Genotypic features of lentivirus transgenic mice.

Lentivector-mediated transgenesis is increasingly used, whether for basic studies as an alternative to pronuclear injection of naked DNA or to test candidate gene therapy vectors. In an effort to characterize the genetic features of this approach, we first measured the frequency of germ line transmission of individual proviruses established by infection of fertilized mouse oocytes. Seventy integrants from 11 founder (G0) mice were passed to 111 first generation (G1) pups, for a total of 255 events corresponding to an average rate of transmission of 44%. This implies that integration had most often occurred at the one- or two-cell stage and that the degree of genotypic mosaicism in G0 mice obtained through this approach is generally minimal. Transmission analysis of eight individual proviruses in 13 G2 mice obtained by a G0-G1 cross revealed only 8% of proviral homozygosity, significantly below the 25% expected from purely Mendelian transmission, suggesting counter-selection due to interference with the functions of targeted loci. Mapping of 239 proviral integration sites in 49 founder animals revealed that about 60% resided within annotated genes, with a marked tendency for clustering in the middle of the transcribed region, and that integration was not influenced by the transcriptional orientation. Transcript levels of a set of arbitrarily chosen target genes were significantly higher in two-cell embryos than in embryonic stem cells or adult somatic cells, suggesting that, as previously noted in other settings, lentiviral vectors integrate preferentially into regions of the genome that are transcriptionally active or poised for activation.

Dépression résistant au traitement: enjeux, thérapie et place des nouvelles approches de neurostimulation [Treatment resistant depression: therapy and novel neuromodulatory treatments].

Unipolar depression is among the leading cause of invalidity and disability-adjusted life-years. Many depressed patients do not respond to several antidepressant treatments. Several treatments have been investigated in resistant depression using electrical or magnetic stimulation of the brain. In this field, electroconvulsivotherapy remains to date the only treatment validated for efficacy and security. Novel neuromodulatory treatments used in neurological conditions are currently under investigation. Vagus nerve stimulation and deep brain stimulation may offer long-term efficacy and therefore justify expensive and highly specialized treatment programs.

The BENEFIT trial: testing the hypothesis that trypanocidal therapy is beneficial for patients with chronic Chagas heart disease

Among the pathophysiological derangements operating in the chronic phase of Chagas disease, parasite persistence is likely to constitute the main mechanism of myocardial injury in patients with chronic chagasic cardiomyopathy. The presence of Trypanosoma cruzi in the heart causes a low-grade, but relentless, inflammatory process and induces myocardial autoimmune injury. These facts suggest that trypanocidal therapy may positively impact the clinical course of patients with chronic Chagas heart disease. However, the experimental and clinical evidence currently available is insufficient to support the routine use of etiologic treatment in these patients. The BENEFIT project - Benznidazole Evaluation for Interrupting Trypanosomiasis - is an international, multicenter, double-blind, placebo-controlled trial of trypanocidal treatment with benznidazole in patients with chronic Chagas heart disease. This project is actually comprised of two studies. The pilot study investigates whether etiologic treatment significantly reduces parasite burden, as assessed by polymerase chain reaction-based techniques and also determines the safety and tolerability profile of the trypanocidal drug in this type of chagasic population. The full-scale study determines whether antitrypanosomal therapy with benznidazole reduces mortality and other major cardiovascular clinical outcomes in patients with chronic Chagas heart disease.

Current status and perspectives of cell therapy in Chagas disease

One century after its discovery, Chagas disease, caused by the protozoan, Trypanosoma cruzi, remains a major health problem in Latin America. Mortality and morbidity are mainly due to chronic processes that lead to dysfunction of the cardiac and digestive systems. About one third of the chronic chagasic individuals have or will develop the symptomatic forms of the disease, with cardiomyopathy being the most common chronic form. This is a progressively debilitating disease for which there are no currently available effective treatments other than heart transplantation. Like in other cardiac diseases, tissue engineering and cell therapy have been investigated in the past few years as a means of recovering the heart function lost as a consequence of chronic damage caused by the immune-mediated pathogenic mechanisms elicited in individuals with chronic chagasic cardiomyopathy. Here we review the studies of cell therapy in animal models and patients with chronic Chagas disease and the perspectives of the recovery of the heart function lost due to infection with T. cruzi.

