New in vivo model to assess macroscopic, histological, and molecular changes in Peyronie's disease.

BACKGROUND: Peyronie's Disease (PD) is a connective tissue disorder that affects the tunica albuginea (TA) of the penis causing curvature and erectile dysfunction. The pathophysiology is not well understood and, for this reason, treatment options are limited. OBJECTIVE: The aim of the present study is to analyze and compare whether single or multiple instillations of plasma in the TA of rats is capable of triggering macroscopic, histopathological, and molecular changes consistent with PD. MATERIAL/METHODS: Fifty male Wistar rats were divided into four groups: Group 1: a single instillation of plasma in the TA; Group 2: a single instillation of distilled water in the TA; Group 3: four instillations of plasma in the TA (1x per week); and Group 4: four instillations of distilled water in the TA (1× per week). Forty-five days after the last instillation a manual inspection of the corpus cavernosum, a penile erection test and a penectomy were performed to obtain material for histopathological and molecular analysis. RESULTS: It was observed that 31.25% of the rats that received repeated instillations of plasma presented penile curvature according to the erection test, while none of the rats from the control group or group with one instillation of plasma presented curvature. In the animals that received four instillations of plasma, the following differences were observed in relation to the control group: increase in fibrosis and the deposition of collagen I. The protein expression of heparanase (HPSE) and TGF-ß increased in the groups that received a single or four instillations of plasma, and the protein expression of heparanase-2 (HPSE-2), metalloproteinases (MMP-2, MMP-9) and metalloproteinase inhibitor (TIMP-2) showed an increase in the group that received four instillations of plasma. There was a significant increase in the gene expression of HPSE, MMP-9, and TGF-ß in the group that received four instillations of plasma. In the analysis of the glycosaminoglycans, an increase was observed in the secretion of galactosaminoglycans chondroitin sulfate and dermatan sulfate (CS/DS) in the group that received four instillations of plasma. DISCUSSION: Previous studies have demonstrated increased protein expression. of HPSE, MMP-9 and TGF-ß with instillation of blood in the TA; however, there was no increase in gene expression. In the present study, the increase in the expression of TGF-ß with plasma instillations, proved to be more reliable. The two models with plasma (one or four instillations) demonstrated significant histopathological and molecular changes when compared to the control group. However, only in the group with four plasma instillations there was a macroscopic change. The idea is that repeatedly extravasation of TGF-ß present in plasma of predisposed individuals acts as a trigger for the development and maintenance of changes in the extracellular matrix that perpetuate an anomalous inflammatory process present in PD. CONCLUSION: The present study shows that the repeated instillation of plasma is a low cost in vivo model for the study of PD.

Layer-by-Layer Biomimetic Microgels for 3D Cell Culture and Nonviral Gene Delivery.

Localized release of nucleic acid therapeutics is essential for many biomedical applications, including gene therapy, tissue engineering, and medical implant coatings. We applied the substrate-mediated transfection and layer-by-layer (LbL) technique to achieve an efficient local gene delivery. In the experiments presented herein, we embeded lipoplexes containing plasmid DNA encoding for enhanced green fluorescent protein (pEGFP) within polyelectrolyte alginate-based microgels composed of poly(allylamine hydrochloride) (PAH), chondroitin sulfate (CS), and poly-l-lysine (PLL) with diameters between 70 and 90 µm. Droplet-based microfluidics was used as the main process to produce the alginate (ALG)-based microgels with discrete size, shape, and low coefficient of variation. The physicochemical and morphological properties of the polyelectrolyte microgels were characterized via optical microscopy, scanning electron microscopy (SEM), and zeta potential analysis. We found that polyelectrolyte microgels provide low cytotoxicity and cell-material interactions (adhesion, spreading, and proliferation). In addition, the microsystem showed the ability to load lipoplexes and a loading efficiency equal to 83%, and it enabled in vitro surface-based transfection of MCF-7 cells. This approach provides a new suitable route for cell adhesion and local gene delivery.

Recent advances in co-delivery nanosystems for synergistic action in cancer treatment.

