Reduction of hyperacute rejection and protection of metabolism and function in hearts of human decay accelerating factor (hDAF)-expressing pigs

OBJECTIVE: The use of pig hearts can solve the problem of shortage of donor hearts for transplantation. However, targeting rejection by single genetic modification was proven to be ineffective, highlighting the requirement for complex genetic modifications and more effective methods for transgenic animal production. We evaluated here whether hearts of hDAF transgenic pigs generated using our technique sperm-mediated gene transfer (SMGT) will be protected from structural damage, metabolic changes, and mechanical dysfunction during perfusion with human blood. METHODS: Hearts from control (C, n = 6) or transgenic (T, n = 5) pigs were perfused ex vivo for 4 h with fresh human blood using the ex vivo working mode system allowing monitoring of the function, metabolism, and structure. RESULTS: Cardiac output (mean+/-SEM) was maintained in T constant throughout the experiment, at 3.58+/-0.36 and 3.83+/-0.14 l/min after 30 min and 4 h, respectively, while cardiac output decreased to 1.95+/-0.35 l/min in C after 30 min of perfusion (p < 0.01 vs. T). The maximum increase in coronary perfusion pressure was reduced in T to 154+/-16% as compared to C (237+/-10%, p < 0.001). Myocardial ATP after 4 h was 21.1+/-1.1 nmol/mg dry wt (similar to initial) in T, while it decreased in C to 17.2+/-1.4 (p < 0.05). Deposition of complement factors C3 and C5b9 was present in C but not in T after perfusion. CONCLUSION: We have shown that hearts from hDAF transgenic pigs produced by SMGT are protected during perfusion with human blood and are metabolically stable and maintain mechanical function above the threshold level for life support.     

Simultaneous pancreas-kidney transplantation: a single-center experience and prospective analysis

In patients with end-stage chronic kidney disease (CKD) and type 1 diabetes mellitus (DM 1), simultaneous pancreas-kidney (SPK) transplantation is currently considered the gold standard therapy. The aim of this study was to analyze and report the long-term clinical outcomes of the 23 SPK transplantations performed at our institution over an 84-month period (January 1, 2000 to December 31, 2006). A prospective analysis of these patients included donor, recipient, and transplantation characteristics. The only requirements for transplantation were blood group compatibility and a negative cross-match. Bladder drainage via pancreaticoduodenocystostomy was performed in all of the patients. Due to a pulmonary embolus 1 patient (4.3%) died at 2 months. The actuarial patient survival rates at 3 months and 1, 3, and 5 years were 95.6%. Causes for the renal graft loss were chronic allograft nephropathy in 3 cases (13%) and death of the patient in 1 case (4.3%). The actuarial censored renal allograft survival rates at 3 months and at 1 year were 100%, and at 3 and 5 years were 91.3%. Causes for the renal graft loss were chronic rejection in 1 case (4.3%) and patient death in 1 case (4.3%). The actuarial censored pancreatic allograft survival rates at 3 months and at 1 and 3 years were 100%, and at 5 years was 95.6%. The results of this work add further evidence that SPK is the gold standard therapy for selected patients with end-stage CKD due to DM 1.     

First-line liver resection and salvage liver transplantation are increasing therapeutic strategies for patients with hepatocellular carcinoma and child a cirrhosis

AIM: The present study focused on nine patients with hepatocellular carcinoma (HCC) associated with Child A liver cirrhosis undergoing first-line liver resection and salvage liver transplantation (SLT) for liver tumor recurrence. PATIENTS AND METHODS: Forty-six patients with HCC underwent liver transplantation (OLT); 37 (80.5%) were primary liver transplantations (PLTs) and 9 (19.5%) were SLTs. All patients who underwent SLT received minor transabdominal liver resections. RESULTS: The posttransplant 1-, 3-, and 5-year overall survival rates for SLT (88.9%, 88.9%, and 88.9%) were similar to those for PLT (78%, 62.7%, and 62.7%). Four (10.8%) patients in the PLT group had HCC recurrence, while there was zero recurrence in the SLT group. The 1-, 3-, 5-year disease-free survival rates for PLT (89%, 74%, and 74%) were similar to those for SLT (100%, 100%, and 100%). The 1-, 3-, 5-year disease-free survival rates after PLT were 89%, 74%, and 74%, and after SLT were 100%, 100%, and 100%, respectively. The operative mortality, intraperioperative bleeding, operative time, intensive care unit stay, in-hospital stay, and overall incidence of postoperative complications were similar in the two groups. CONCLUSIONS: In our experience, SLT for HCC is a feasible procedure with similar results in terms of overall survival, disease-free survival, and postoperative complications to those reported for patients who underwent PLT at our institute. An important role exists for SLT as shown by the fact that such a strategy has been used in the 20% of the patients undergoing OLT for HCC.     

