Detection and molecular staging of bladder cancer using real-time RT-PCR for gelatinases (MMP-2, MMP-9) and TIMP-2 in peripheral blood

IntroductionMolecular staging of bladder cancer based on the detection of mRNA of urothelial specific genes in circulating cancer cells has been inconclusive. We analyze whether real-time RT-PCR evaluation of gelatinases (MMP-9, MMP-2) and TIMP-2 in peripheral blood to diagnose and characterize patients with bladder neoplasm.Material and methodTotal RNA is extracted from circulating blood cells in 42 individuals (11 healthy controls, 31 patients with bladder cancer of different stages) and real-time RT-PCR performed using specific primers for MMP-9, MMP-2, TIMP-2 and ribosomal 18S. The quantification values of mRNA are described as relative to 18S mRNA (ΔΔCt method) and the results are blindly compared with data obtained from histological diagnosis and clinical staging.ResultsNormalized levels of MMP-9 and MMP-2 mRNA are higher in patients with cancer than controls (1.82 ± 0.6-fold and 2.7 ± 0.6-fold, respectively; P < .05). Patients with metastatic disease also have increased MMP-9, MMP-2 and TIMP-2 mRNA levels (9.6 ± 0,20-fold, 5.22 ± 0.26-fold and 1,97 ± 0,22-fold, respectively; P < .05). MMP-9 and MMP-2 are also associated with advanced clinical stage and grade (P < .05). A ratio between variables that increases the ability to segregate patients with Ta, T1, T2-4M0 and T2-4M1 tumours is proposed.ConclusionsBoth non-invasive bladder tumor recognition and molecular staging of the disease is possible using real-time RT-PCR-based detection of gelatinases and TIMP-2 in peripheral blood. The ability to distinguish metastatic disease is higher for MMP-9 but MMP-2 discriminates better levels of tumour invasion. Further investigation in this field could yield promising results regarding molecular evaluation of bladder neoplasia.     

No differences of carotid intima-media thickness between young patients with ankylosing spondylitis and healthy controls

ObjectiveAccelerated atherosclerosis in inflammatory rheumatic diseases such as ankylosing spondylitis (AS) stands out among the leading causes of morbidity and mortality. We assessed the correlation between subclinical carotid atherosclerosis and its related clinical parameters in AS patients.MethodsTwenty-eight patients (23 males, 5 females) with AS and 27 sex- and age-matched controls were consecutively recruited to this study. We estimated the carotid intima–media thickness (IMT) and parameters related to arterial elastic properties, including the distensibility coefficient (DC), stiffness index (β), and incremental elastic modulus (E_inc) using high-resolution ultrasonography. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) were measured using enzyme-linked immunosorbent assay (ELISA).ResultsCarotid IMT values and arterial elastic parameters in AS patients showed no statistical significance compared to those of controls (0.57 ± 0.07 vs 0.55 ± 0.05, p = 0.387 for IMT, 28.45 ± 9.23 vs 31.93 ± 9.52, p = 0.175 for DC, 2.32 ± 0.18 vs 2.29 ± 0.15, p = 0.559 for stiffness index (β), and 0.14 ± 0.05 vs 0.12 ± 0.03, p = 0.116 for E_inc). The serum level of IL-6 in AS patients was significantly different compared with controls (p = 0.001), but not in serum levels of TNF-α and MCP-1 (p = 0.162, p = 0.087, respectively). Carotid IMT and all arterial elastic parameters calculated in this study were not found to be associated with serum levels of TNF-α, IL-6, and MCP-1.ConclusionThis cross-sectional study showed that carotid IMT and parameters related with arterial elastic properties in young AS patients without clinically evident cardiovascular risk factors were not different from those of sex- and age-matched healthy controls. Serum levels of TNF-α, IL-6, and MCP-1 did not reflect the degree of carotid subclinical atherosclerosis. However, these findings should be confirmed further in a larger population.     

Auto-antibodies do not influence development of atherosclerotic plaques in rheumatoid arthritis

