Flavonoid-Labeled Biopolymer in the Structure of Lipid Membranes to Improve the Applicability of Antioxidant Nanovesicles.

Nanovesicles produced with lipids and polymers are promising devices for drug and bioactive delivery and are of great interest in pharmaceutical applications. These nanovesicles can be engineered for improvement in bioavailability, patient compliance or to provide modified release or enhanced delivery. However, their applicability strongly depends on the safety and low immunogenicity of the components. Despite this, the use of unsaturated lipids in nanovesicles, which degrade following oxidation processes during storage and especially during the proper routes of administration in the human body, may yield toxic degradation products. In this study, we used a biopolymer (chitosan) labeled with flavonoid (catechin) as a component over a lipid bilayer for micro- and nanovesicles and characterized the structure of these vesicles in oxidation media. The purpose of this was to evaluate the in situ effect of the antioxidant in three different vesicular systems of medium, low and high membrane curvature. Liposomes and giant vesicles were produced with the phospholipids DOPC and POPC, and crystalline cubic phase with monoolein/DOPC. Concentrations of chitosan-catechin (CHCa) were included in all the vesicles and they were challenged in oxidant media. The cytotoxicity analysis using the MTT assay (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) revealed that concentrations of CHCa below 6.67 µM are non-toxic to HeLa cells. The size and zeta potential of the liposomes evidenced the degradation of their structures, which was minimized by CHCa. Similarly, the membrane of the giant vesicle, which rapidly deteriorated in oxidative solution, was protected in the presence of CHCa. The production of a lipid/CHCa composite cubic phase revealed a specific cubic topology in small-angle X-ray scattering, which was preserved in strong oxidative media. This study demonstrates the specific physicochemical characteristics introduced in the vesicular systems related to the antioxidant CHCa biopolymer, representing a platform for the improvement of composite nanovesicle applicability.

Advanced Microfluidic Technologies for Lipid Nano-Microsystems from Synthesis to Biological Application.

Microfluidics is an emerging technology that can be employed as a powerful tool for designing lipid nano-microsized structures for biological applications. Those lipid structures can be used as carrying vehicles for a wide range of drugs and genetic materials. Microfluidic technology also allows the design of sustainable processes with less financial demand, while it can be scaled up using parallelization to increase production. From this perspective, this article reviews the recent advances in the synthesis of lipid-based nanostructures through microfluidics (liposomes, lipoplexes, lipid nanoparticles, core-shell nanoparticles, and biomimetic nanovesicles). Besides that, this review describes the recent microfluidic approaches to produce lipid micro-sized structures as giant unilamellar vesicles. New strategies are also described for the controlled release of the lipid payloads using microgels and droplet-based microfluidics. To address the importance of microfluidics for lipid-nanoparticle screening, an overview of how microfluidic systems can be used to mimic the cellular environment is also presented. Future trends and perspectives in designing novel nano and micro scales are also discussed herein.

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.

Hybrid polymer/lipid vesicle synthesis: Association between cationic liposomes and lipoplexes with chondroitin sulfate.

The association of cationic carriers with different anionic mucoadhesive biopolymers has been widely explored as an alternative to improve their delivery routes and specific targeting. This work presents a complete analysis of the association between chondroitin sulfate (CS) and cationic liposomes (CLs)/lipoplex (CL-pDNA). In this study, plasmid DNA (pDNA) was used as a genetic cargo for association with carriers. Firstly, we measured the stoichiometry of pseudo complexes and evaluated their colloidal properties, structural and morphological characteristics. Optimized CL-pDNA lipoplexes (positive z-potential) and CL-CS / CL-pDNA-CS (negative z-potential with CS mass ratio of 9% (w/w)) were further studied in detail. Small-angle X-ray scattering analysis and cryo-transmission electron microscopy micrographs revealed that the electrostatic interaction between CS and CL / CL-pDNA easily reorganized the lipid bilayers resulting in nanoscale uni/multilamellar vesicles. A high CS mass ratio (9% (w/w)) led to the reassembly of liposomal structure, wherein the pDNA was easily exchanged for CS chains, forming more than 50% of dense multilamellar vesicles. This data evidenced that the association between CS and CLs is not a conventional coating process since it generates complex and hybrid structures. We believe that these obtained colloidal data may be used in the future to investigate polymer-tailored nanocarriers and their production process. In brief, the colloidal study of hybrid structures may open interesting perspectives for developing novel carriers for drug and gene delivery applications.