Nanocarrier delivery systems have been widely studied to carry unique or dual chemical drugs. The major challenge of chemotherapies is to overcome the multidrug-resistance (MDR) of cells to antineoplastic medicines. In this context, nano-scale technology has allowed researchers to develop biocompatible nano-delivery systems to overcome the limitation of chemical agents. The development of nano-vehicles may also be directed to co-deliver different agents such as drugs and genetic materials. The delivery of nucleic acids targeting specific cells is based on gene therapy principles to replace the defective gene, correct genome errors or knock-down a particular gene. Co-delivery systems are attractive strategies due to the possibility of achieving synergistic therapeutic effects, which are more effective in overcoming the MDR of cancer cells. These combined therapies can provide better outcomes than separate delivery approaches carrying either siRNA, miRNA, pDNA, or drugs. This article reviews the main design features that need to be associated with nano-vehicles to co-deliver drugs, genes, and gene-drug combinations with efficacy. The advantages and disadvantages of co-administration approaches are also overviewed and compared with individual nanocarrier systems. Herein, future trends and perspectives in designing novel nano-scale platforms to co-deliver therapeutic agents are also discussed.

Sequence length dependence in arginine/phenylalanine oligopeptides: Implications for self-assembly and cytotoxicity.

We present a detailed study on the self-assembly and cytotoxicity of arginine-rich fragments with general form [RF]n (n=1-5). These highly simplified sequences, containing only two l-amino acids, provide suitable models for exploring both structure and cytotoxicity features of arginine-based oligopeptides. The organization of the sequences is revealed over a range of length scales, from the nanometer range down to the level of molecular packing, and their cytotoxicity toward C6 rat glioma and RAW264.7 macrophage cell lines is investigated. We found that the polymorphism is dependent on peptide length, with a progressive increase in crystalline ordering upon increasing the number of [RF] pairs along the backbone. A dependence on length was also found for other observables, including critical aggregation concentrations, formation of chiral assemblies and half maximum inhibitory concentrations (IC50). Whereas shorter peptides self-assemble into fractal-like aggregates, clear fibrillogenic capabilities are identified for longer sequences with octameric and decameric chains exhibiting crystalline phases organized into cross-ß structures. Cell viability assays revealed dose-dependent cytotoxicity profiles with very similar behavior for both glioma and macrophage cell lines, which has been interpreted as evidence for a nonspecific mechanism involved in toxicity. We propose that structural organization of [RF]n peptides plays a paramount role regarding toxicity due to strong increase of local charge density induced by self-assemblies rich in cationic groups when interacting with cell membranes.

Preventive DNA vaccination against CEA-expressing tumors with anti-idiotypic scFv6.C4 DNA in CEA-expressing transgenic mice.

Carcinoembryonic antigen (CEA) is expressed during embryonic life and in low level during adult life. Consequently, the CEA is recognized by the immune system as a self-antigen and thus CEA-expressing tumors are tolerated. Previously, we constructed a single chain variable fragment using the 6.C4 (scFv6.C4) hybridoma cell line, which gave rise to antibodies able to recognize CEA when C57/Bl6 mice were immunized. Here, the scFv6.C4 ability to prevent the CEA-expressing tumor growth was assessed in CEA-expressing transgenic mice CEA2682. CEA2682 mice immunized with the scFv6.C4 expressing plasmid vector (uP/PS-scFv6.C4) by electroporation gave rise to the CEA-specific AB3 antibody after the third immunization. Sera from immunized mice reacted with CEA-expressing human colorectal cell lines CO112, HCT-8, and LISP-1, as well as with murine melanoma B16F10 cells expressing CEA (B16F10-CEA). Cytotoxic T lymphocytes (CTL) from uP/PS-scFv6.C4 immunized mice lysed B16F10-CEA (56.7%) and B16F10 expressing scFv6.C4 (B16F10-scFv6.C4) (46.7%) cells, against CTL from uP-immunized mice (10%). After the last immunization, 5 × 105 B16F10-CEA cells were injected into the left flank. All mice immunized with the uP empty vector died within 40 days, but uP/PS-scFv6.C4 vaccinated mice (40%) remained free of tumor for more than 100 days. Splenocytes obtained from uP/PS-scFv6.C4 vaccinated mice showed higher T-cell proliferative activity than those from uP vaccinated mice. Collectively, DNA vaccination with the uP-PS/scFv6.C4 plasmid vector was able to give rise to specific humoral and cellular responses, which were sufficient to retard growth and/or eliminate the injected B16F10-CEA cells.