BackgroundMany questions remain unanswered about premature atherosclerosis in rheumatoid arthritis (RA). Besides inflammation, some studies have suggested the role of autoantibodies on its pathogenesis.ObjectiveThe aim of this study was to investigate the presence of antibodies against phospholipids, beta2-glycoprotein1 (beta2-gp1), lipoprotein lipase, and heat shock proteins (Hsp) in RA patients and to evaluate their possible association with subclinical carotid atherosclerosis.MethodsSeventy-one RA patients and 53 age- and sex-matched controls were selected to perform anticardiolipin antibodies (aCL) (IgG and IgM), anti-beta2-gp1 (IgG, IgM, and IgA), anti-lipoprotein lipase (anti-LPL), anti-Hsp 60, and anti-Hsp 65 by ELISA tests. Intima-medial thickness (IMT) of common carotid and presence of plaques were assessed by high-resolution B-mode ultrasonography. Exclusion criteria were smoking, diabetes, and arterial hypertension. Lipoproteins, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and fibrinogen levels, as well as health assessment questionnaire (HAQ) and disease activity score (DAS) 28 were also evaluated.ResultsAge (48.93 ± 12.31 vs. 45.37 ± 9.37 years; p = 0.20) and body mass index (BMI) (p = 0.69) were similar in RA and controls, as well as female gender (p = 0.56). The mean IMT was similar between RA and controls (0.721 ± 0.16 vs. 0.667 ± 0.14 mm, p = 0.07) but the frequency of plaques was higher in RA (14.1% vs. 1.9%; p = 0.02). In RA patients, IMT measurements did not differ according to the presence or absence of these antibodies: IgG aCL (0.62 ± 0.64 vs. 0.72 ± 0.17 mm, p = 0.24), IgM aCL (0.65 ± 0.79 vs. 0.73 ± 0.17 mm, p = 0.33), anti-Hsp 60 (0.78 ± 0.20 vs. 0.71 ± 0.16 mm, p = 0.27), anti-Hsp 65 (0.73 ± 0.16 vs. 0.72 ± 0.17 mm, p = 0.77), IgG anti-beta2-gp1 (0.73 ± 0.16 vs. 0.71 ± 0.17 mm, p = 0.72), and anti-CCP (0.71 ± 0.16 vs. 0.76 ± 0.20 mm, p = 0.36). In addition, IMT did not correlate with antibodies titers: IgG aCL (r = ? 0.09, p = 0.47), IgM aCL (r = ? 0.15, p = 0.21), anti-Hsp 60 (r = 0.10, p = 0.42), anti-Hsp 65 (r = 0.05, p = 0.69), IgG anti-beta2-gp1 (r = ? 0.07, p = 0.57), IgM anti-beta2-gp1 (r = ? 0.05, p = 0.69), IgA anti-beta2-gp1 (r = 0.03, p = 0.79), and anti-CCP (r = ? 0.07, p = 0.57). RA patients with plaques had a significantly higher age compared to those without plaques (p = 0.001), as well as higher mean IMT (p < 0.001), total cholesterol (p = 0.001), and LDL (p = 0.003).ConclusionsIn RA a clear association between all autoantibodies studied herein and increased IMT or presence of plaques was not observed. The great prevalence of carotid atherosclerosis in RA was related to age, total and LDL cholesterol, as identified in normal population.     

Transplant long-surviving induced by CD40-CD40 ligand costimulation blockade is dependent on IFN-γ through its effect on CD4(+)CD25(+) regulatory T cells

BackgroundIFN-γ was documented to be commonly associated with acute rejection. In the present study, we investigated the role of IFN-γ in the transplant long-surviving induced by blocking CD40–CD40 ligand (CD40–CD40L) costimulation and its mechanisms.MethodsIFN-γ expression in cardiac allografts and spleens from syngeneic and allogeneic recipients with or without anti-CD40L monoclonal antibody (MR-1) treatment was examined by real-time RT-PCR. The grafts survival time in Wild type (IFN-γ^+/+) and IFN-γ deficient (IFN-γ^?/?) recipients was investigated. Mixed lymphocyte reaction (MLR) of CD4⁺ T cells and cytotoxic T lymphocyte (CTL) assay of CD8⁺ T cells were also studied. FoxP3 expression in allografts and spleens from IFN-γ^+/+ or IFN-γ^?/? recipients with MR-1 treatment was examined. Furthermore, FoxP3, IL-10 and CTLA-4 expressions and the suppressive capability of CD4⁺CD25⁺ regulatory T cells were examined.ResultsRejected allografts showed significantly higher IFN-γ expression than long-surviving allografts. Allograft survival was not prolonged in nonimmunosuppressed IFN-γ^?/? mice. Administration of MR-1 induced long-term survival in 90.1% of IFN-γ^+/+ recipients (98 ± 6.6 days) but failed to do so in IFN-γ^?/? group (16.2 ± 4.0 days). IFN-γ^?/? recipients facilitated the proliferation and CTL generation of T cells. The allografts and spleens from IFN-γ^+/+ recipients contained higher FoxP3 expression than IFN-γ^?/? recipients. Moreover, CD4⁺CD25⁺ T cells from IFN-γ^+/+ recipients displayed a higher FoxP3 and IL-10 expression and suppressive capability.ConclusionIFN-γ plays an important role in the long-surviving induced by blocking CD40–CD40L through inhibiting the function of activated T cells and increasing suppressive capability of CD4⁺CD25⁺ regulatory T cells.     