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.

Amyloid-like Self-Assembly of a Hydrophobic Cell-Penetrating Peptide and Its Use as a Carrier for Nucleic Acids.

Cell-penetrating peptides (CPPs) are a topical subject potentially exploitable for creating nanotherapeutics for the delivery of bioactive loads. These compounds are often classified into three major categories according to their physicochemical characteristics: cationic, amphiphilic, and hydrophobic. Among them, the group of hydrophobic CPPs has received increasing attention in recent years due to toxicity concerns posed by highly cationic CPPs. The hexapeptide PFVYLI (P, proline; F, phenylalanine; V, valine; Y, tyrosine; L, leucine; and I, isoleucine), a fragment derived from the C-terminal portion of α1-antitrypsin, is a prototypal example of hydrophobic CPP. This sequence shows reduced cytotoxicity and a capacity of nuclear localization, and its small size readily hints at its suitability as a building block to construct nanostructured materials. In this study, we examine the self-assembling properties of PFVYLI and investigate its ability to form noncovalent complexes with nucleic acids. By using a combination of biophysical tools including synchrotron small-angle X-ray scattering and atomic force microscopy-based infrared spectroscopy, we discovered that this CPP self-assembles into discrete nanofibrils with remarkable amyloidogenic features. Over the course of days, these fibrils coalesce into rodlike crystals that easily reach the micrometer range. Despite lacking cationic residues in the composition, PFVYLI forms noncovalent complexes with nucleic acids that retain ß-sheet pairing found in amyloid aggregates. In vitro vectorization experiments performed with double-stranded DNA fragments indicate that complexes promote the internalization of nucleic acids, revealing that tropism toward cell membranes is preserved upon complexation. On the other hand, transfection assays with splice-correction oligonucleotides (SCOs) for luciferase expression show limited bioactivity across a narrow concentration window, suggesting that the propensity to form amyloidogenic aggregates may trigger endosomal entrapment. We anticipate that the findings presented here open perspectives for using this archetypical hydrophobic CPP in the fabrication of nanostructured scaffolds, which potentially integrate properties of amyloids and translocation capabilities of CPPs.

Arginine-modified chitosan complexed with liposome systems for plasmid DNA delivery.

In order to make more efficient chitosan-based nanoparticles for transfection in physiological condition, chitosomes composed of chitosan modified with arginine and complexed with DOTAP/DOPE lipids are synthesized (named chitosomes) by reverse phase evaporation technique. Structure analyses of chitosomes with or without plasmid DNA (pDNA) are performed by electrophoresis, zeta potential, dynamic light scattering, small angle X-ray scattering and isothermal titration calorimetry, and transfection efficiency and cytotoxicity are performed in HEK293 T cells. Chitosomes have a positive surface charge (X¯= 52 mV) with an average size of 116 nm, and interaction with pDNA are favored thermodynamically and do not suffer aggregation significantly. In our experimental conditions, the transfection efficiency average reaches 86% ±â€¯3, while the Lipofectamine® reaches 87% ±â€¯5 in vitro. Cytotoxicity of chitosomes are tolerable. Structural analyses show that that chitosomes-pDNA complexes appear to have multilamellar vesicle structures hosting pDNA in-between bilayers which favor interaction with cell membrane and delivery of pDNA. Results show that synthesized chitosomes are promising carriers for gene delivery.

Self-assembly and intracellular delivery of DNA by a truncated fragment derived from the Trojan peptide Penetratin.

Penetratin is a short Trojan peptide that attracts great interest in biomedical research for its capacity to translocate biological membranes. Herein, we study in detail both self-assembly and intracellular delivery of DNA by the heptamer KIWFQNR, a truncated peptide derived from Penetratin. This shortened sequence possesses a unique design with bolaamphiphilic characteristics that preserves the longest noncationic amino acid portion found in Penetratin. These features convey amphipathicity to assist self-assembly and make it a suitable model for exploring the role of hydrophobic residues for peptide interaction and cell uptake. We show that the fragment forms peptiplexes (i.e., peptide-DNA complexes), and aggregates into long nanofibers with clear ß-sheet signature. The supramolecular structure of nanofibers is likely composed of DNA cores surrounded by a peptide shell to which the double helix behaves as a template and induces fibrillization. A nucleation and growth mechanism proceeding through liquid-liquid phase separation of coacervates is proposed for describing the self-assembly of peptiplexes. We also demonstrate that peptiplexes deliver double-stranded 200 bp DNA into HeLa cells, indicating its potential for preparing non-viral vectors for oligonucleotides through noncovalent strategies. Since the main structural features of native Penetratin are conserved in this simpler fragment, our findings also highlight the role of uncharged amino acids for structuration, and thus for the ability of Penetratin to cross 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.