Effects of oral N-acetylcysteine on walking capacity, leg reactive hyperemia, and inflammatory and angiogenic mediators in patients with intermittent claudication.

Increased oxidative stress and inflammation contribute to impaired walking capacity and endothelial dysfunction in patients with intermittent claudication (IC). The goal of the study was to determine the effects of oral treatment with the antioxidant N-acetylcysteine (NAC) on walking capacity, leg postocclusive reactive hyperemia, circulating levels of inflammatory mediators, and whole blood expression of angiogenic mediators in patients with IC. Following a double-blinded randomized crossover design, 10 patients with IC received NAC (1,800 mg/day for 4 days plus 2,700 mg before the experimental session) and placebo (PLA) before undergoing a graded treadmill exercise test. Leg postocclusive reactive hyperemia was assessed before and after the test. Blood samples were taken before and after NAC or PLA ingestions and 5 and 30 min after the exercise test for the analysis of circulating inflammatory and angiogenic markers. Although NAC increased the plasma ratio of reduced to oxidized glutathione, there were no differences between experimental sessions for walking tolerance and postocclusive reactive hyperemia. Plasma concentrations of soluble vascular cell adhesion protein-1, monocyte chemotactic protein-1, and endothelin-1 increased similarly following maximal exercise after PLA and NAC (P < 0.001). Whole blood expression of pro-angiogenic microRNA-126 increased after maximal exercise in the PLA session, but treatment with NAC prevented this response. Similarly, exercise-induced changes in whole blood expression of VEGF, endothelial nitric oxide synthase and phosphatidylinositol 3-kinase R2 were blunted after NAC. In conclusion, oral NAC does not increase walking tolerance or leg blood flow in patients with IC. In addition, oral NAC prevents maximal exercise-induced increase in the expression of circulating microRNA-126 and other angiogenic mediators in patients with IC.

Effects of N-acetylcysteine on skeletal muscle structure and function in a mouse model of peripheral arterial insufficiency.

OBJECTIVE: Abnormalities in skeletal muscle structure and function are important contributors to exercise intolerance and functional decline in peripheral arterial disease. In this study, we tested the hypothesis that administration of N-acetylcysteine (NAC) would improve fatigue resistance and ameliorate the histopathological changes in skeletal muscle in a mouse model of peripheral arterial disease. We also anticipated that NAC treatment would lower the levels of biomarkers of oxidative damage in the ischemic muscle. METHODS: Male Balb/c mice were subjected to bilateral ligation of the femoral artery and, after 2 weeks of recovery, received daily intraperitoneal injections of either NAC (150 mg/kg) or saline for 15 days. At the end of the treatment, the extensor digitorium longus (EDL) and soleus muscles were excised for assessment of contractile function in vitro and histological analysis. Free malondialdehyde and protein carbonyl levels were measured in the gastrocnemius muscle. RESULTS: In the soleus muscle, force after 10 minutes of submaximal tetanic stimulation (60 Hz, 300 ms trains, 0.3 trains/s) was higher (P < .05) in NAC-treated animals (45% ± 3% of the initial value; n = 7) when compared with controls (30.3% ± 3%; n = 8). No differences were found in fatigue development between groups in the EDL muscle (ligated NAC, 35.7% ± 1.9%; ligated saline, 37.5% ± 1.1%). In addition, there was a tendency for lower levels of connective tissue deposition in the soleus of animals treated with NAC (n = 6) when compared with those that received only saline (n = 9) (ligated NAC, 16% ± 2% vs ligated saline, 24% ± 2%; P = .057). No differences were found in lipid peroxidation or protein carbonyl levels between ligated saline and ligated NAC groups. CONCLUSIONS: Taken together, these results indicate that treatment with NAC improves fatigue resistance in the soleus but not the EDL muscle in a model of peripheral arterial insufficiency. CLINICAL RELEVANCE: Despite the increasing burden of peripheral arterial disease (PAD) and its detrimental consequences on the quality of life of the patients, few pharmacological therapies have shown to evoke meaningful effects on functional performance in these individuals. N-acetylcysteine is approved for clinical use, has minimal side effects and most important, has shown to consistently improve exercise performance in animals and humans. In this study, we showed, for the first time, that treatment with this drug at a dose amenable for clinical application evoked marked effects on fatigue resistance in the soleus muscle in a mouse model of PAD. These encouraging findings set the stage for translational studies to determine the acute and long-term impact of this drug on walking capacity in patients with PAD.

