Silvia Neves

Cationic Liposomes for Gene Delivery: From Biophysics to Biological Applications

The use of an efficient carrier for nucleic acid-based medicines is considered to be a determinant factor for the successful application of gene therapy. The drawbacks associated with the use of viral vectors, namely those related with safety problems, have prompted investigators to develop alternative methods for gene delivery, cationic lipid-based systems being the most representative. Despite extensive research in the last decade on the use of cationic liposomes as gene transfer vectors and the development of elegant strategies to enhance their biological activity, these systems are still far from being viable alternatives to the use of viral vectors in gene therapy. In this review considerations are made regarding the structure-activity relationships of cationic liposome/DNA complexes and the key formulation parameters influencing the features of lipoplexes are presented and discussed in terms of their effect on biological activity. Particular emphasis is given to the interaction of the lipoplexes with serum components as well as to novel strategies developed to circumvent difficulties that may emerge upon iv administration of the complexes. Finally, since the ability of the lipoplexes to be stored while preserving their transfection activity is a crucial issue for the repeated use of such carriers, approaches reported on the improvement of their physical stability are also reviewed.

Transferrin lipoplex-mediated suicide gene therapy of oral squamous cell carcinoma in an immunocompetent murine model and mechanisms involved in the antitumoral response.

Suicide gene therapy has been used for the treatment of a variety of cancers. We reported previously the in vitro efficacy of the Herpes Simplex Virus Thymidine kinase (HSV-tk)/ganciclovir (GCV) system to mediate cytotoxicity in oral squamous cancer cells, using transferrin (Tf)-lipoplexes, prepared from cationic liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and cholesterol. In the present study, we evaluated the antitumoral efficacy mediated by this lipoplex formulation in two suicide gene therapy strategies, HSV-tk/GCV and cytosine deaminase (CD)/5-fluorocytosine (5-FC), using a syngeneic, orthotopic murine model for head and neck squamous cell carcinoma. The cellular and molecular events associated with the antitumoral response elicited by both the therapeutic approaches were investigated by analyzing tumor cell death, tumor-infiltrating immune cells and tumor cytokine microenvironment. Significant tumor reduction was achieved upon intratumoral delivery of HSV-tk or CD genes mediated by Tf-lipoplexes, followed by intraperitoneal injection of GCV or 5-FC, respectively. Enhanced apoptosis, the recruitment of NK cells, CD4 and CD8 T-lymphocytes and an increase in the levels of several cytokines/chemokines were observed within the tumors. These observations suggest that suicide gene therapy with lipoplexes modifies the tumor microenvironment, and leads to the recruitment of immune effector cells that can act as adjuvants in reducing the tumor size.

Cellular and molecular events associated with the antitumor response induced by the cytosine deaminase/5-fluorocytosine suicide gene therapy system in a rat liver metastasis model.

The bacterial cytosine deaminase (CD) gene converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil. We have previously shown, in a rat liver metastasis model from colon carcinoma, that intratumoral injection of a CD-expressing plasmid into the animals followed by 5-FC treatment results in the regression of the treated tumor as well as distant uninjected tumors. The aim of this study was to further analyze the mechanisms associated with tumor regression induced upon application of suicide CD/5-FC strategy. Tumor regression was associated with an increased apoptosis, the recruitment of natural killer cells, CD4- and CD8 T lymphocytes within the tumors and an increased expression of several cytokines/chemokines mRNAs. These data indicate that the CD/5-FC suicide strategy is associated with the triggering of cellular and molecular events leading to an efficient antitumor immune response involving both innate and acquired immunity.

Requirements of circulatory support during liver transplantation : Are patients with familial amyloidosis different from other patients?

