptosis. Thus, here we provide evidence for a role of soluble mediators of inflammation (i.e. cytokines) in the regulation of Notch signaling and for the implication of a dysregulated Notch pathway to endothelial and vascular dysfunction.

Expression and release of soluble HLA-E is an immunoregulatory feature of endothelial cell activation.

Human leukocyte antigen (HLA)-E belongs, with HLA-G and HLA-F, to the non-classic major histocompatibility complex (MHC) class I (Ib) molecules, broadly defined by a limited polymorphism and a restricted pattern of cellular expression. In contrast to HLA-G, the expression and function of HLA-E and HLA-F in physiologic and pathologic processes remain poorly established. In the present study, we show that HLA-E protein expression in normal human nonlymphoid organs is mainly restricted to endothelial cells (ECs). HLA-E is also basally expressed by B and T lymphocytes, natural killer (NK) cells and by macrophages. We demonstrate that tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and interferon gamma (IFNgamma) up-regulate the cell-surface expression of HLA-E on ECs in vitro and induce the release of soluble HLA-E (sHLA-E). HLA-E up-regulation protects IFN-gamma-activated ECs from NK-mediated cell lysis, while sHLA-E protects bystander cells. Finally, sHLA-E is not detected in normal sera, and increased serum levels correlate with disease activity in patients with antineutrophil cytoplasmic antibody-associated systemic vasculitis. Thus, HLA-E expression and release of sHLA-E are features of EC activation and emphasize immunoregulatory functions of the endothelium. The present identification of soluble HLA-E molecules may have important implications in understanding the pathogenesis of immune-mediated vascular diseases and for the diagnosis and monitoring of patients.

The effect of a first kidney transplant on a subsequent transplant outcome: an experimental and clinical study.

BACKGROUND: Second kidney transplantations have a roughly similar clinical outcome to first transplantations. Nevertheless, the effect of the presence of the first, nonfunctional transplant at the time of the second transplantation may also influence its outcome and has not yet been specifically studied. METHODS: We analyzed the effect of the presence of a first graft on the outcome of a second graft in a rodent allograft model and in a cohort of 240 human second kidney allograft recipients. RESULTS: In rodents, 100 days subsequent to the rejection of the first graft, we observed an increase in blood but not spleen CD4(+)CD25(+) T cells, whereas no differences were observed in transcriptional patterns. Adoptive transfer of day 100 splenocytes did not prolong graft survival. Moreover, the presence of a first rejected graft does not prolong the survival of a second graft performed at a later date. In the human context, a higher incidence of patients with anti-HLA immunization and a higher % of PRA were observed in retransplant recipients with primary allograft nephrectomy. Despite a relatively low statistical power, our data do not suggest significant differences in graft outcome between recipients of second transplants with primary allograft nephrectomy and those without. CONCLUSION: Collectively, the data from both an experimental model and a large cohort of human recipients of a second graft do not suggest a beneficial effect of the presence of a first rejected graft at the time of a second transplantation.

RhoA activation mediates phosphatidylinositol 3-kinase-dependent proliferation of human vascular endothelial cells: an alloimmune mechanism of chronic allograft nephropathy.

HLA class I ligation on graft endothelial cells (EC) has been shown to promote graft arteriosclerosis and chronic allograft nephropathy. This study investigated transcriptional and functional changes mediated by anti-HLA antibodies (Ab), developed by transplant recipient, on vascular renal EC. For mimicking interactions that occur between alloantibodies and graft endothelium, HLA-typed primary cultures of human EC were incubated in vitro in the presence of monomorphic or polymorphic anti-HLA class I Ab. Gene expression analysis identified the upregulation of several molecules involved in cell signaling and proliferation, including the GTP-binding protein RhoA. It was demonstrated further that HLA class I ligation on EC induced a rapid translocation of RhoA to the cell membrane associated with F-actin stress fiber formation and cytoskeleton reorganization. Western blot analysis showed that anti-HLA class I Ab induced, in addition to RhoA, the activation of phosphatidylinositol 3-kinase, reflected by the phosphorylation of Akt (Ser473) and GSK3beta (Ser9), in EC. C3 exoenzyme, an inhibitor of RhoA, inhibited RhoA translocation in response to HLA class I ligation and reduced phosphatidylinositol 3-kinase activation. EC proliferation and cell cycle progression, examined by 5,6-carboxyfluorescein diacetate succinimidyl ester staining, demonstrated that anti-HLA-induced EC proliferation was efficiently prevented by the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor simvastatin (0.1 micromol/L) through inhibition of RhoA geranylgeranylation. Taken together, these findings support the conclusion that RhoA is a key mediator of signaling pathways that lead to cytoskeletal reorganization and EC proliferation in response to alloantibodies that bind to HLA class I and demonstrate the specific and potent inhibitory effect of simvastatin on allostimulated EC growth.

Non-HLA-type endothelial cell reactive alloantibodies in pre-transplant sera of kidney recipients trigger apoptosis.