Serum levels of MMP-3 and cathepsin K in patients with ankylosing spondylitis: effect of TNFalpha antagonist therapy

ObjectiveTo measure serum levels of MMP-3 and cathepsin K in patients with ankylosing spondylitis (AS) and in controls and to look for changes in these variables during TNFα antagonist therapy.MethodsWe prospectively studied a group of patients who met New York criteria for AS and a group of healthy volunteers. We recorded age, disease duration, main features of the disease, BASDAI, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). Serum MMP-3 and cathepsin K were assayed in duplicate using ELISA kits (Quantikine MMP-3, R&;D Systems; and Cathepsin K, Biomedica). We also assayed IL-17 (Quantikine IL-17, R&;D Systems) and BMP-7 (human BMP-7 DuoSet, R&;D Systems). In patients treated with TNFα antagonists, the assays were repeated 10 weeks after treatment initiation. The Mann–Whitney test was used for between-group comparisons and the Wilcoxon test for evaluations of changes under treatment. Correlation testing was performed. P-values less than 0.05 were considered significant.ResultsWe studied 23 outpatients with AS and 21 controls, with mean age of 39.9 years and 41.2 years, respectively (NS). Disease duration was 10.1 years (1.3); most patients had axial disease (n = 21) and carried HLA-B27 (n = 19). At baseline, the mean BASDAI was 44.1 mm (4.1) and the mean CRP level was 22.3 mg/L (4.7). Serum MMP-3 levels were significantly higher in the patients than in the controls (4.71 vs. 2.79 ng/ml, P = 0.04); levels were also higher for cathepsin K (6.4 vs. 3.6 pg/ml) and IL-17 (60.4 vs. 32 pg/ml), but the differences were not statistically significant. No difference was noted for BMP-7. The only positive correlation was between the ESR and the CRP level (P = 0.0002). Thirteen patients were evaluated 10 weeks into TNFα antagonist therapy (adalimumab, n = 7; etanercept, n = 4; or infliximab, n = 2). Serum MMP-3 decreased significantly (P = 0.04); significant decreases were also noted for the ESR, CRP, and BASDAI.ConclusionMMP-3 is significantly increased in patients with active AS but fails to correlate significantly with conventional variables used to assess disease activity. TNFα antagonist therapy induces a significant decrease in MMP-3 levels, together with decreases in conventional variables (ESR, CRP, and BASDAI). MMP-3 may be a biomarker for disease activity in AS but supplies no additional information to the clinician.     

25 mg etanercept once weekly in rheumatoid arthritis and spondylarthropathy

ObjectiveTo assess the clinical results at 6 months of etanercept 25 mg once weekly (half-dose), and etanercept 25 mg twice weekly (full-dose), in patients with rheumatoid arthritis or spondylarthropathy.MethodsCase records of all patients treated by etanercept for at least 6 months in the same rheumatoloy unit were retrospectively studied, to assess the mean values of DAS-28 and BASDAI, just before (J0), and after 6 months (M6) of treatment, in patients with rheumatoid arthritis or spondylarthropathy treated with etanercept 25 mg given either once or twice weekly.Results112 patients had been treated for at least 6 months (44 at half-dose, and 68 at full-dose). Values of DAS-28 or BASDAI both at J0 and M6 were available in 92 patients. DAS-28 dramatically improved both in the half-dose group (from 5.2 ± 0.8 to 3.5 ± 0.8) and in the full-dose group (from 5.5 ± 1.0 to 4.1 ± 1.0). BASDAI also strikingly improved both in the half-dose group (from 60 ± 13 to 25 ± 18), and in the full-dose group (from 58 ± 15 to 37 ± 23).ConclusionAlthough this was not a double-blind, prospective, randomised study, the strong improvement noticed in the half-dose group suggests that etanercept 25 mg once a week can induce major clinical and biological relief in some patients with RA or spondylarthropathy.     

DIO2 modifies inflammatory responses in chondrocytes

ObjectiveSelenium neutralizes interleukin-1β (IL-1β) induced inflammatory responses in chondrocytes. We investigated potential mechanisms for this through in vitro knock down of three major selenoproteins, Iodothyronine Deiodinase-2 (DIO2), Glutathione Peroxidase-1 (GPX1), and Thioredoxin Reductase-1 (TR1) in primary human chondrocytes.MethodsPrimary human chondrocytes were transfected with scrambled small interfering ribonucleic acid (siRNA) or siRNA specific for DIO2, GPX1 and TR1. After 48 h, transfected cells were cultured in serum free media for 48 h, with or without 10 pg/ml IL-1β for the final 24 h. The efficiency of siRNAs was confirmed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot analysis. The gene expression, by qRT-PCR, of cyclooxygenase-2 (COX2), IL-1β, and Liver X receptor (LXR) alpha and beta was evaluated to determine the impact of selenoprotein knockdown on inflammatory responses in chondrocytes.ResultsThe messenger RNA (mRNA) expression of DIO2, GPX1, and TR1 was significantly decreased by the specific siRNAs (reduced 56%, P = 0.0004; 96%, P < 0.0001; and 66%, P < 0.0001, respectively). Suppression of DIO2, but not GPX1 or TR1, significantly increased (～2-fold) both basal (P = 0.0005) and IL-1β induced (P < 0.0001) COX2 gene expression. Similarly, suppression of DIO2 significantly increased (～9-fold) IL-1β induced IL-1β gene expression (P = 0.0056) and resulted in a 32% (P = 0.0044) decrease in LXRα gene expression but no effect on LXRβ.ConclusionsSuppression of the selenoprotein DIO2 resulted in strong pro-inflammatory effects with increased expression of inflammatory mediators, IL-1β and COX2, and decreased expression of LXRα suggesting that this may be the upstream target through which the anti-inflammatory effects of DIO2 are mediated.     