A multimodal MRI investigation of the subventricular zone in mild cognitive impairment and Alzheimer's disease patients

The subventricular zone (SVZ) is a region that lies immediately beneath the ependymal layer on the lateral wall of the lateral ventricles, and is separated from the caudate nucleus by a layer of myelin. It contains multipotent neural stem cells. The aim of this study was to investigate the tissue around the SVZ, with the hypothesis that multimodal MRI is able to highlight the progressive disruption of tissue caused by the neurodegenerative disease in this area. We combined volumetric and diffusion tensor (DTI) imaging using a 3 T imager in a cross-sectional study including 30 patients with amnestic-mild cognitive impairment (a-MCI), 30 patients with Alzheimer's disease (AD) and 30 age- and gender-matched healthy controls (HC). Our data indicate that mean diffusivity (MD) values increase continuously from HC through a-MCI to AD in the bilateral SVZ, where most of the proliferating stem cells in the adult brain are located. This result was specific for the SVZ and could not be observed in other periventricular areas. Multimodal MRI, being able to highlight structural changes of microscopic tissue in humans in vivo, could represent a precious tool to complement histological studies of neurogenesis.     

AMPK activators inhibit the proliferation of human melanomas bearing the activated MAPK pathway

Raf/MEK/ERK signaling can inhibit the liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) pathway, thus rendering melanoma cells resistant to energy stress conditions. We evaluated whether pharmacological reactivation of the AMPK function could exert antitumor effects on melanoma cells bearing this pathway constitutively active because of a mutation in NRAS or BRAF genes. Nine melanoma cell lines were treated with the AMPK activators 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and phenformin. The activation of AMPK enzymatic activity, phosphorylation of AMPK and acetyl-CoA carboxylase kinase, in-vitro proliferation, cell cycle, and in-vivo growth of xenografts in nude mice were evaluated. AICAR and phenformin promoted phosphorylation and enzymatic activity of AMPK, as well as phosphorylation of the AMPK downstream target acetyl-CoA carboxylase. Drug treatment of either BRAF-mutant or NRAS-mutant melanomas, at doses not inducing cell death, was accompanied by a dose-dependent decrease in melanoma cell proliferation because of cell cycle arrest in either the G0/G1 or the S phase, associated with an increased expression of the p21 cell cycle inhibitor. Melanomas isolated from subcutaneously implanted mice, 25 days from treatment with AICAR, showed increased staining of the senescence-associated marker β-galactosidase, high p21 expression, and evidence of necrosis. Altogether, these results indicate that pharmacological activators of AMPK-dependent pathways inhibit the cell growth of melanoma cells with active Raf/MEK/ERK signaling and provide a rationale for further investigation on their use in combination therapies.     

Reduced Occipital and Prefrontal Brain Volumes in Dysbindin-Associated Schizophrenia

A three-marker C–A–T dysbindin haplotype identified by Williams et al (PMID: 15066891) is associated with increased risk for schizophrenia, decreased mRNA expression, poorer cognitive performance, and early sensory processing deficits. We investigated whether this same dysbindin risk haplotype was also associated with structural variation in the gray matter volume (GMV). Using voxel-based morphometry, whole-volume analysis revealed significantly reduced GMVs in both the right dorsolateral prefrontal and left occipital cortex, corresponding to the behavioral findings of impaired spatial working memory and EEG findings of impaired visual processing already reported. These data provide important evidence of the influence of dysbindin risk variants on brain structure, and suggest a possible mechanism by which disease risk is being increased.     

Combined effects of botulinum toxin and casting treatments on lower limb spasticity after stroke

Optimal treatment of spasticity requires a combination of pharmacotherapy and muscle lengthening. We evaluated 13 stroke patients with equinovarus foot randomized to treatment with either botulinum toxin A (BTA) injection plus ankle-foot casting (n=6) or BTA alone (n=7). The tibialis posterior and calf muscles (range of BTA injection: 190 to 320 U) were treated in each patient. Castings were worn at night for four months. Each patient was examined before, and at two and four months after BTA injection using the static and dynamic baropodometric tests, the Modified Ashworth Scale and the 10-meter walking test. At two months, therapeutic effects were observed in both groups. At four months, the study group showed further clinical improvement, while the control group returned to baseline performance. Thus, prolonged stretching of spastic muscles after BTA injection affords long-lasting therapeutic benefit, enhancing the effects of the toxin alone.     