AMILIAL amyloidotic polyneuropathy (FAP) is a genetic dominant disease with a meaningful prevalence in Portugal, Sweden, and Japan. A large number of biochemistry anomalies could underlie this disease but by far the most frequent is the transthyretin methionine 30 anomaly (FAP ATTR Met 30) 1 whose symptoms start in adult age and death ensues commonly 10 to 15 years after the initial complaints. Liver transplantation is the only therapy that has proved to stop this disease. As opposed to transplant recipients with liver diseases, FAP patients present without preoperative coagulation disorders and portal hypertension, and the transplant tends to be technically easier, with less blood loss and shorter operating time. 2,3 Nevertheless the circulatory involvement is always present in the natural course of this disease with autonomic dysfunction being a characteristic finding and cardiac arrhythmias and diastolic dysfunction being frequently observed. 4 A high incidence of severe hypotension is reported during liver transplant in FAP 2,5 and rhythm and intracardiac conduction anomalies have also been reported as a troublesome problem during FAP anesthesia. 6 The aim of this study was to assess the intraoperative interventions on the circulatory system and to correlate them with the findings of preoperative routine examinations and intra-operative events. PATIENTS Group I included all 90 FAP ATTR Met 30 recipients of first liver transplant during a 7-year period, 50 male and 40 female, with age of 35.6 6 7.2 years, weight of 56.4 6 11.9 kg, body mass index (BMI) of 20.8 6 4.1 kg/m 2 , disease duration (since the first symptom) of 4.8 6 2.5 years, and a neurologic score of 33.9 6 12.6 in the scale of Macedo and col. Group II included all the 125 adults (79 male, 46 female) with other diseases that received a first liver transplant during the same period. Groups were not different with regard to height and sex. Group II was older (42.3 6 12.8 years, P , .001), and had a greater weight (65.7 6 12.0 kg, P , .001) and BMI (23.8 6 4.3 kg/m 2 , P , .001). Hematologic and liver function tests were normal in group I. Group II presented lower hemoglobin (P , .001), fibrinogen (P , .001), and albumin (P , .001) and higher prothrombin time (P , .001), direct (P , .001) and total bilirubin (P , .001), AST (P , .001), ALT (P , .001), and creatinine (P , .001). The transplant was done by classic technique in 27 patients (30.0%) of group I and 39 (31.2%) of group II (P 5 ns) and by piggy-back technique in the remaining. Seventeen patients (18.9%) in group I and none in group II had a permanent pacemaker. METHOD Anaesthesia, monitoring, and circulatory support protocols were identical in the two groups and did not change during the years of the study. Low-dose dopamine (3 mg/kg/min or less) was used in all patients. Higher doses of dopamine were used as a first line circulatory support. Dobutamine was used to treat low cardiac output with adequate filling pressures and phenylephrine to treat vasodilatation. Antiarrhythmic agents or vasodilators were used as standard in general practice. Emergency transcutaneous pacing was always available. Group I values were compared with group II values by Mann-Whitney U test or chi-square test followed by Fisher test, as appropriate. Within each group, if one drug was used in a number of patients that allowed subsequent statistics, patients that used it and patients that did not use it were compared with the same statistical tests. Data as mean 6 standard deviation. RESULTS Venovenous bypass was used in 31.1% of group I and 33.6% of group II (P 5 ns). Group I patients needed 5.2 6 9.4 units of red blood cells (RBC), 12.7 6 16.6 units of fresh frozen plasma (FFP), and 3.4 6 8.9 units of platelets. Blood products consumption was significantly higher in group II: 13.1 6 12.7 units of RBC (P , .001), 26.4 6 21.4 units of FFP (P , .001), and 9.0 6 8.6 units of platelets (P , .001). Except for low-dose dopamine, at least one sympathomimetic drug was used in 75.6% of group I and 38.4% of group II patients (P , .001). Dopamine (5 mg/kg/min or more), dobutamine, phenylephrine, and epinephrine were respectively used in 43.3%, 2.2%, 48.9%, and 7.8% of group I patients. The respective values in group II patients were 27.2% (P , .05), 12.8% (P 5 ns), 3.2% (P , .001), and 4.0% (P 5 ns). Lidocaine was used in 7.8% of group I and 3.2% of group II patients (P 5 ns). Norepinephrine, digoxin, verapamil, amiodarone, sodium nitroprusside, or nitroglycerine were used in a maximum of one patient in each group (P 5 ns). Emergency pacing was never used. Within group II, the patients that needed inotropic drugs had lower albumin (P , .05) and higher creatinine

Local immunosuppression in clinical small bowel transplantation (report of two cases).