Impact of polyclonal anti-thymocyte globulins on the expression of adhesion and inflammation molecules after ischemia–reperfusion injury

BackgroundPolyclonal anti-thymocyte globulins (ATGs) are immunosuppressive agents used for the treatment and prevention of acute organ rejection after transplantation. ATGs induce apoptosis and complement-mediated cell death in peripheral T-lymphocytes and have shown a reduction of leukocyte adhesion after ischemia–reperfusion (IRI). We analyzed the impact of different ATGs upon the expression of adhesion and inflammation molecules after IRI.Materials and methodsThe major arteries and veins of the extremities of cynomolgus monkeys were surgically isolated and flushed with Ringer's lactate at 4 °C. After 60 min of ischemia the limbs were reperfused with matching human blood. ATGs were added to the blood 30 min prior to the reperfusion, forming four groups: Tecelac-ATG group (n = 16), Fresenius(S)-ATG group (n = 16), Thymoglobulin-ATG group (n = 12) and a control group (n = 16). Biopsies from muscular tissue were obtained after the experiments. The expression of adhesion (ICAM-1, VCAM, PECAM, CD11b, CD62E) and inflammation (IL-1, IL-6, TNF-α) molecules on endothelium, leukocytes, and reperfused tissue was analyzed by means of immunohistochemistry.ResultsThe expression of the studied adhesion molecules (ICAM-1, VCAM, PECAM, CD11b, and CD62E) was significantly increased in the control group when compared with the treated groups. The expression of IL-1, IL-6, and TNF-α was reduced in the ATG-groups in comparison to the control group.DiscussionOur results showed that ATGs caused a reduction of the expression of adhesion and inflammation molecules both in endothelium and reperfused tissue. The inhibition of the expression of molecules required for firm cellular adhesion, may contribute to decreasing cellular graft infiltration after post-ischemic reperfusion.     

Inflammatory and growth factor response to continuous and intermittent exercise in youth with cystic fibrosis

BackgroundChildren with cystic fibrosis (CF) tend to suffer from chronic systemic inflammation and may have impaired growth associated with muscle catabolism. Therefore, investigating which type of exercise can elicit an anabolic response with minimal inflammation is of clinical value.MethodsTwelve children with CF (mean ± SD; age: 14.7 ± 2.3 years, predicted FEV₁: 90.0 ± 21.6%) and biological age-matched controls (age: 13.9 ± 2.1 years) completed moderate-intensity, continuous exercise (MICE) and high-intensity, intermittent exercise (HIIE) on separate days. During each exercise, blood was drawn at various time points and analyzed for immune cells, inflammatory cytokines, and growth mediators.ResultsAt rest, children with CF had higher concentrations of neutrophils and IL-6 compared with controls. In children with CF, HIIE did not affect immune cell subsets or cytokines: TNF-α, IL-6, and tumor necrosis factor-like weak inducer of apoptosis (TWEAK). All immune cell subsets and IL-6 increased significantly with MICE in both groups. Growth hormone (GH) increased with both types of exercise, with a greater change from rest during MICE.ConclusionsHIIE was a sufficient stimulus to increase GH in children with CF, without affecting systemic inflammation.     

Blockade of CD27/CD70 pathway to reduce the generation of memory T cells and markedly prolong the survival of heart allografts in presensitized mice

