A long-term physical activity training program increases strength and flexibility, and improves balance in older adults.

PURPOSE: Physical activity training programs in older adults have recognized health benefits. Evidence suggests that training should include a combination of progressive resistance, balance, and functional training. Our aim was to assess the effects of a simple physical activity program working on strength, flexibility, cardiovascular fitness, and balance in older adults, as well as the effects of a detraining period, at various different ages. METHODS: This was longitudinal prospective study, including a convenience sample of 227 independent older adults (54 men, 173 women) who completed a simple 9-month training program and 3-month detraining follow-up. The subjects were categorized into two age groups (65-74 [n = 180], and >74 years [n = 47]). At the beginning of the study (baseline), the end of the training period, and 3 months later (postdetraining), body mass index, body fat percentage, triceps skinfold thickness, hand grip strength, lower limb and trunk flexibility, resting heart rate, heart rate after exercise, and balance were measured, while VO(2 max) was estimated using the Rockport fitness test and/or measured directly. RESULTS: Significant improvements in strength (p < .0001), flexibility (p < .0001), heart rate after exercise (p < .0001), and balance (p < .0001) were observed at the end of the training program. Flexibility and balance (p < .0001) improvements were maintained at the end of the detraining. CONCLUSION: A simple long-term physical activity training program increases strength in both sexes, improves flexibility in women, and improves balance in older adults. The results also indicate the importance of beginning early in old age and maintaining long-term training.

Heat makes a difference in activity-based anorexia: a translational approach to treatment development in anorexia nervosa.

OBJECTIVE: To test the effect of raising ambient temperature (AT) on activity-based anorexia (ABA) and to extend to female rats previous findings reported in male animals. METHOD: Two studies are reported in which female rats were submitted to food restriction and free access to an activity wheel either separately or in combination under changing (21-32 °C) or constant AT (21 °C). RESULTS: Warming ABA animals reversed running activity, preserved food-intake, and enabled female rats to recover from acute weight loss. Moreover, sedentary food-restricted warmed rats maintained a body weight equivalent to the levels of animals housed at standard AT in spite of 20% reduced food-intake. DISCUSSION: The replication on female rats corroborates the effect previously reported for males, which is indicative of the robust effect of AT in recovering rats from ABA. The findings reported here represent strong preclinical evidence in favor of heat supply as a useful adjunctive component for the treatment of anorexia nervosa (AN).

Clinical Implications of (Pro)renin Receptor (PRR) Expression in Renal Tumours.

(1) Background: Renal cancer is one of the most frequent malignancies in Western countries, with an unpredictable clinical outcome, partly due to its high heterogeneity and the scarcity of reliable biomarkers of tumour progression. (Pro)renin receptor (PRR) is a novel receptor of the renin-angiotensin system (RAS) that has been associated with the development and progression of some solid tumours by RAS-dependent and -independent mechanisms. (2) Methods: In this study, we analysed the immunohistochemical expression of PRR at the centre and border in a series of 83 clear-cell renal cell (CCRCCs), 19 papillary (PRCC) and 7 chromophobe (ChRCC) renal cell carcinomas, and the benign tumour renal oncocytoma (RO, n = 11). (3) Results: PRR is expressed in all the tumour subtypes, with higher mean staining intensity in ChRCCs and ROs. A high expression of PRR at the tumour centre and at the infiltrative front of CCRCC tissues is significantly associated with high grade, tumour diameter, local invasion and stage, and with high mortality risk by UCLA integrated staging system (UISS) scale. (4) Conclusions: These findings indicate that PRR is associated with the development and progression of renal tumours. Its potential as a novel biomarker for RCC diagnosis/prognosis and as a promising therapeutic target should be taken into account in the future.

Soluble PD-L1 Is an Independent Prognostic Factor in Clear Cell Renal Cell Carcinoma.