Injectable alginate hydrogel for enhanced spatiotemporal control of lentivector delivery in murine skeletal muscle.

Hydrogels are an especially appealing class of biomaterials for gene delivery vehicles as they can be introduced into the body with minimally invasive procedures and are often applied in tissue engineering and regenerative medicine strategies. In this study, we show for the first time the use of an injectable alginate hydrogel for controlled delivery of lentivectors in the skeletal muscle of murine hindlimb. We propose to alter the release rates of lentivectors through manipulation of the molecular weight distribution of alginate hydrogels. The release of lentivector was tested using two different ratios of low and high molecular weight (MW) alginate polymers (75/25 and 25/75 low/high MW). The interdependency of lentivector release rate and alginate degradation rate was assessed in vitro. Lentivector-loaded hydrogels maintained transduction potential for up to one week in vitro as demonstrated by the continual transduction of HEK-293T cells. Injection of lentivector-loaded hydrogel in vivo led to a sustained level of transgene expression for more than two months while minimizing the copies of lentivirus genome inserted into the genome of murine skeletal muscle cells. This strategy of spatiotemporal control of lentivector delivery from alginate hydrogels may provide a versatile tool to combine gene therapy and biomaterials for applications in regenerative medicine.

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.

NAADP-sensitive two-pore channels are present and functional in gastric smooth muscle cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as an important modulator of Ca(2+) release from the endo-lysosomal system in a variety of cells by a new and ubiquitous class of endo-lysosomal ion channels known as the two-pore channels (TPCs). However, the role of TPCs in NAADP action in smooth muscle is not known. In the present work, we investigated the effects of NAADP in gastric smooth muscle cells and its ability to release Ca(2+) by TPCs. We show that Ca(2+) signals mediated by NAADP were inhibited by disrupting Ca(2+) handling by either acidic organelles (using bafilomycin A1) or the Endoplasmic Reticulum (using thapsigargin, ryanodine or 2-APB). Transcripts for endogenous TPC1 and TPC2 were readily detected and recombinant TPCs localized to the endosomes and/or lysosomes. Overexpression of wild-type TPCs but not pore mutants enhanced NAADP-mediated cytosolic Ca(2+) signals. Desensitizing the NAADP pathway inhibited Ca(2+)-responses to extracellular stimulation with carbachol but not ATP. Taken together, these results indicate that NAADP likely induces Ca(2+) release from the endolysosomal system through TPCs which is subsequently amplified via the ER in an agonist-specific manner. Thus, we suggest a second messenger role for NAADP in smooth muscle cells.

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.

Overuse of training increases mechanoreceptors in supraspinatus tendon of rats SHR.

The presence of mechanoreceptors in tendon after overuse activities can be a further step to learn about tendinopathy and overuse. Studies of tendons mechanoreceptors in rats are rare. We studied 12 isogenic spontaneous hypertensive rats (SHR), which underwent an overuse protocol consisting of an hour per daily session of treadmill running at a speed of 17 m/min, 5 times/week for 4 months. Supraspinatus tendons were evaluated with immunohistochemistry using S100 protein antibodies and histological protocol. Supraspinatus tendons at the end of 4 months of overuse protocol had a high number of media mechanoreceptors (4.3) than controls (0.6). The overexpression of S100 protein antibody in overuse activities maybe could represent a adaptative effort to tendon before the tear.

Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates autophagy in cultured astrocytes.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca(2+)-mobilizing messenger that in many cells releases Ca(2+) from the endolysosomal system. Recent studies have shown that NAADP-induced Ca(2+) mobilization is mediated by the two-pore channels (TPCs). Whether NAADP acts as a messenger in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that intracellular delivery of NAADP evokes Ca(2+) signals from acidic organelles in rat astrocytes and that these signals are potentiated upon overexpression of TPCs. We also show that NAADP increases acidic vesicular organelle formation and levels of the autophagic markers, LC3II and beclin-1. NAADP-mediated increases in LC3II levels were reduced in cells expressing a dominant-negative TPC2 construct. Our data provide evidence that NAADP-evoked Ca(2+) signals mediated by TPCs regulate autophagy.

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.

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.