BackgroundAlloreactive memory T cells are a major obstacle to transplantation acceptance due to their capacity for accelerated rejection.MethodsC57BL/6 mice that had rejected BALB/c skin grafts 4 weeks earlier were used as recipients. The recipient mice were treated with anti-CD154/LFA-1 with or without anti-CD70 during the primary skin transplantation and anti-CD154/LFA-1 or not during the secondary transplantation of BALB/c heart. We evaluated the impact of combinations of antibody-mediated blockade on the generation of memory T cells and graft survival after fully MHC-mismatched transplantations.ResultsOne month after the primary skin transplantation, the proportions of CD4⁺ memory T cells/CD4⁺ T cells and CD8⁺memory T cells/CD8⁺ T cells in the anti-CD154/LFA-1 combination group were 47.32 ± 4.28% and 23.18 ± 2.77%, respectively. In the group that included anti-CD70 treatment, the proportions were reduced to 34.10 ± 2.71% and 12.19 ± 3.52% (P < 0.05 when comparing the proportion of memory T cells between the two groups). The addition of anti-CD70 to the treatment regimen prolonged the mean survival time following secondary heart transplantation from 10 days to more than 90 days (P < 0.001). Furthermore, allogenic proliferation of recipient splenic T cells and graft-infiltrating lymphocytes were significantly decreased. Meanwhile, the proportion of regulatory T cells was increased to 9.46 ± 1.48% on day 100 post-transplantation (P < 0.05).ConclusionsThe addition of anti-CD70 to the anti-CD154/LFA-1 combination given during the primary transplantation reduced the generation of memory T cells. This therapy regimen provided a potential means to alleviate the accelerated rejection mediated by memory T cells during secondary heart transplantation and markedly prolong the survival of heart allografts.     

The Association between Iliac Fixation and Proximal Stent-graft Migration during EVAR Follow-up: Mid-term Results of 154 Talent Devices

ObjectiveThis study investigated the importance of iliac fixation to secure endograft fixation.Materials and methodsComputed tomography (CT) scans of patients who underwent endovascular aneurysm repair with an endoprosthesis of great columnar strength (Talent? stent graft) were analysed retrospectively. Patients were enrolled consecutively between June 2000 and January 2007 and prospectively followed up with serial CT imaging. The superior mesenteric artery was used as a reference point to determine endograft migration (centerline endograft displacement of ≥10 mm). Proximal and distal fixation lengths were defined as the length of the endograft that was in full apposition to the aortic neck or common iliac arteries, respectively.ResultsProximal endograft migration occurred in 32 of 154 patients (21%) at a follow-up duration of 32 ± 14 months; 13 migrations required treatment (8%). Migration was more frequent in patients treated with aorto-uniiliac devices than bifurcation devices (p < 0.008). The migrator and non-migrator groups had similar demographic and abdominal aortic aneurysm (AAA) characteristics. The migrator group had significantly shorter proximal (30 ± 12 mm vs. 41 ± 13 mm, P < 0.001) and distal endograft fixation lengths (31 ± 18 mm vs. 47 ± 15 mm, P < 0.001). By multivariate regression analysis, proximal and distal endograft fixations were significant predictors for endograft migration at follow-up (P < 0.001).ConclusionIliac endograft fixation, along with proximal fixation, is a significant predictor for endograft migration.     

The effectiveness of hypertonic saline and pentoxifylline (HTS-PTX) resuscitation in haemorrhagic shock and sepsis tissue injury: comparison with LR, HES, and LR-PTX treatments

PurposeTo compare lung and liver injury and laboratory results in haemorrhagic shock and sepsis models treated with combinations of lactated Ringer's solution (LR), 7.5% hypertonic saline (HTS), hydroxyethyl starch (HES), and pentoxifylline (PTX).MethodsMale Sprague-Dawley rats (200–290 g) were assigned randomly to one of four treatment groups (n = 16 per group): (1) LR; (2) HES; (3) LR–PTX; and (4) HTS–PTX. Each group was subdivided into (1) haemorrhagic shock (n = 8) and (2) sepsis (n = 8) model groups. A venous catheter was used to inject resuscitation fluids, and an arterial catheter was used to withdraw blood and monitor mean arterial pressure (MAP). Lung and liver histology, bronchoalveolar lavage (BAL) fluid, and cytokine levels were evaluated.ResultsThe mean lung injury score was 1.7. At 24 h after treatment, the total leucocyte count in the BAL fluid was significantly (p < 0.05) higher with LR treatment (10 × 10⁶ ± 0.8) than with other treatments in the sepsis model groups (HES, 6 × 10⁶ ± 1.2; LR–PTX, 5 × 10⁶ ± 1.5; HTS–PTX, 5 × 10⁶ ± 0.6). The higher total leucocyte count after LR treatment was attributable to a greater increase in the number of neutrophils (17 ± 1.5%) compared with increases after the other treatments (HES, 6 ± 0.8%; LR–PTX, 10 ± 1.3%; HTS–PTX, 5 ± 0.4%). In the sepsis model groups, the total hepatic injury score was also significantly (p < 0.05) higher with LR treatment (9.9 ± 0.5) than with the other treatments (HES, 6.7 ± 0.8; LR–PTX, 5.6 ± 0.7; HTS–PTX, 3.1 ± 0.9). This also occurred in the shock model (LR, 10.6 ± 2.1; HES, 5.8 ± 0.9; LR–PTX, 7.3 ± 0.9; HTS–PTX, 3.5 ± 0.9). As compared with LR treatment, HTS–PTX resuscitation resulted in a 49% decrease in TNF-α, 29% decrease in IL-1β, and 58% decrease in IL-6 in the shock model at 24 h (p < 0.05), and the respective decreases were 45, 24, and 35% in the sepsis model (p < 0.05).ConclusionHTS–PTX was superior to HES, LR–PTX, and LR for treating shock and sepsis, and LR–PTX and HES gave better results than LR therapy alone.     