The vascular endothelium of transplanted organs represents an important target for allograft-directed immune responses. Although HLA antigens expressed on graft endothelial cells (EC) can become targets of the host immune response, the role of other, non-HLA-encoded EC antigens has been proposed but is still unclear. The aim of this study was to investigate the presence of and to characterize anti-EC antibodies (AECA) in 57 kidney transplant recipients according to their HLA-immunization status. Flow cytometry in pretransplant sera was used to detect AECA reactive with surface antigens on ABO and HLA-typed primary cultures of arterial ECs, stimulated or not with tumor necrosis factor-alpha (TNFalpha) or interferon-gamma (IFNgamma). FACS analysis revealed the presence of AECA in 47% of HLA-sensitized (PRA = 10%: mostly IgG) vs. 16.0% in nonsensitized patients (PRA < 10%) (p < 0.02). No significant correlation was found between the presence of AECA and acute rejection occurrence and graft outcome. Non-HLA reactive AECA are directed against TNFalpha- and IFNgamma-inducible membrane molecule(s), and react with two predominant antigens of approximately 35 kDa and approximately 50 kDa expressed on ECs but not on B cells. Binding of AECA decreases in vitro EC viability by 50-60% by promoting EC apoptosis, as demonstrated by DNA fragmentation assays.

Rapid selection of differentially expressed genes in TNF[alpha]-activated endothelial cells.

BACKGROUND: RNA differential display (DD) RT-PCR is a useful method to identify and clone differentially expressed genes. However, the rate of false positives and redundancy associated with this PCR-based method as well as laborious downstream screening steps constitute major limitations. Here we present DD RT-PCR and reverse northern (RN) protocols allowing rapid and acurate identification of genes upregulated in porcine endothelial cells (EC) in response to TNFalpha. MATERIALS AND METHODS: The housekeeping gene beta-actin was used to investigate mispriming and to set up optimal conditions for DD-RT-PCR and RN. In this study DD was performed to compare resting and TNFalpha-activated ECs. Selection of DD-fragments was performed following 30-cycles of PCR using serial dilutions of template cDNA and regulation of 6 out of 17 candidates genes were first confirmed by semi-quantitative RN. RESULTS: Using this protocol, 5 out of 6 DD-fragments were further confirmed to be upregulated by Northern blot, and 3 novel porcine cDNAs were cloned including the pro-apoptotic member of the Bcl-2 family, Noxa. CONCLUSION: In this study we demonstrate that the combination of DD-RT-PCR and RN, which efficiently reduces the number of false positive candidates derived from mispriming at the screening step, allows a rapid identification of differentially expressed genes.

Acute graft pyelonephritis and long-term kidney allograft outcome.

BACKGROUND: Long-term graft function is the result of multiple parameters, including both immune and non-immune components, which have a beneficial or detrimental potential. Among these, despite its frequency and theoretical interest (expression of "danger signals" in the graft itself), the effects of acute graft pyelonephritis (AGPN) on immediate and long-term outcome have not been studied in a large series. This article reviews a cohort of 1387 consecutive primary renal transplant recipients. METHODS: The objective of the study was to define the risk factor for AGPN, the risk profile for recurrence, and the impact of AGPN on long-term graft survival. According to a higher risk for AGPN in females during their follow-up, statistical analyses (Cox model, and multiple regression analysis) were performed by recipient sex strata. RESULTS: Multivariate analysis showed that CMV infection was the only risk factor for AGPN occurrence. AGPN occurred in 13% of the graft recipients during their follow-up. Taken as a whole, AGPN was not associated with a significantly poor long-term outcome. However, when assessed in more detail, the outcome of this population was found to be more complex and to depend on several factors. Early AGPN (during the first 3 months) was significantly detrimental for graft outcome, independently of acute rejection episodes. Moreover, E. coli involvement in a first episode was linked to an increased AGPN recurrence. CONCLUSION: This analysis did not support the concept that with current immunosuppression, strong "danger signals" such as those derived from bacteria within an allograft, are instrumental in initiating acute or chronic rejection.

Ten-year survival of second kidney transplants: impact of immunologic factors and renal function at 12 months.

BACKGROUND: The aim of the present study was to assess long-term survival of cadaveric second kidney allografts performed in our center and to determine risk factors predictive of long-term graft outcome. METHODS: Of 1704 kidney transplantations performed between January 1985 and March 1998, 233 were second grafts. The majority of the recipients were sensitized. All patients were treated with the same quadruple immunosuppressive regimen. RESULTS: Kaplan-Meier analysis documented graft survival of 89% at 1 year, 76% at 5 years, and 53% at 10 years. Graft survival was similar for second and primary kidney transplants performed during the same period of time. When long-term second graft survival was examined, only two risk factors were found to be significant: (1) the degree of human leukocyte antigen (HLA) DR mismatch (MM) and (2) the number of acute rejection episodes. Multivariate analysis of several pre- and posttransplant variables also confirmed the importance of HLA MM (DR> A), but also, identified serum creatinine at 12 months as the most significant predictor of graft survival. In addition, the Cox proportional hazards model revealed that only the year of transplantation had an independent significant effect on acute rejection occurrence (RR = 0.591, 95%CI 0.437 to 0.801, P < 0.0007). Indeed, the incidence of acute rejection was found to decrease over time (44% of patients experienced at least one episode of acute rejection before 1990 vs. 17% after 1990). CONCLUSION: Finally, second graft long-term outcome shows an improved evolution according to the time period resulting from a strong decrease in acute rejection incidence and the impact of creatinine at 12 months.