Therapeutic effects of the transplantation of VEGF overexpressing bone marrow mesenchymal stem cells in the hippocampus of murine model of Alzheimer's disease.

Alzheimer's disease (AD) is clinically characterized by progressive memory loss, behavioral and learning dysfunction and cognitive deficits, such as alterations in social interactions. The major pathological features of AD are the formation of senile plaques and neurofibrillary tangles together with neuronal and vascular damage. The double transgenic mouse model of AD (2xTg-AD) with the APPswe/PS1dE9 mutations shows characteristics that are similar to those observed in AD patients, including social memory impairment, senile plaque formation and vascular deficits. Mesenchymal stem cells (MSCs), when transplanted into the brain, produce positive effects by reducing amyloid-beta (Aß) deposition in transgenic amyloid precursor protein (APP)/presenilins1 (PS1) mice. Vascular endothelial growth factor (VEGF), exhibits neuroprotective effects against the excitotoxicity implicated in the AD neurodegeneration. The present study investigates the effects of MSCs overexpressing VEGF in hippocampal neovascularization, cognitive dysfunction and senile plaques present in 2xTg-AD transgenic mice. MSC were transfected with vascular endothelial growth factor cloned in uP vector under control of modified CMV promoter (uP-VEGF) vector, by electroporation and expanded at the 14th passage. 2xTg-AD animals at 6, 9 and 12 months old were transplanted with MSC-VEGF or MSC. The animals were tested for behavioral tasks to access locomotion, novelty exploration, learning and memory, and their brains were analyzed by immunohistochemistry (IHC) for vascularization and Aß plaques. MSC-VEGF treatment favored the neovascularization and diminished senile plaques in hippocampal specific layers. Consequently, the treatment was able to provide behavioral benefits and reduce cognitive deficits by recovering the innate interest to novelty and counteracting memory deficits present in these AD transgenic animals. Therefore, this study has important therapeutic implications for the vascular damage in the neurodegeneration promoted by AD.

Activation of the Janus kinase/signal transducer and activator of transcription pathway in multiple myeloma is not related to point mutations in kinase and pseudokinase domains of JAK1.

Abstract Considering the recent impact of tyrosine kinase inhibitors in the treatment of myeloproliferative disorders carrying a recurrent JAK2 mutation not identified in multiple myeloma (MM), this study aimed to search for mutations in kinase and pseudokinase domains of the JAK1 gene in an attempt to define any critical and recurring change that can be used as a therapeutic target. We obtained CD138 + purified cells from 27 bone marrow aspirates of untreated MM, four normal controls and four MM cell lines. After amplification of kinase and pseudokinase domains of JAK1 in cDNA samples, the fragments were automatically sequenced. Seventy-eight percent of MM cases showed at least one polymorphism, all being synonymous single nucleotide polymorphisms (SNPs), with allele frequencies consistent with previous studies in normal European, African American and Asian populations. The four cell lines also showed only synonymous SNPs. Mutations in the kinase and pseudokinase domains of the JAK1 gene do not seem to be important for activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway because we were not able to find any recurrent mutation in a case series of 27 patients and four MM cell lines.

High doses of vascular endothelial growth factor 165 safely, but transiently, improve myocardial perfusion in no-option ischemic disease.