BIS response to tamponade and dobutamine in swine varies with hypnotic/opiate ratio

ObjectivesThis study in swine assessed BIS stability in response to decreases and increases in cardiac output under two propofol/remifentanil dosage combinations, both producing the same depth of surgical anaesthesia.MethodsEight anaesthetized-paralyzed ventilated adult swine were studied using a random-order cross-over design. Four received a P low/R high combination (P, 8.4 ± 0.9 mg/kg/h; and R, 0.54 ± 0.02 μg/kg/min) and then a P high/R low combination (P, 26.7 ± 2.1 mg/kg/h; and R, 0.34 ± 0.01 μg/kg/min). The other four had these two combinations in the reverse order. Under each P/R combination, and after a 60-minutes steady state, a 15-minute stable cardiac tamponade was induced by intrapericardial gelatine infusion. Then, after returning to pre tamponade condition, a 15 minutes period with dobutamine was allowed.ResultsTamponade induced falls in average mean arterial pressure (MAP) (from 79 ± 18 to 47 ± 9 mmHg; p < 0.05) and cardiac output (Qc) (from 1.90 ± 0.46 l/min to 1.20 ± 0.38 l/min, p < 0.05). Conversely, dobutamine increased both MAP and Qc (p < 0.05). During each type of hemodynamic challenges, changes in anaesthesia depth as assessed by BIS differed dramatically between the two drug combinations, despite observing the same percent change in P and R effect-site concentration. With P high/R low and tamponade, BIS fell from 65 ± 5 to 29 ± 10 (p < 0.05); dobutamine produced opposite effects. With P low/R high, in contrast, BIS was not influenced by either of the hemodynamic challenges.ConclusionConversely to a high propofol/low remifentanil combination, a low propofol/high remifentanil combination allows constant anaesthetic depth in the face of haemodynamic challenges.     

¿Existe un intervalo de tiempo de isquemia fría seguro para el injerto renal?

ObjectiveIt is aimed to characterize the true relationship of the cold ischemia time (CIT) with graft survival and with the principal post-transplantation events.Material and methodsWe analyzed 378 kidney transplants, studying the relationship of the CIT with graft survival using a univariate analysis according to the COX model and seeking the optimum cutoff according to the Kaplan-Meier method and log-rank test. The relationship between CIT and the principal events of the post-transplant was studied using the binary logistic regression.ResultsThe mean follow-up of all the group was 77.8 months (± 51 SD) and the mean CIT was 14.8 hours (± 5.1 SD). The univariate analysis revealed that the CIT was not related with the graft survival as a continuous variable (OR = 1.04; 95% CI: 0.9-1.08; p > 0.05). On establishing the cutoff at 18 hours, we found differences in the actuarial survival. Survival at 5 years was 91% with CIT < 18 h versus 84% with CIT >18 h. Each hour of cold ischemia increased risk of delay in the graft function by 10% (OR = 1.1; 95% CI: 1.05-1.15; p < 0.001) and also conditioned a greater incidence of acute rejection (41.5% vs. 55.3%; p = 0.02) and less time to the first rejection episode (72.6 days ± 137 vs. 272.2 days ± 614.8; p = 0.023) after 18 hours. The CIT did not seem to be related (p < 0.05) with the rest of the post-transplantation events, such as surgical complications or hospital admissions.ConclusionsIn our experience, cold ischemia under 18 hours does not seem to negatively affect graft survival.     

A novel neutrophil derived inflammatory biomarker of pulmonary exacerbation in cystic fibrosis

BackgroundThe focus of this study was to characterize a novel biomarker for cystic fibrosis (CF) that could reflect exacerbations of the disease and could be useful for therapeutic stratification of patients, or for testing of potential drug treatments. This study focused exclusively on a protein complex containing alpha-1 antitrypsin and CD16b (AAT:CD16b) which is released into the bloodstream from membranes of pro-inflammatory primed neutrophils.MethodsNeutrophil membrane expression and extracellular levels of AAT and CD16b were quantified by flow cytometry, Western blot analysis and by 2D-PAGE. Interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and AAT:CD16b complex were quantified in CF plasma (n = 38), samples post antibiotic treatment for 14 days (n = 10), chronic obstructive pulmonary disease (n = 10), AAT deficient (n = 10) and healthy control (n = 14) plasma samples by ELISA.ResultsCell priming with IL-8 and TNF-alpha caused release of the AAT:CD16b complex from the neutrophil cell membrane. Circulating plasma levels of IL-8, TNF-alpha and AAT:CD16b complex were significantly higher in patients with CF than in the other patient groups or healthy controls (P < 0.05). Antibiotic treatment of pulmonary exacerbation in patients with CF led to decreased plasma protein concentrations of AAT:CD16b complex with a significant correlation with improved FEV1 (r = 0.81, P = 0.003).ConclusionThe results of this study have shown that levels of AAT:CD16b complex present in plasma correlate to the inflammatory status of patients. The AAT:CD16b biomarker may become a useful addition to the clinical diagnosis of exacerbations in CF.     