LOCAL IMMUNOSUPPRESSION (L-IMS) has been used for many years, particularly in animal experimental organ transplantation. It has also been used in human kidney transplantation. The rationale for this practice is that the most important immunologic events leading to rejection occur in the graft. For instance, it has been demonstrated that local expansion of donor-specific T cells may mediate graft rejection after the first posttransplant days, without the participation of the circulating pool of lymphocytes. Thus, it should be expected that local intraarterial delivery of the immunosuppressive agents reaches higher levels of efficacy for the same doses or even for lower doses than when they are systemically administered. To our knowledge, despite the encouraging results of many experimental studies, no clinical use of L-IMS has yet been carried out in human organ transplantation, with the exception of some data from the 1960s. Small bowel transplantation (SBTx) still carries a high incidence of severe cellular rejection, with serious and devastating consequences. Faced with two particularly difficult patients, the present investigators used L-IMS for several weeks posttransplantation, with lower doses of immunosuppressive drugs and obvious success. These two cases and the technique of L-IMS are reported herein.

Microenvironment influence on human colon adenocarcinoma phenotypes and matrix metalloproteinase-2, p53 and β-catenin tumor expressions from identical monoclonal cell tumor in the orthotopic model in athymic nude rats

Abstract The present study aims to identify differences between left and right colon adenocarcinoma arising from identical clonal cell and to find out if microenvironment has any influence on matrix metalloproteinase-2 (MMP2), p53 and β-catenin tumor expressions. Material and methods. Rats (RNU) were submitted to cecostomy to obtain the orthotopic model of right colon tumor (n = 10), while for the left colon model (n = 10), a colon diversion and distal mucous fistula in the descending colon was used. Cultivated human colon adenocarcinoma cells (WiDr) were inoculated in stomas submucosa. Histopathological analysis, real-time reverse transcription-PCR for β-catenin, p53 and MMP2, as well as immunohistochemical analysis for p53 and β-catenin expression were conducted. Central tendency, variance analysis and the Livak delta-delta–CT method were used for statistical analysis, adopting a 5% significance level. Results. All tumors from the left colon exhibited infiltrative ulceration, while in the right colon tumor growth was predominantly exophytic (67%). In the left colon, tumor growth was undifferentiated (100%), while it was moderately differentiated in the right colon (83%). In right colon tumors, MMP2, p53, and β-catenin gene expressions were higher than compared to left colon (p = 4.59354E-05, p = 0.0035179, p = 0.00093798, respectively, for MMP2, p53 and β-catenin). β-catenin and p53 results obtained by real-time polymerase chain reaction were confirmed by immunohistochemistry assay (p = 0.01 and p = 0.001, respectively, for β-catenin and p53). Conclusion. Left and right human colon adenocarcinomas developed in animal models have distinct phenotypes even when they have the same clonal origin. Microenvironment has influenced p53, β-catenin, and MMP2 expression in animal models of colon cancer.

A novel model of distal colon cancer in athymic mice

PURPOSE The present a novel adenocarcinoma model in athymic mice. METHODS Seven athymic mice were used. Colon diversion and distal fistula were made. Adenocarcinoma cells were inoculated in the submucosa of fistula. Tumor growth was monitored daily. Scintigraphy with 99mTc-MIBI was performed to identify the tumor. RESULTS The model of distal colon cancer is feasible. Tumor detection was possible by both, macroscopically and molecular imaging. All resections demonstrated poorly differentiated tumors. Colon obstruction occurred in one case, similarly to evolution in human tumors of distal colon. CONCLUSION The proposed model of distal colon cancer is feasible, allows for easy monitoring of tumoral growth by both, macroscopically and molecular imaging, and is suitable for studying the evolution of tumor with implementation of cytotoxic therapy in vivo.

Intraoperative management of liver transplantation for familial amyloid polyneuropathy Met30: what has changed in the last 10 years?