UNLABELLED: Gene therapy can induce angiogenesis in ischemic tissues. The aim of this study was to assess safety, feasibility, and results, both clinical and on myocardial perfusion, of gene therapy in refractory angina. This was a phase I/II, prospective, temporal-controlled series, clinical trial. Thirteen patients were maintained for minimum 6 months under optimized clinical management, and then received intramyocardial injections of 2000 µg plasmid vascular endothelial growth factor 165 and were followed by single-photon emission computed tomography (SPECT), treadmill tests, Minnesota quality of life questionnaire (QOL), and New York Heart Association (NYHA) functional plus Canadian Cardiovascular Society (CCS) angina classifications. There were no deaths, early or late. During the optimized clinical treatment, we observed worsening of rest ischemia scores on SPECT (p<0.05). After treatment, there was a transitory increase in myocardial perfusion at the third-month SPECT under stress (pre-operative [pre-op] 18.38 ± 7.51 vs. 3 months 15.31 ± 7.30; p<0.01) and at the sixth month under rest (pre-op 13.23 ± 7.98 vs. 6 months: 16.92 ± 7.27; p<0.01). One year after, there were improvements in treadmill test steps (pre-op 2.46 ± 2.07 vs.12 months 4.15 ± 2.23; p<0.01) and oxygen consumption (pre-op 7.66 ± 4.47 vs.12 months 10.89 ± 4.65; p<0.05), QOL (pre-op 48.23 ± 18.35 vs.12 months 28.31 ± 18.14; p<0.01) scores, and CCS (pre-op 3 [3-3.5] vs.12 months 2 [1-2.5]; p<0.01) and NYHA (pre-op 3 [3-3] vs. 2 [2-2] vs. 12 months 2 [1-2]; p<0.01) classes. Gene therapy demonstrated to be feasible and safe in this advanced ischemic cardiomyopathy patient sample. There were improvements in clinical evaluation parameters, and a transitory increase in myocardial perfusion detectable by SPECT scintigraphy. CLINICAL TRIAL REGISTRATION: NCT00744315 http://clinicaltrials.gov/

Feasibility of robotic laparoendoscopic single-site partial nephrectomy for renal tumors >4 cm.

BACKGROUND: Laparoendoscopic single-site (LESS) urologic procedures have gained significant interest worldwide in an attempt to further reduce morbidity and minimize scarring associated with conventional laparoscopic surgery. The robotic technology has overcome some of the limitations of manual single-incision surgery relating to lack of triangulation, instrument collision, and surgical exposure. There are no data on robotic LESS partial nephrectomy (PN) for renal tumors >4 cm. OBJECTIVES: To evaluate the feasibility of robotic LESS PN for renal tumors >4 cm. DESIGN, SETTING, AND PARTICIPANTS: Data from 67 consecutive patients who underwent robotic LESS PN were collected between May 2009 to January 2011. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Patients were stratified into two groups: 20 patients with renal tumors >4 cm (group 1) and 47 patients with renal tumors ≤ 4 cm (group 2). Perioperative data were recorded and comparisons between the two groups were analyzed using the Mann-Whitney U test for continuous variables and Fisher exact test for categorical variables. RESULTS AND LIMITATIONS: No statistically significant differences were found between the two groups in demographic information, operative complications, pathologic characteristics, mean decline in estimated glomerular filtration rate, estimated blood loss, operative times, conversion rate, or positive surgical margins. However, group 1 had a higher mean nephrometry score (p<0.01), longer warm ischemia time (p = 0.007), and longer length of stay (p = 0.046). Its retrospective design and being conducted at a single center were the main limitations of this study. CONCLUSIONS: This study demonstrated the feasibility and safety of robotic LESS PN for tumors >4 cm. Patients with tumors >4 cm had a statistically significant, higher mean nephrometry score, longer warm ischemia time, and longer length of stay, but there was no increased risk of adverse outcomes. A long-term study is needed to confirm the durable renal preservation and oncologic outcomes for patients with larger tumor burden.

Psychological and behavioral effects of chloral hydrate in day-case pediatric surgery: a randomized, observer-blinded study.