Detection and analysis of MMP-2 and MMP-9 in seminal plasma

BackgroundThe aim of this study was to explore the association between semen parameters and matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expression in human seminal plasma samples.MethodsConventional semen analysis was performed on semen samples (n = 64). MMP-2 and MMP-9 expression in seminal plasma was determined using gelatin zymography.ResultsCompared to semen samples with a normal sperm count, semen samples with a low sperm count (≤ 19 × 10⁶/ml) showed reduced sperm viability, a reduced percentage of Grade A sperm, a reduced percentage of morphologically-normal sperm, and lower proMMP-9 and MMP-9 but higher proMMP-2 and MMP-2 levels (P < 0.05). There were correlations between MMP-2 and MMP-9 expression and the percentage of Grade A sperm and morphologically-normal sperm (P < 0.05).ConclusionsMMP-2 and MMP-9 are both present in human semen, and low-concentration semen samples have increased MMP-2 and decreased MMP-9. We also observed relationships between MMP-2, MMP-9 and seminal parameters.     

Ischemia-reperfusion injury in rat steatotic liver is dependent on NFκB P65 activation

BackgroundSteatotic liver grafts tolerate ischemia–reperfusion (I/R) injury poorly, contributing to increased primary graft nonfunction following transplantation. Activation of nuclear factor kappa-B (NFκB) following I/R injury plays a crucial role in activation of pro-inflammatory responses leading to injury.MethodsWe evaluated the role of NFκB in steatotic liver injury by using an orthotopic liver transplant (OLT) model in Zucker rats (lean to lean or obese to lean) to define the mechanisms of steatotic liver injury. Obese donors were treated with bortezomib to assess the role of NF-κB in steatotic liver I/R injury. Hepatic levels of NF-κB and pro-inflammatory cytokines were analyzed by ELISA. Serum transaminase levels and histopathological analysis were performed to assess associated graft injury.ResultsI/R injury in steatotic liver results in significant increases in activation of NF-κB (40%, p < 0.003), specifically the p65 subunit following transplantation. Steatotic donor pretreatment with proteasome inhibitor bortezomib (0.1 mg/kg) resulted in significant reduction in levels of activated NF-κB (0.58 ± 0.18 vs. 1.37 ± 0.06 O.D./min/10 μg protein, p < 0.003). Bortezomib treatment also reduced expression of pro-inflammatory cytokines MIP-2 compared with control treated steatotic and lean liver transplants respectively (106 ± 17.5 vs. 443.3 ± 49.9 vs. 176 ± 10.6 pg/mL, p = 0.02), TNF-α (223.8 ± 29.9 vs. 518.5 ± 66.5 vs. 264.5 ± 30.1 pg/2 μg protein, p = 0.003) and IL-1β (6.0 ± 0.91 vs. 19.8 ± 5.2 vs. 5 ± 1.7 pg/10 μg protein, p = 0.02) along with a significant reduction in ALT levels (715 ± 71 vs. 3712.5 ± 437.5 vs. 606 ± 286 U/L, p = 0.01).ConclusionThese results suggest that I/R injury in steatotic liver transplantation are associated with exaggerated activation of NFκB subunit p65, leading to an inflammatory mechanism of reperfusion injury and necrosis. Proteasome inhibition in steatotic liver donor reduces NFκB p65 activation and inflammatory I/R injury, improving transplant outcomes of steatotic grafts in a rat model.     

Quality of food intake after bariatric surgery: vertical gastrectomy versus gastric bypass

