Cardiac AAV9-S100A1 gene therapy rescues post-ischemic heart failure in a preclinical large animal model.

As a prerequisite for clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated virus (AAV)-S100A1 gene therapy in a preclinical large animal model of heart failure. S100A1, a positive inotropic regulator of myocardial contractility, becomes depleted in failing cardiomyocytes in humans and animals, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium (Ca(2+)) handling in acutely and chronically failing hearts in small animal models. We induced heart failure in domestic pigs by balloon occlusion of the left circumflex coronary artery, resulting in myocardial infarction. After 2 weeks, when the pigs displayed significant left ventricular contractile dysfunction, we administered, by retrograde coronary venous delivery, AAV serotype 9 (AAV9)-S100A1 to the left ventricular, non-infarcted myocardium. AAV9-luciferase and saline treatment served as control. At 14 weeks, both control groups showed significantly decreased myocardial S100A1 protein expression along with progressive deterioration of cardiac performance and left ventricular remodeling. AAV9-S100A1 treatment prevented and reversed these functional and structural changes by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca(2+) cycling, sarcoplasmic reticulum calcium handling, and energy homeostasis. Transgene expression was restricted to cardiac tissue, and extracardiac organ function was uncompromised. This translational study shows the preclinical feasibility of long-term therapeutic effectiveness of and a favorable safety profile for cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our results present a strong rationale for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure.

Neuronal estrogen receptor-alpha mediates neuroprotection by 17beta-estradiol.

17beta-Estradiol (E(2)) was shown to exert neuroprotective effects both in in vitro and in vivo models of stroke. Although these effects of E(2) are known to require estrogen receptor-alpha (ER alpha), the cellular target of estrogen-mediated neuroprotection remains unknown. Using cell type-specific ER mutant mice in an in vivo model of stroke, we specifically investigated the role of ER alpha in neuronal cells versus its role in the microglia in the mediation of neuroprotection by estrogens. We generated and analyzed two different tissue-specific knockout mouse lines lacking ER alpha either in cells of myeloid lineage, including microglia, or in the neurons of the forebrain. Both E(2)-treated and E(2)-untreated mutant and control mice were subjected to a permanent middle cerebral artery occlusion for 48 h, and the infarct volume was quantified. Although the infarct volume of E(2)-treated female myeloid-specific ER alpha knockout mice was similar to that of E(2)-treated control mice, both male and female neuron-specific ER alpha mutant mice had larger infarcts than did control mice after E(2) treatment. We conclude that neuronal ER alpha in female and male mice mediates neuroprotective estrogen effects in an in vivo mouse model of stroke, whereas microglial ER alpha is dispensable.

Danshen protects liver grafts from ischemia/reperfusion injury in experimental liver transplantation in rats.

Reperfusion injury remains one of the major problems in transplantation. Free radicals and disturbance of microcirculation are the supposed main contributors. Recent evidence shows that Danshen, a traditional Chinese drug used in vascular diseases, can scavenge radicals and improve microcirculation. This study investigates its effect on liver transplantation (LTx). Before organ recovery, female Sprague-Dawley rats (210-240 g) received intravenous Danshen or the same volume of Ringer solution as control. LTx was performed after 1 h of cold storage. Microperfusion, leukocyte-endothelium interaction and latex-bead phagocytosis were evaluated with in vivo microscopy. Survival, transaminases and histology were assessed. Immunohistology was used for TNF-alpha levels. anova and Fisher's exact test were employed for statistical analyses as appropriate. Survival increased from 60% in controls to 100% (P < 0.05). AST and LDH decreased from 3969 +/- 1255 U/l and 15444 +/- 5148 U/l in controls to 1236 +/- 410 U/l and 5039 +/- 1594 U/l, respectively (P < 0.05). In vivo microscopy revealed decreased leukocyte-adherence and increased blood flow velocity in sinusoidal zones after administration of Danshen (P < 0.05), while latex-bead phagocytosis was found in 60% of controls (P < 0.05). The TNF-alpha index decreased from 2.08 +/- 0.09 in controls to 1.09 +/- 0.09 (P < 0.05). This study clearly demonstrates hepatoprotective effects after experimental LTx, which can be explained via anti-oxidative effects, improved microcirculation and decreased Kupffer cell activation.

Danshen protects kidney grafts from ischemia/reperfusion injury after experimental transplantation.

Danshen (DS) is used for treatment of various ischemic events in the traditional Chinese medicine. Hence, this study was designed to investigate its effect on ischemia/reperfusion injury (IRI) after experimental kidney transplantation (eKTx). Nephrectomized Sprague-Dawley rats underwent eKTx. Some animals were infused with 1.5 ml DS 10 min before surgery. Kidney grafts were transplanted after cold storage for 20 h in Histidine-Tryptophane-Ketoglutarate solution. After reperfusion blood samples were collected for blood urinary nitrogen (BUN), creatinine, lactate dehydrogenase (LDH), and alanine transaminase. Further, tissue was assessed for morphologic and pathophysiologic changes. Donor preconditioning with DS (DS-d) significantly decreased BUN, creatinine, LDH, and aspartate aminotransferase to 65-97% of controls while preconditioning of the recipient (DS-r) decreased values to 58-82% (P < 0.05). Tubular damage and caspase-3 decreased significantly in both DS-d and DS-r (DS-d: 96% and 67%, DS-r: 83% and 75% of controls) while heat shock protein 72 and superoxide dismutase increased significantly (DS-d: 143% and 173%, DS-r: 166% and 194% of controls). Further, inducible nitric oxide synthase and tumor necrosis factor-alpha decreased (DS-d: 84% and 61%, DS-r: 79% and 67% of controls) after DS. Preconditioning of both donors and recipients with DS significantly reduces IRI and thus improves graft function after eKTx.