BACKGROUND/PURPOSE: This prospective, randomized, and observer-blinded study was performed to evaluate the effects of oral chloral hydrate on perioperative psychological and behavioral phenomena in children. METHODS: In total, 100 boys (age, 1-5 years) scheduled for day-case unilateral orchiopexy were randomly allocated into 2 groups and orally administered either 40 mg/kg of chloral hydrate (CH group) or placebo (control group) 30 minutes before surgery, followed by assessment of anxiety, induction compliance, emergence delirium, postoperative pain, and maladaptive behavioral changes. RESULTS: Anxiety scores were significantly lower in the CH group compared with the control group (45.7 vs 28.8). The induction compliance of the CH group was better than that of the control group (3.2 vs 4.8). Postoperative sedation was more frequent (62.7% vs 20.4%); however, the incidence of vomiting was lower (2.0% vs 14.3%) in the CH group than in the control group. Postoperative emergence delirium and maladaptive behavior changes were similar between the 2 groups. CONCLUSION: Decreasing preoperative anxiety with oral chloral hydrate improves induction compliance and reduces postoperative pain intensity without delaying recovery in young boys. However, chloral hydrate had little impact on emergence delirium and postoperative maladaptive behavior.

The idiotype (Id) cascade in mice elicited the production of anti-R24 Id and anti-anti-Id monoclonal antibodies with antitumor and protective activity against human melanoma.

Gangliosides have been considered as potential targets for immunotherapy because they are overexpressed on the surface of melanoma cells. However, immunization with purified gangliosides results in a very poor immune response, usually mediated by IgM antibodies. To overcome this limitation, we immunized mice with R24, a monoclonal antibody (mAb) that recognizes the most tumor-restricted ganglioside (GD3); our goal was to obtain anti-idiotype (Id) antibodies bearing the internal image of GD3. Animals produced anti-Id and anti-anti-Id antibodies. Both anti-Id and anti-anti-Id antibodies were able to inhibit mAb R24 binding to GD3. In addition, the anti-anti-Id antibodies were shown to recognize GD3 directly. Anti-Id and anti-anti-Id mAb were then selected from two fusion experiments for evaluation. The most interesting finding emerged from the characterization of the anti-anti-Id mAb 5.G8. It was shown to recognize two different GD3-expressing human melanoma cell lines in vitro and to mediate tumor cell cytotoxicity by complement activation and antibody-dependent cellular cytotoxicity. The biological activity of the anti-anti-Id mAb was also tested in a mouse tumor model, in which it was shown to be a powerful growth inhibitor of melanoma cells. Thus, activity of the anti-anti-Id mAb 5.G8 matched that of the prototypic anti-GD3 mAb R24 both in vitro and in vivo. Altogether, our results indicate that the idiotype approach might produce high affinity, specific and very efficient antitumor immune responses.

Effect of gene therapy with vascular endothelial growth factor after abdominoplasty on TRAM flap viability in a rat model.

BACKGROUND: The transverse rectus abdominis musculocutaneous (TRAM) flap may develop necrosis, especially in patients with risk factors such as previous abdominoplasty, caused by damage to perforating vessels during surgical procedures. This study was designed from the perspective of using vascular endothelial growth factor (VEGF) gene therapy with plasmid vector after abdominoplasty to stimulate neovascularization of the TRAM flap, thus increasing flap viability. METHODS: Thirty-two Wistar rats were divided into four groups (n = 8). A right inferiorly based TRAM flap was constructed in all animals and was the only procedure performed in group I (TRAM flap). Animals from groups II (abdominoplasty) and III (plasmid) underwent abdominoplasty and were injected intramuscularly with physiologic saline solution and empty plasmid, respectively. Group IV (VEGF) received intramuscular injection of naked plasmid DNA encoding VEGF-165 during abdominoplasty. The TRAM flap was created 30 days after abdominoplasty. RESULTS: The mean necrosis was 24.65 +/- 18.13 percent in group I, 62.49 +/- 28.06 percent in group II, 57.80 +/- 25.43 percent in group III, and 18.33 +/- 16.20 percent in group IV. The number of vessels in the TRAM flap was determined by immunohistochemistry using the antibody human heart factor. Groups I and IV had a similar number of vessels, as did groups II and III. Groups I and IV had greater viability and number of vessels than groups II and III. CONCLUSION: VEGF gene therapy increased viability and vessel number in the TRAM flap created after abdominoplasty in a rat model.

Electroporation of vascular endothelial growth factor gene in a unipedicle transverse rectus abdominis myocutaneous flap reduces necrosis.