IntroductionThe different bariatric surgical techniques have an influence on food tolerance and the presence of vomiting. There have been few studies on the impact of these techniques on the quality of food intake.Patients and methodA prospective and comparative study was performed on a consecutive patient cohort operated on due to morbid obesity between May 2008 and November 2010. The quality of the diet was evaluated before and at 3, 6, 12 and 24 months postoperatively, using the questionnaire described by Suter et al.ResultsOne hundred and five patients (64 vertical gastrectomy [VG] and 41 gastric bypass [GB]) completed the questionnaire before the surgery, and 87 at 3 months, 79 at 6 months, 53 at 12 months, and 18 at 24 months after surgery. The overall score of the questionnaire before surgery was 23.5 ± 2.6, with a significant difference at 3 months (20.4 ± 3.8, P<.001), at 6 months (21.3 ± 4.6, P<.001) and at 12 months (22.4 ± 3.3, P<.044), and with no difference at 24 months (23.2 ± 2.5, P<.622), after surgery. On comparing food intake of VG versus GB, the scores were similar before surgery (23.8 ± 2.4 vs 23.0 ± 2.8, P<.125) as well as in the post-surgical follow up at 3 months (20.5 ± 3,9 vs 20.2 ± 3.7, P<.599), 6 months (21.1 ± 5.3 vs 21.7 ± 3.4, P<.243), 12 months (22.3 ± 3.3 vs 22.7 ± 3.4, P<.140) and 24 months (22.9 ± 3.0 vs 23.6 ± 2.2, P = 1.00).ConclusionsThe worsening of the quality of food intake is common in the first months after bariatric surgery, gradually improving and with no differences being seen between VG and GB.     

Anti-inflammatory effects of DX-890, a human neutrophil elastase inhibitor

BackgroundNeutrophil elastase (NE)-mediated inflammation contributes to lung damage in cystic fibrosis (CF). We investigated if DX-890, a small-protein NE inhibitor, could reduce neutrophil trans-epithelial migration and reduce activity released from neutrophils and NE-induced cytokine expression in airway epithelial cells.MethodsActivated blood neutrophils (CF and healthy) treated ± DX-890 were assayed for NE activity. Transmigration of calcein-labeled neutrophils was studied using a 16HBE14o⁻ epithelial monolayer. IL-8 release from primary nasal epithelial monolayers (CF and healthy) was measured after treatment ± DX-890 and NE or CF sputum.ResultsDX-890 reduced NE activity from neutrophils (CF and healthy) and reduced neutrophil transmigration. DX-890 pre-treatment reduced IL-8 release from epithelial cells of healthy or CF subjects after stimulation with NE and CF sputum sol. All improvements with DX-890 were statistically significant (p < 0.05).ConclusionsDX-890 reduces NE-mediated transmigration and inflammation. NE inhibition could be useful in managing neutrophilic airway inflammation in CF.     

Systemic effects of epidural and intra-articular glucocorticoid injections in diabetic and non-diabetic patients

IntroductionWhereas the systemic effects of glucocorticoid therapy have been extensively reported, little is known about those of local glucocorticoid injections. The objective of this study was to look for systemic effects of local glucocorticoid injections at two sites in diabetic and non-diabetic patients.MethodsWe studied 29 patients (18 women and 11 men with an age range of 18–86 years). The injection site was the epidural space in 18 patients (4 with and 14 without diabetes) with disk-related sciatica and the shoulder in 11 patients (8 with and 3 without diabetes) with frozen shoulder. Each patient was given three injections of 1.5 ml cortivazol (5.625 mg of cortivazol or about 85 mg prednisone-equivalent per injection and about 250 mg prednisone-equivalent in all), at 3-day intervals. Of the 12 patients with diabetes, 2 were on insulin therapy. At baseline and at the post-treatment visits 1, 7, and 21 days after the third injection, the following tests were done: plasma cortisol and ACTH at 8 am, urinary free cortisol excretion in 24 hours, fasting and postprandial blood glucose, serum cholesterol and triglycerides, and serum sodium and potassium. Blood pressure was measured at each visit.ResultsMean systolic blood pressure increased significantly between baseline (123 ± 10 mmHg) and the first two post-treatment visits (day 1, 127 ± 9 mmHg; and day 7, 128 ± 10 mmHg) but returned to baseline values by the third post-treatment visit (day 21). Mean postprandial blood glucose was significantly higher at the day 1 post-treatment visit (10.1 ± 5.4 mmol/l) than at baseline (7.5 ± 2.9 mmol/l). At the day 7 post-treatment visit, blood glucose remained significantly elevated compared to baseline in the 12 diabetic patients (13.9 ± 4.8 mmol/l versus 9.4 ± 3.3 mmol/l at baseline). In both the overall population and the various subgroups, plasma cortisol and ACTH and urinary free cortisol were markedly reduced at the day 1 and day 7 post-treatment visits, compared to baseline. At the day 21 visit, these variables were diminished in the group given epidural injections, whereas plasma cortisol and ACTH were normal in the group treated intra-articularly. No significant variations were noted for fasting blood glucose or for serum levels of cholesterol, triglycerides, sodium, and potassium.ConclusionThe administration of three local cortivazol injections was followed by suppression of the corticotropic axis that persisted beyond 21 days after epidural injection and recovered more rapidly after intra-articular injection. Systolic blood pressure increased transiently. Elevations in postprandial glucose levels lasted longer in diabetic than non-diabetic patients.