Evaluation of the serum-free light chain test in untreated patients with AL amyloidosis.

We evaluated the Serum Free Light Chain (FLC) test in a series of 133 untreated patients with systemic AL amyloidosis. The FLC test detected the monoclonal gammopathy in 87% compared with 92% for immunofixation of serum and urine in combination. However, both tests proved complementary. The FLC test was also a valuable tool in patients with advanced renal failure in spite of uninvolved light chain retention. Higher FLC levels were associated with higher bone marrow plasmocytosis, poorer Karnofsky index and heart involvement, and therefore reflected disease severity.

Donor preconditioning with taurine protects kidney grafts from injury after experimental transplantation.

BACKGROUND: Ischemia/reperfusion injury is a major problem in clinical transplantation (Tx). Taurine has been shown to protect liver grafts from ischemia/reperfusion injury after Tx. Thus, this study was designed to evaluate its effect on kidney grafts after transplantation. MATERIALS AND METHODS: Various concentrations of taurine were infused before donor nephrectomy (1.5 mL; 30, 100, 300 mM). Controls were given the same volume of Ringers' solution. Subsequently, grafts were cold-stored for 19 h in histidine-tryptophan-ketoglutarate solution and transplanted. Six hours after Tx, graft function and injury were assessed with blood urea nitrogen/creatinine and aspartate aminotransferase/lactate dehydrogenase. Graft biopsies were taken to evaluate tubular damage, caspase-3, superoxide dismutase, and heat shock protein 72 (HSP-72) to index necrosis, apoptosis, antioxidative capacity, and regeneration, respectively. RESULTS: Taurine significantly decreased blood urea nitrogen, creatinine, aspartate aminotransferase, and lactate dehydrogenase in a dose-dependent manner to up to 71%, 69%, 51%, and 53% of controls, respectively. Further, tubular damage and caspase-3 expression decreased to 44% and 18% of control values (P < 0.01), while superoxide dismutase and heat shock protein 72 expression increased by 95% and 77% of controls, respectively (P < 0.05). CONCLUSIONS: This study demonstrates that donor preconditioning with taurine protects kidney grafts from injury (apoptosis, necrosis), improves graft function, and increases the regenerative potential most likely via mechanisms including antioxidation.

Contribution of increased oral bioavailability and reduced nonglomerular renal clearance of digoxin to the digoxin-clarithromycin interaction.

AIMS: A clinically important interaction between the cardiac glycoside digoxin and the antibiotic clarithromycin has been suggested in earlier reports. The aim of this study was to investigate the extent of the interaction and the relative contribution of different mechanisms. METHODS: In a randomized, placebo-controlled, double-blind cross-over design single oral doses of 0.75 mg digoxin with oral coadministration of placebo or 250 mg clarithromycin twice daily for 3 days were administered to 12 healthy men. Additionally, three of the subjects received single intravenous doses of 0.01 mg x kg(-1) digoxin with oral placebo or clarithromycin. Digoxin plasma and urine concentrations were determined by a highly sensitive radioimmunoassay. RESULTS: Oral coadministration of clarithromycin resulted in a 1.7-fold increase of the area under the digoxin plasma concentration-time curve [mean AUC(0,24) +/- SD 23 +/- 5.2 vs. 14 +/- 2.9 microg x L(-1) x h; 95% confidence interval (CI) on the difference 7.0, 12; P = 0.002] and in a reduction of the nonglomerular renal clearance of digoxin [mean ClRng(0, 24) +/- SD 34 +/- 39 vs. 57 +/- 41 mL min-1; 95% CI on the difference 7.2, 45; P = 0.03]. The ratios of mean digoxin plasma concentrations with and without clarithromycin were highest during the absorption period of clarithromycin. After intravenous administration digoxin AUC(0,24) increased only 1.2-fold during coadministration of clarithromycin. CONCLUSIONS: Increased oral bioavailability and reduced nonglomerular renal clearance of digoxin both contribute to the interaction between digoxin and clarithromycin, probably due to inhibition of intestinal and renal P-glycoprotein.

Anti-p53 in breast cancer: concordance of different assay procedures and association with p53 antigen expression.

OBJECTIVE: Anti-p53 levels detected by different methods were compared in a predefined group of patients with breast cancer and correlated with p53 antigen expression in the corresponding tumors. METHODS: P53 autoantibodies were investigated in 165 patients with primary breast cancer using ELISAs with recombinant or native p53. Immunoblot and indirect immunofluorescence (Huh7) were used for confirmation, p53 antigen expression in the tumor was determined immunohistochemically. RESULTS: Using ELISA, overall 18/165 positives (11%) were detected, with only partly concordant results between the assays. Five positive sera were confirmed by immunoblot, and three also by indirect immunofluorescence. Anti-p53-positive patients detected by more than two assays showed accumulated p53 in the tumor (6/6) and mostly suffered from recurrent tumors (4/6; p = 0.02). In these cases, a trend towards a shortened disease-free interval was found (26 vs. 49 months; n.s.). In patients with a positive or borderline result in only one of the serological methods, there was no increased rate of p53 accumulation compared to anti-p53-negative patients (4/19 versus 35/126). CONCLUSIONS: Lack of assay standardization may partly explain the divergence in reports on anti-p53 and its clinicopathological associations. We speculate that, in different groups of patients, anti-p53 might be induced by different mechanisms.