Necrosis in TRAM (transverse rectus abdominis myocutaneous) still occurs in flap breast reconstruction. Blood flow may be improved by vascular endothelial growth factor (VEGF), an endogenous protein that stimulates neovascularization. Experimental studies of gene therapy with plasmid vector expressing human VEGF (hVEGF) presented inadequate results. Low level of gene expression could be the cause. To prove that high level of VEGF gene expression can minimize necrosis of TRAM flap, electroporation of VEGF plasmid was tested.Forty-two adult, male, Wistar-EPM rats were randomly distributed in 6 groups of 7 animals and 50 microg of vectors were injected in the intradermal layer of TRAM flap donor region, by electroporation: LacZ (beta-galactosidase gene); CG (no substance injected and flap elevated); P2G (empty gT plasmid in area 2); PV2G (gT-VEGF165 in area 2); P4G (empty gT plasmid in area 4); PV4G (gT-VEGF165 in area 4). Five days after flap elevation, the animals were euthanized and the degree of necrosis was analyzed by histology and paper template method.Dermal X-gal staining after electroporation with pSV2lacZ proved high rate of gene transfer. Mean values of necrosis by the paper template method were: CG (74.5%), P2G (62.2%), PV2G (41.1%), P4G (76.6%), and PV4G (59%). Degree of necrosis, preservation of muscle layer, and degree of infiltrates seen by histology were in accordance with mean values of necrosis.Intradermal injection of gT-VEGF165 in area 2, by electroporation, was effective in reducing unipedicle TRAM flap necrosis, in rats.

Tissue inhibitor of metalloproteinase 1 expression associated with gene demethylation confers anoikis resistance in early phases of melanocyte malignant transformation.

Although anoikis resistance has been considered a hallmark of malignant phenotype, the causal relation between neoplastic transformation and anchorage-independent growth remains undefined. We developed an experimental model of murine melanocyte malignant transformation, where a melanocyte lineage (melan-a) was submitted to sequential cycles of anchorage blockade, resulting in progressive morphologic alterations, and malignant transformation. Throughout this process, cells corresponding to premalignant melanocytes and melanoma cell lines were established and show progressive anoikis resistance and increased expression of Timp1. In melan-a melanocytes, Timp1 expression is suppressed by DNA methylation as indicated by its reexpression after 5-aza-2'-deoxycytidine treatment. Methylation-sensitive single-nucleotide primer extension analysis showed increased demethylation in Timp1 in parallel with its expression along malignant transformation. Interestingly, TIMP1 expression has already been related with negative prognosis in some human cancers. Although described as a MMP inhibitor, this protein has been associated with apoptosis resistance in different cell types. Melan-a cells overexpressing Timp1 showed increased survival in suspension but were unable to form tumors in vivo, whereas Timp1-overexpressing melanoma cells showed reduced latency time for tumor appearance and increased metastatic potential. Here, we demonstrated for the first time an increment in Timp1 expression since the early phases of melanocyte malignant transformation, associated to a progressive gene demethylation, which confers anoikis resistance. In this way, Timp1 might be considered as a valued marker for melanocyte malignant transformation.

A comparison of high volume/low concentration and low volume/high concentration ropivacaine in caudal analgesia for pediatric orchiopexy.

BACKGROUND: It is unclear whether the volume or concentration of local anesthetic influences its spread and quality of caudal analgesia when the total drug dose is fixed. METHODS: We performed this study in a prospective, randomized, observer-blind manner. Children aged 1-5 yr received a constant dose of 2.25 mg/kg of ropivacaine prepared as either 1.0 mL/kg of 0.225% (low volume/high concentration [LVHC], n = 37) or 1.5 mL/kg of 0.15% solution (high volume/low concentration [HVLC], n = 36). Both solutions contained radiopaque dye. RESULTS: The median spread levels with ranges in the HVLC group (confirmed by fluoroscopic examination) were significantly higher (T6, T3-11) than in the LVHC group (T11, T8-L2). There were no significant differences in recovery times, postoperative pain scores, or side effects between the two groups. After discharge, fewer children in the HVLC group required rescue oral acetaminophen compared with the LVHC group (50.0% vs 75.7%). First oral acetaminophen time was found to be significantly longer with HVLC patients than LVHC patients (363.0 min vs 554.5 min). CONCLUSIONS: We confirmed (with fluoroscopy) that a caudal block with 1 mL/kg ropivacaine spreads to T11 and to T6 with 1.5 mL/kg. If the total dose is fixed, caudal analgesia with a larger volume of diluted ropivacaine (0.15%) provides better quality and longer duration after discharge than a smaller volume of more concentrated ropivacaine (0.225%) in children undergoing day-case orchiopexy. The spread level of ropivacaine correlated significantly with the first oral acetaminophen time after discharge.