FAMILIAL amyloid polyneuropathy (FAP) is a genetic disorder inevitably lethal without liver transplantation. This procedure has been performed for FAP since 1990 and this disease is now, by far, the most frequent metabolic indication for liver transplantation, with more than 700 patients having undergone transplantation worldwide. In the last years, results improved significantly, and FAP Met30 1-year survival rates reached 90% to 98%. Because these patients had no liver insufficiency, their livers have been used as grafts in transplantation of other patients, since October 1995, in the so-called “domino” or sequential liver transplantation.

Delocalized lipophilic cations as a new therapeutic approach in cancer

Delocalized lipophilic cations (DLCs) penetrate plasma and mitochondrial membranes and accumulate in mitochondria. The higher mitochondrial membrane potentials of neoplastic vs normal cells, in general, account for greater uptake and may be a way to selectively target these cells since. Dequalinium (DQA) is a DLC and so our goal is to evaluate the therapeutic potential of DQA in cancer, namely in B-cell Chronic Lymphocytic Leukaemia (B-CLL), Acute Promyelocytic Leukaemia (APL) and Hepatocellular Carcinoma (HCC). For this we used 3 cell lines, EHEB (B-CLL), HL-60 (APL) and HUH-7 (HCC), to evaluate the effect of different concentrations of DQA either by single dose administration, by daily dose administration and by association with conventional anticarcinogenic agents. Cell viability and death was determined by the resazurin assay, optical microscopy and by flow cytometry. The latter was also used to evaluate the mitochondrial membrane potential, the levels of ROS (H2O2; O2•-) and the antioxidant defense, Reduced Glutathione (GSH), using fluorescent probes. We found that DQA induced a decrease in cell viability inducing cell death by late apoptosis/necrosis in a time, dose and cell type dependent manner, with and IC50 of 2.5, 4.7 and 7.5 μM at 48h of exposure, respectively to HL-60, HUH-7 and EHEB. These effects may be mediated by oxidative stress as we have observed and increase in ROS production and a decrease in GSH levels and in mitochondrial membrane potential. We also observed that if DQA is administered on a daily basis a much lower concentration is required to induce the same effect. On the other hand, the association of DQA with the conventional drug induces a synergistic effect, because lower concentration of both drugs is required to obtain the some effect.

Evaluation of the role of mir-34b in modulation of radioresistance in non-small cell lung cancer

Radiotherapy is a major therapeutic weapon in lung cancer. However, the resistance to radiotherapy is frequent. The microRNAs of the miR-34 family, miR34a, miR-34b and miR-34c, described as effector molecules in the cellular response to activation of P53, have low expression levels in lung cancer [1]. The mRNA of the anti-apoptotic protein BCL-2 is among the targets of miR-34 family. The aims of our study were to clarify the involvement of miR-34b over-expression in the modulation of radiation response in NSCLC cell lines and the mechanisms involved. For these purposes we used two radioresistant NSCLC cell lines, A549, expressing P53, and H1299, not expressing P53. Cells transfected with pre-miR-34b or with a transfection control were exposed to different irradiation doses. The response to irradiation was assessed by cell survival curves obtained by clonogenic assay, and flow citometry allowed the characterization of cell death and the quantification of BCL-2, BAX and P53 protein expression levels. Our results showed that both cell lines had low expression levels of miR-34 family members, especially for miR-34b/c. Over-expression of miR-34b sensitized A549 cells to low doses of radiation and decreased BCL-2 expression, but without changing apoptosis levels. H1299 cells remained unchanged. These results suggest that in NSCLC expressing P53, response to radiotherapy is dependent on BCL-2 levels and may be modulated by over-expression of miRNA34b. Other cell death mechanisms than apoptosis, but also involving BCL-2, like autophagy, could to be involved. Author details Department of Molecular Biology, Faculty of Medicine, University of Coimbra (FMUC), Coimbra, Portugal. IBILI, FMUC, Coimbra, Portugal. Medical Genetics, FMUC, University of Coimbra, Coimbra, Portugal. CIMAGO, FMUC, Coimbra, Portugal. Center for Neuroscience and Cell Biology, University of Coimbra, Portugal.