5-Fluorouracil-loaded poly(ε-caprolactone) nanoparticles combined with phage E gene therapy as a new strategy against colon cancer

This work aimed to develop a new therapeutic approach to increase the efficacy of 5-fluorouracil (5-FU) in the treatment of advanced or recurrent colon cancer. 5-FU-loaded biodegradable poly(ε-caprolactone) nanoparticles (PCL NPs) were combined with the cytotoxic suicide gene E (combined therapy). The SW480 human cancer cell line was used to assay the combined therapeutic strategy. This cell line was established from a primary adenocarcinoma of the colon and is characterized by an intrinsically high resistance to apoptosis that correlates with its resistance to 5-FU. 5-FU was absorbed into the matrix of the PCL NPs during synthesis using the interfacial polymer disposition method. The antitumor activity of gene E from the phage ϕX174 was tested by generating a stable clone (SW480/12/E). In addition, the localization of E protein and its activity in mitochondria were analyzed. We found that the incorporation of 5-FU into PCL NPs (which show no cytotoxicity alone), significantly improved the drug's anticancer activity, reducing the proliferation rate of colon cancer cells by up to 40-fold when compared with the nonincorporated drug alone. Furthermore, E gene expression sensitized colon cancer cells to the cytotoxic action of the 5-FU-based nanomedicine. Our findings demonstrate that despite the inherent resistance of SW480 to apoptosis, E gene activity is mediated by an apoptotic phenomenon that includes modulation of caspase-9 and caspase-3 expression and intense mitochondrial damage. Finally, a strongly synergistic antiproliferative effect was observed in colon cancer cells when E gene expression was combined with the activity of the 5-FU-loaded PCL NPs, thereby indicating the potential therapeutic value of the combined therapy.

Tumor microenvironment and immune effects of antineoplastic therapy in lymphoproliferative syndromes.

Lymphomas represent a wide group of heterogenic diseases with different biological and clinical behavior. The underlying microenvironment-specific composition seems to play an essential role in this scenario, harboring the ability to develop successful immune responses or, on the contrary, leading to immune evasion and even promotion of tumor growth. Depending on surrounding lymphoid infiltrates, lymphomas may have different prognosis. Moreover, recent evidences have emerged that confer a significant impact of main lymphoma's treatment over microenvironment, with clinical consequences. In this review, we summarize these concepts from a pathological and clinical perspective. Also, the state of the art of lymphoma's anti-idiotype vaccine development is revised, highlighting the situations where this strategy has proven to be successful and eventual clues to obtain better results in the future.

Angiogenesis in Wounds Treated by Microdeformational Wound Therapy.

BACKGROUND:: Mechanical forces play an important role in tissue neovascularization and are a constituent part of modern wound therapies. The mechanisms by which vacuum assisted closure (VAC) modulates wound angiogenesis are still largely unknown. OBJECTIVE:: To investigate how VAC treatment affects wound hypoxia and related profiles of angiogenic factors as well as to identify the anatomical characteristics of the resultant, newly formed vessels. METHODS:: Wound neovascularization was evaluated by morphometric analysis of CD31-stained wound cross-sections as well as by corrosion casting analysis. Wound hypoxia and mRNA expression of HIF-1α and associated angiogenic factors were evaluated by pimonidazole hydrochloride staining and quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively. Vascular endothelial growth factor (VEGF) protein levels were determined by western blot analysis. RESULTS:: VAC-treated wounds were characterized by the formation of elongated vessels aligned in parallel and consistent with physiologically function, compared to occlusive dressing control wounds that showed formation of tortuous, disoriented vessels. Moreover, VAC-treated wounds displayed a well-oxygenated wound bed, with hypoxia limited to the direct proximity of the VAC-foam interface, where higher VEGF levels were found. By contrast, occlusive dressing control wounds showed generalized hypoxia, with associated accumulation of HIF-1α and related angiogenic factors. CONCLUSIONS:: The combination of established gradients of hypoxia and VEGF expression along with mechanical forces exerted by VAC therapy was associated with the formation of more physiological blood vessels compared to occlusive dressing control wounds. These morphological changes are likely a necessary condition for better wound healing.