(1). Background: Immunohistochemical (IHC) evaluation of programmed death-1 (PD-1) and its ligand (PD-L1) is being used to evaluate advanced malignancies with potential response to immune checkpoint inhibitors. We evaluated both plasma and tissue expression of PD-1 and PD-L1 in the same cohort of patients, including non-metastatic and metastatic clear cell renal cell carcinoma (CCRCC). Concomitant plasma and tissue expression of PD-1 and PD-L1 was evaluated with emphasis on diagnostic and prognostic implications. (2) Methods: we analyzed PD-1 and PD-L1 IHC expression in tumor tissues and soluble forms (sPD-1 and sPD-L1) in plasma from 89 patients with CCRCC, of which 23 were metastatic and 16 received systemic therapy. The primary endpoint was evaluation of overall survival using Kaplan-Meier analysis and the Cox regression model. Plasma samples from healthy volunteers were also evaluated. (3) Results: Interestingly, sPD-1 and sPD-L1 levels were lower in cancer patients than in controls. sPD-1 and sPD-L1 levels and their counterpart tissue expression both at the tumor center and infiltrating front were not associated. Higher expression of both PD-1 and PD-L1 were associated with tumor grade, necrosis and tumor size. PD-1 was associated to tumor stage (pT) and PD-L1 to metastases. sPD-1 and sPD-L1 were not associated with clinico-pathological parameters, although both were higher in patients with synchronous metastases compared to metachronous ones and sPD-L1 was also higher for metastatic patients compared to non-metastatic patients. sPD-1 was also associated with the International Metastatic Renal Cell Cancer Database Consortium (IMDC) prognostic groups in metastatic CCRCC and also to the Morphology, Attenuation, Size and Structure (MASS) response criteria in metastatic patients treated with systemic therapy, mainly tyrosine-kinase inhibitors. Regarding prognosis, PD-L1 immunostaining at the tumor center with and without the tumor front was associated with worse survival, and so was sPD-L1 at a cut-off >793 ng/mL. Combination of positivity at both the tissue and plasma level increased the level of significance to predict prognosis. (4) Conclusions: Our findings corroborate the role of PD-L1 IHC to evaluate prognosis in CCRCC and present novel data on the usefulness of plasma sPD-L1 as a promising biomarker of survival in this neoplasia.

Synovial fluid peptidase activity as a biomarker for knee osteoarthritis clinical progression.

AIMS: To analyze the potential role of synovial fluid peptidase activity as a measure of disease burden and predictive biomarker of progression in knee osteoarthritis (KOA). METHODS: A cross-sectional study of 39 patients (women 71.8%, men 28.2%; mean age of 72.03 years (SD 1.15) with advanced KOA (Ahlbäck grade ≥ 3 and clinical indications for arthrocentesis) recruited through the (Orthopaedic Department at the Complejo Asistencial Universitario de León, Spain (CAULE)), measuring synovial fluid levels of puromycin-sensitive aminopeptidase (PSA), neutral aminopeptidase (NAP), aminopeptidase B (APB), prolyl endopeptidase (PEP), aspartate aminopeptidase (ASP), glutamyl aminopeptidase (GLU) and pyroglutamyl aminopeptidase (PGAP). RESULTS: Synovial fluid peptidase activity varied significantly as a function of clinical signs, with differences in levels of PEP (p = 0.020), ASP (p < 0.001), and PGAP (p = 0. 003) associated with knee locking, PEP (p = 0.006), ASP (p = 0.001), GLU (p = 0.037), and PGAP (p = 0.000) with knee failure, and PEP (p = 0.006), ASP (p = 0.001), GLU (p = 0.037), and PGAP (p < 0.001) with knee effusion. Further, patients with the greatest functional impairment had significantly higher levels of APB (p = 0.005), PEP (p = 0.005), ASP (p = 0.006), GLU (p = 0.020), and PGAP (p < 0.001) activity, though not of NAP or PSA, indicating local alterations in the renin-angiotensin system. A binary logistic regression model showed that PSA was protective (p = 0.005; Exp (B) 0.949), whereas PEP (p = 0.005) and GLU were risk factors (p = 0.012). CONCLUSION: These results suggest synovial fluid peptidase activity could play a role as a measure of disease burden and predictive biomarker of progression in KOA. Cite this article: Bone Joint Res 2020;9(11):789-797.