Autonomous growth of BALB/MK keratinocytes transfected with a retroviral vector carrying the human epidermal growth factor gene

Epidermal growth factor (EGF), which promotes epidermal regeneration and wound closure, is important for the proliferation and differentiation of epidermal and epithelial tissues in animals. Exogenous EGF is a promising therapeutic agent for wound healing, but its general use is restricted by the limited availability of this protein. In this work, we show that the transfection of mouse BALB/MK keratinocytes, which are totally dependent on EGF for growth and migration, with mature cDNA for human EGF via a retroviral vector abolished the cells requirement for exogenous EGF. The transformed cells had normal morphology and a growth rate that varied according to the source of the retroviral vector used. Keratinocyte transfection with EGF cDNA provides a time- and cost-efficient means of culturing keratinocytes and yields cells that may be useful for skin grafting.

Prepuncture ultrasound-measured distance: an accurate reflection of epidural depth in infants and small children.

BACKGROUND AND OBJECTIVES: Epidural cannulation is technically difficult in children who have small anatomic structures. Ultrasound information regarding the distance of skin-to-ligament flavum may be useful, leading to an increase in success rate without dural puncture. This study was performed to assess whether ultrasound-measured, skin-to-ligament flavum distance would reflect the needle depth during epidural puncture in infants and children. METHODS: The study compromised 180 children, aged 2 to 84 months, undergoing urologic surgery. After induction of anesthesia, ultrasound images of the longitudinal median and transverse views were acquired from L4-L5 in lateral decubitus position. Measured distance of skin-to-ligament flavum in each view was compared with the perpendicular skin-to-epidural depth, which was obtained from needle depth and angle by use of a trigonometric ratio equation. Additionally, we evaluated the ultrasound visibility of the ligament flavum and dura mater, number of puncture attempts, and complications. RESULTS: The correlation coefficient between measured distance and perpendicular epidural depth was slightly higher in longitudinal median view (R2 = 0.848) than in transverse view (R2 = 0.788). The visibility of ligament flavum and dura mater was "good" in 91 and 170 of 180 patients, respectively, and "sufficient" in the remaining subjects. The epidural space was located on first puncture attempt in 179 of 180 cases (99.4%). No incidents of dural puncture or bloody tap occurred. CONCLUSIONS: Ultrasound, particularly in the longitudinal median view, provides accurate information on the distance of skin-to-ligament flavum in infants and children. With reference to the measured distance, epidural puncture can be performed with minimal risk of dural puncture (upper limit of 95% CI = 1.67%).

Angiotensin II AT1 receptor mutants expressed in CHO cells caused morphological change and inhibition of cell growth.

To assess the importance of the leucine residues in positions 262 and 265 of the angiotensin AT(1) receptor for signaling pathways and receptor expression and regulation, we compared the properties of CHO cells transfected with the wild type or the L262D or L265D receptor point mutants. It was found that the two mutants significantly increased the basal intracellular cyclic AMP (cAMP) formation in an agonist-independent mode. The morphology transformation of CHO cells was correlated with the increased cAMP formation, since forskolin, a direct activator of adenylate cyclase mimicked this effect on WT-expressing CHO cells. DNA synthesis was found to be inhibited in these cell lines, indicating that cAMP may also have determined the inhibitory effect on cell growth, in addition to the cell transformation from a tumorigenic to a non-tumorigenic phenotype. However a role for an increased Ca2+ influx induced by the mutants in non-stimulated cells cannot be ruled out since this ion also was shown to cause transformed cells to regain the morphology and growth regulation.