Structured intermittent interruption of chronic HIV infection treatment with highly active antiretroviral therapy: Effects on leptin and TNF-alpha.

The changes in nutritional parameters and adipocytokines after structured intermittent interruption of highly active antiretroviral treatment of patients with chronic HIV infection are analyzed. Twenty-seven patients with chronic HIV infection (median CD4+ T cell count/microl: nadir, 394; at the beginning of structured interruptions, 1041; HIV viral load: nadir, 41,521 copies/ml; at the beginning of structured interruptions <50 copies/ml; median time of previous treatment: 60 months) were evaluated during three cycles of intermittent interruptions of therapy (8 weeks on/4 weeks off). CD4+ T cell count, HIV viral load, anthropometric measures, and serum concentrations of triglycerides, cholesterol, leptin, and tumor necrosis factor and its soluble receptors I and II were determined. After the three cycles of intermittent interruptions of therapy, no significant differences in CD4+ T cell count/microl, viral load, or serum concentrations of cholesterol or triglycerides with reference to baseline values were found. A near-significant higher fatty mass (skinfold thicknesses, at the end, 121 mm, at the beginning, 100 mm, p = 0.100), combined with a significant increase of concentration of leptin (1.5 vs. 4.7 ng/ml, p = 0,044), as well as a decrease in serum concentrations of soluble receptors of tumor necrosis factor (TNFRI, 104 vs. 73 pg/ml, p = 0.022; TNFRII 253 vs. 195 pg/ml, p = 0.098) were detected. Structured intermittent interruption of highly active antiretroviral treatment of patients with chronic HIV infection induces a valuable positive modification in markers of lipid turnover and adipose tissue mass.

Identification of tumor-associated antigens by large-scale analysis of genes expressed in human colorectal cancer.

Despite the high prevalence of colon cancer in the world and the great interest in targeted anti-cancer therapy, only few tumor-specific gene products have been identified that could serve as targets for the immunological treatment of colorectal cancers. The aim of our study was therefore to identify frequently expressed colon cancer-specific antigens. We performed a large-scale analysis of genes expressed in normal colon and colon cancer tissues isolated from colorectal cancer patients using massively parallel signal sequencing (MPSS). Candidates were additionally subjected to experimental evaluation by semi-quantitative RT-PCR on a cohort of colorectal cancer patients. From a pool of more than 6000 genes identified unambiguously in the analysis, we found 2124 genes that were selectively expressed in colon cancer tissue and 147 genes that were differentially expressed to a significant degree between normal and cancer cells. Differential expression of many genes was confirmed by RT-PCR on a cohort of patients. Despite the fact that deregulated genes were involved in many different cellular pathways, we found that genes expressed in the extracellular space were significantly over-represented in colorectal cancer. Strikingly, we identified a transcript from a chromosome X-linked member of the human endogenous retrovirus (HERV) H family that was frequently and selectively expressed in colon cancer but not in normal tissues. Our data suggest that this sequence should be considered as a target of immunological interventions against colorectal cancer.

Effects of highly active antiretroviral therapy with nelfinavir in vertically HIV-1 infected children: 3 years of follow-up. Long-term response to nelfinavir in children.

BACKGROUND Antiretroviral treatment (ART) in children has special features and consequently, results obtained from clinical trials with antiretroviral drugs in adults may not be representative of children. Nelfinavir (NFV) is an HIV-1 Protease Inhibitor (PI) which has become as one of the first choices of PI for ART in children. We studied during a 3-year follow-up period the effects of highly active antiretroviral therapy with nelfinavir in vertically HIV-1 infected children. METHODS Forty-two vertically HIV-infected children on HAART with NFV were involved in a multicentre prospective study. The children were monitored at least every 3 months with physical examinations, and blood sample collection to measure viral load (VL) and CD4+ cell count. We performed a logistic regression analysis to determinate the odds ratio of baseline characteristics on therapeutic failure. RESULTS Very important increase in CD4+ was observed and VL decreased quickly and it remained low during the follow-up study. Children with CD4+ <25% at baseline achieved CD4+ >25% at 9 months of follow-up. HIV-infected children who achieved undetectable viral load (uVL) were less than 40% in each visit during follow-up. Nevertheless, HIV-infected children with VL >5000 copies/ml were less than 50% during the follow-up study. Only baseline VL was an important factor to predict VL control during follow-up. Virological failure at defined end-point was confirmed in 30/42 patients. Along the whole of follow-up, 16/42 children stopped HAART with NFV. Baseline characteristics were not associated with therapeutic change. CONCLUSION NFV is a safe drug with a good profile and able to achieve an adequate response in children.