Altered Tissue and Plasma Levels of Fibroblast Activation Protein-α (FAP) in Renal Tumours.

(1) Background: Renal cell carcinoma (RCC) is a heterogeneous and complex disease with only partial response to therapy, high incidence of metastasis and recurrences, and scarce reliable biomarkers indicative of progression and survival. Cancer-associated fibroblasts (CAFs) play an important role supporting and promoting renal cancer progression. (2) Methods: In this study, we analysed fibroblast activation protein-α (FAP) immunohistochemical expression and its soluble isoform (sFAP) in tumour tissues and plasma from 128 patients with renal tumours. (3) Results: FAP is expressed in the cell surface of CAFs of the tumour centre and infiltrating front from clear cell renal cell carcinomas (CCRCC, n = 89), papillary renal cell carcinomas (PRCC, n = 21), and chromophobe renal cell carcinomas (ChRCC, n = 8), but not in the benign tumour renal oncocytoma (RO, n = 10). A high expression of FAP and low levels sFAP are significantly associated with high tumour diameter, high grade, and high pT stage, lymph node invasion, development of early metastases, and worse 5-year cancer specific survival of CCRCC patients. (4) Conclusions: These findings corroborate the potential usefulness of FAP immunohistochemistry and plasma sFAP as a biomarker of CCRCC progression and point to CAF-related proteins as promising immunohistochemical biomarkers for the differential diagnosis of ChRCC and RO.

Altered expression of fibroblast activation protein-α (FAP) in colorectal adenoma-carcinoma sequence and in lymph node and liver metastases.

Colorectal cancer (CRC) is a major health problem in elderly people because of its high incidence and high mortality rate. Despite early screening programs, more than half of CRC patients are diagnosed at advanced stages. Fibroblast activation protein-α (FAP) expression in cancer-associated fibroblasts (CAFs) has been associated with a higher risk of metastases and poor survival. Here, we have analyzed the immunohistochemical expression of FAP in 41 adenoma-carcinoma sequences. In addition, FAP expression was analyzed individually and in combination with ß-catenin (BCAT), CD44 and Cyclin-D1 expression in primary tumors and in their corresponding lymph node and liver metastases (n=294). Finally, soluble FAP (sFAP) levels in plasma from CRC patients (n=127) were also analyzed by ELISA. FAP was expressed only in CRC tissue and its expression level was found to be higher in tumors exhibiting deeper local invasion and poorer cancer cell differentiation. FAP and concomitant nuclear BCAT expression in cancer cells at the infiltrating front of primary tumors and in lymph node metastases was independently associated with 5- and 10-year cancer specific and disease-free survival. Moreover, lower sFAP levels correlated with poorer survival. These findings support the potential importance of FAP as a biomarker of CRC development and progression.

A study of the toxic effect of oxidized sunflower oil containing 4-hydroperoxy-2-nonenal and 4-hydroxy-2-nonenal on cortical TrkA receptor expression in rats.

Sunflower oil at a specific oxidation stage (when several oxygenated alpha,beta-unsaturated aldehydes are generated, mainly 4-hydroperoxy-trans-2-alkenals and 4-hydroxy-trans-2-alkenals), caused at 70 degrees C with aeration for 7 days, was administered intraperitoneally to rats. This oil was studied by means of solid phase micro-extraction followed by gas chromatography/mass spectrometry (SPME-GC/MS) and by proton nuclear magnetic resonance ((1)H-NMR). Oxidized sunflower oil (3 ml/kg/day) was administered to male Sprague-Dawley rats for 21 days. The control group was administered non-oxidized sunflower oil in the same volume and for the same duration as the experimental group. A significant decrease in the number of neural cells positively immunostained for TrkA receptor was detected in the frontal cortex of the experimental group, with respect to controls, suggesting both neuronal damage as well as a deficit in neuronal survival signalling at this level. This could lead to apoptosis of cholinergic neurons, which play a key role in memory and attention function. These results indicate that toxic substances present in the oxidized sunflower oil, among them 4-hydroxy-trans-2-nonenal (HNE) and 4-hydroperoxy-trans-2-nonenal (HPNE), could disrupt survival signalling of frontal cortex cholinergic neurons, which could lead to apoptosis and neurodegenerative diseases. In the case of humans, this fact reinforces the necessity of avoiding the re-utilization of oxidized sunflower oil, in order to contribute to long-term neurodegenerative diseases prevention.