Surgical treatment of extraperitoneal rectal cancer

The aim of the study was to evaluate the results of open surgery with sphincter preservation and nerve-sparing total mesorectal excision and a fast-track protocol, without a protective stoma in a consecutive series of patients with extraperitoneal rectal cancer. From 1998 to 2007, 89 patients with extraperitoneal rectal cancer were treated according to a prospective protocol. Eight-six patients were submitted to anterior resection with a low or ultra-low anastomosis and nerve-sparing total mesorectal excision. Fifty-four patients received neoadjuvant therapy. Twenty-eight patients were treated according to a fast-track postoperative protocol. Primary protective colostomies were performed in 6 cases (6.9%), while a secondary colostomy was necessary in 3 patients (3.4%). There was just one postoperative death (1.1%) and major morbidity occurred in 12.3%. Seven patients developed anastomotic dehiscence; 3 were successfully treated with a secondary colostomy and 4 were treated conservatively. 68.4% of the patients treated with the fast-track protocol could be discharged on postoperative day 4. 73% of patients were still surviving at a 5-year follow-up (48 patients). The incidence of local recurrences was 3.1%. Anterior resection in the form of open nerve-sparing total mesorectal excision with selective use of neoadjuvant therapy can be successfully performed without a protective stoma in more than 80% of patients. The fast-track protocol seems to increase the quality of the patient's postoperative condition and reduce the hospital stay.     

Relevance of apoptosis in influencing recovery of hibernating myocardium

BACKGROUND: Hibernating myocardium (HM) is viable but dysfunctional myocardium which can recover following revascularization. Myocyte necrosis is virtually absent in HM; however, cellular loss may take place by apoptosis, although this is controversial. AIM: To assess the presence of apoptosis and its relevance in HM. METHODS: During coronary artery by-pass surgery (CABG), 21 patients underwent transmural biopsy in the dysfunctional left anterior descending artery tributary area of the left ventricle (LV), with kinetic recovery at follow-up, thus fulfilling the HM criteria. All patients underwent echocardiographic follow-up at 12 months. All biopsies were evaluated by light microscopy, electron microscopy (EM), and molecular analysis. RESULTS: All biopsies were structurally altered, showing increased fibrosis and myocytes with variable size. Myocyte dedifferentiation was not detected by immunohistochemistry or EM. On stepwise linear regression, 1 year LVEF was predicted by the apoptotic index (beta=-0.973, p=0.002), the normotrophic cell percentage (beta=0.449, p=0.038), and mean fibrosis (beta=-0.412, p=0.51). CONCLUSIONS: Our biopsy study detected a wide range of morphological substrate heterogeneity in HM with degenerative features. We have demonstrated for the first time in humans that myocyte apoptosis is an important phenomenon in HM, negatively influencing LV functional recovery after CABG.     

The glucose clamp reveals an association between adiponectin gene polymorphisms and insulin sensitivity in obese subjects

Results concerning the association of adiponectin gene polymorphisms (single-nucleotide polymorphisms, SNPs) with obesity, type 2 diabetes (T2DM), metabolic disorders and insulin resistance have not lead to definite conclusions. The aim of our study was to investigate a possible association between the -11391G>A and -11377C>G SNPs of adiponectin gene and measure of insulin sensitivity evaluated by the hyperinsulinemic-euglycemic clamp in a group of 'uncomplicated' obese subjects (with no associated comorbidities) (n=99, mean age 35 years) with a history of obesity lasting at least 10 years. The study of uncomplicated obese subjects, free of possible confounding factors that could interfere with insulin sensitivity, such as pharmacological treatment, provides a good model to assess insulin sensitivity per se. We observed that subjects homozygous for the G allele at locus -11391 had lower M (mg/kg min)/fat-free mass (FFM) index and adiponectin levels compared to subjects with GA+AA genotypes (P=0.002 and P=0.03, respectively) and subjects carrying the -11377G variant had lower M (mg/kg min)/FFM index and adiponectin levels compared to noncarriers (P=0.003 and P=0.03, respectively). Our results imply that the two promoter SNPs, -11391G>A and -11377C>G, of the adiponectin gene are associated with a reduced insulin sensitivity evaluated by hyperinsulinemic-euglycemic clamp in obese subjects.