Different profiles of immune reconstitution in children and adults with HIV-infection after highly active antiretroviral therapy.

BACKGROUND Recent advances in characterizing the immune recovery of HIV-1-infected people have highlighted the importance of the thymus for peripheral T-cell diversity and function. The aim of this study was to investigate differences in immune reconstitution profiles after highly active antiretroviral therapy (HAART) between HIV-children and adults. METHODS HIV patients were grouped according to their previous clinical and immunological status: 9 HIV-Reconstituting-adults (HIV-Rec-adults) and 10 HIV-Reconstituting-children (HIV-Rec-children) on HAART with viral load (VL) or=500 cells/microL at least during 6 months before the study and CD4+

Anti-TNF therapy in acute sciatica reduces the long-term need for surgery: A three-year follow-up of a randomized double blind placebo controlled trial

Introduction: Two subcutaneous injections of adalimumab in severeacute sciatica have demonstrated a significant benefit on the numberof back surgeries in a short-term randomized controlled clinical trial[1]. This 3-year follow-up study aimed to determine whether theshort-term benefit was sustained over a longer period of time.Methods: Information on surgery was retrieved in 56/61 patients(93%). We used a Cox proportional hazard models to determinefactors predisposing to surgery.Results: Twenty-three (41%) patients had back surgery within 3 years,8/29 (28%) in the adalimumab group and 15/ 27 (56%) in the placebogroup, p = 0.038. Adalimumab injections reduced the need for backsurgery by 61% (Hazard Ratio (HR): 0.39 (95% CI: 0.17-0.92). In amultivariate model, treatment with a TNF-α antagonist remained thestrongest protective factor (HR 0.17, p = 0.002). Other significantpredictors of surgery were a good correlation between symptomsand MRI findings (HR = 11.6, p = 0.04), baseline intensity of leg pain(HR = 1.3, p = 0.06), intensity of back pain (HR = 1.4, p = 0.03)and duration of sickness leave (HR = 1.01 per day, p = 0.03).Conclusion: A short course of adalimumab in patients with severeacute sciatica significantly reduces the need for back surgery.

Timing of adequate antibiotic therapy is a greater determinant of outcome than are TNF and IL-10 polymorphisms in patients with sepsis.

INTRODUCTION Genetic variations may influence clinical outcomes in patients with sepsis. The present study was conducted to evaluate the impact on mortality of three polymorphisms after adjusting for confounding variables, and to assess the factors involved in progression of the inflammatory response in septic patients. METHOD The inception cohort study included all Caucasian adults admitted to the hospital with sepsis. Sepsis severity, microbiological information and clinical variables were recorded. Three polymorphisms were identified in all patients by PCR: the tumour necrosis factor (TNF)-alpha 308 promoter polymorphism; the polymorphism in the first intron of the TNF-beta gene; and the IL-10-1082 promoter polymorphism. Patients included in the study were followed up for 90 days after hospital admission. RESULTS A group of 224 patients was enrolled in the present study. We did not find a significant association among any of the three polymorphisms and mortality or worsening inflammatory response. By multivariate logistic regression analysis, only two factors were independently associated with mortality, namely Acute Physiology and Chronic Health Evaluation (APACHE) II score and delayed initiation of adequate antibiotic therapy. In septic shock patients (n = 114), the delay in initiation of adequate antibiotic therapy was the only independent predictor of mortality. Risk factors for impairment in inflammatory response were APACHE II score, positive blood culture and delayed initiation of adequate antibiotic therapy. CONCLUSION This study emphasizes that prompt and adequate antibiotic therapy is the cornerstone of therapy in sepsis. The three polymorphisms evaluated in the present study appear not to influence the outcome of patients admitted to the hospital with sepsis.