Brain endocannabinoid system is involved in fluoxetine-induced anorexia.

In order to describe the effects of chronic fluoxetine administration on the brain endocannabinoid system in lean and obese Zucker rats, brain immunostaining for the CB1 and CB1-phosphorylated cannabinoid receptors was carried out. Obese Zucker rats showed significantly increased the numbers of neural cells positively immunostained for the CB1-phosphorylated receptor in the striatum, compared to their lean litter-mates. Chronic fluoxetine administration decreased the number of neural cells immunostained for CB1-phosphorylated receptor in several striatal and hippocampal regions of obese Zucker rats, compared to controls treated with saline. In contrast, no change in CB1-phosphorylated receptor immunostaining was observed in fluoxetine-treated lean rats, with respect to controls. Taken together, these results suggest the involvement of the hippocampal and striatal endocannabinoid receptor system in fluoxetine-induced anorexia in lean and obese Zucker rats.

The surge in biosimilars: considerations for effective pharmacovigilance and EU regulation.

The development of biosimilars is growing rapidly, especially in Europe. They are a cost-effective alternative to original biological medicines and can help improve patient access to these therapies. The European Medicines Agency (EMA) has been the first to issue scientific guidelines related to regulatory requirements for the approval of biosimilars. These guidelines have been being updated in line with advances in analytical techniques and growing experience in the clinical use of these drugs. Given the complex nature of biological medicines, they pose a greater potential risk of immunogenicity than nonbiological medicines, and hence warrant special consideration. The risk management plan for biopharmaceuticals (innovator and biosimilar drugs) should be based on strengthening ongoing pharmacovigilance activities, especially in the post-approval period. This paper addresses regulatory issues related to the approval of biosimilars in Europe associated with safety considerations linked to the development and use of these medicines. We also discuss the issues of immunogenicity, interchangeability and traceability of biological medicines.

Effects of conjugated linoleic acid on liver composition and fatty acid oxidation are isomer-dependent in hamster.

OBJECTIVE: The present work was designed to study the effects of the two main isomers of conjugated linoleic acid (CLA), cis-9,trans-11 and trans-10,cis-12, on liver composition and hepatic fatty acid oxidation in hamsters. METHODS: Animals were divided into three groups that were fed atherogenic diets supplemented with 0.5% linoleic acid, cis-9,trans-11 CLA, or trans-10,cis-12 CLA for 6 wk. Liver lipids, protein, water and DNA contents, and histologic structure were analyzed. Hepatic carnitine palmitoyltransferase-I and acyl coenzyme A oxidase activities were assessed. Triacylglycerol concentration, and aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, and alkaline phosphatase activities were evaluated in serum. CLA isomer contents were analyzed by gas chromatography in hepatic triacylglycerols. Peroxisome proliferator-activated receptor-alpha mRNA was determined by reverse transcriptase polymerase chain reaction. RESULTS: Trans-10,cis-12 CLA led to significantly greater weight, lower levels of triacylglycerol, cholesterol, and phospholipid, and larger total cell number in liver. Carnitine palmitoyltransferase-I and acyl coenzyme A oxidase activities were significantly increased by this isomer. No changes were induced by cis-9,trans-11 CLA. Trans-10,cis-12 CLA was recovered in significantly lower proportions than cis-9,trans-11 in liver triacylglycerols. Histopathologic analysis showed no abnormalities. No significant differences in serum aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, and alkaline phosphatase activities or in hepatic mRNA peroxisome proliferator-activated receptor-alpha expression were found among the three experimental groups. CONCLUSIONS: These results suggest that the addition of 0.5% of these CLA isomers to the diet do not induce toxic effects in liver after 6 wk of feeding. Intake of trans-10,cis-12 isomer but not of cis-9,trans-11 CLA increases liver fatty acid oxidation. This effect leads to decreased hepatic and serum triacylglycerols.

Plasma peptidases as prognostic biomarkers in patients with first-episode psychosis.

The plasma activity of nine aminopeptidases was monitored over a year in first-episode psychotic patients. We observed significant differences in aminopeptidase B (APB), aminopeptidase N (APN) and dipeptidyl peptidase IV (DPPIV), but not in puromycin-sensitive aminopeptidase (PSA), prolyl endopeptidase (PEP), cysteine aminopeptidase (Cys-AP), aspartate aminopeptidase (Asp-AP), glutamate aminopeptidase (Glu) or piroglutamate aminopeptidase (PGI) in these patients compared to controls, and also a progressive increase in plasma activity, correlated to changes in scores on clinical scales, Global Assessment of Functioning scale (GAF) and Hamilton Depression Rating Scale (HDRS), at 1 month of follow-up. At 1 month after diagnosis, the median score obtained by patients on the GAF was negatively associated with the plasma activity of APB and PEP measured at the beginning of the psychotic episode, indicating a role as a negative prognostic factor that can predict psychiatric symptomatology. In the case of HDRS, scores at 1 month after diagnosis were found to be positively associated with the initial plasma activity of DPPIV, APN and PSA, indicating that their initial elevation is a negative prognostic factor that can predict subsequent depressive symptomatology. Taken together, these results suggest a pathophysiological involvement of plasma peptidases and indicate that aminopeptidase activity can predict the course of first-episode psychosis patients, acting as a prognostic indicator.

Effect of the antidepressant nefazodone on the density of cells expressing mu-opioid receptors in discrete brain areas processing sensory and affective dimensions of pain.

RATIONALE: The principal use of antidepressants is in the treatment of depression and affective disorders. Antidepressants have also been used as an adjuvant to analgesics in pain treatment. However, in chronic treatment, their antinociceptive and antidepressive effects coexist simultaneously. Antidepressants can interact with the opioid system, which is also involved in regulating nociceptive processing and affective state. Chronic antidepressants could act by increasing mu-opioid receptor expression in many brain areas involved in the regulation of nociception and affective state. OBJECTIVES: The aim of this study was to evaluate the antinociceptive and antidepressant-like effects and the possible variations in mu-opioid receptor expression induced by a chronic nefazodone treatment in brain areas related to pain and affective state. METHODS: Wistar rats were chronically treated with nefazodone (10 and 25 mg/kg IP, twice a day, for 14 days). Twelve hours after the last day 14 dose of nefazodone, a tail-flick test was performed. After the administration of a daily dose of nefazodone, Porsolt's test was carried out 12 h after last dose. Two hours after completion of 14 days treatment, other animals were processed for mu-opioid receptor immunocytochemistry using polyclonal antisera raised in rabbits. Several brain regions were analyzed: the frontal and cingulate cortex, the dorsal raphe nucleus and the periaqueductal gray. RESULTS: Chronic nefazodone treatment induced a significant increase in tail-flick latency and a significant decrease in immobility time at total doses of 20 and 50 mg/kg per day ( P<0.05). In treated animals, the density of neural cells immunostained for mu-opioid receptor in the frontal and cingulate cortices, dorsal raphe nucleus and periaqueductal gray had increased after chronic nefazodone compared to controls. CONCLUSION: Therefore, chronic nefazodone induces antinociceptive and antidepressant-like effects in rats and increases mu-opioid receptor expression in brain areas related to pain and affective state. These results suggest that antidepressants could be effective on somatic and affective dimensions of pain and this action could be related to its influence on the opioid system.

Toluene alters appetite, NPY, and galanin immunostaining in the rat hypothalamus.

Toluene is a neurotoxic organic solvent widely used in industry and extensively inhaled by solvent abusers. Toluene, diluted 1:1 in olive oil, was administered intraperitoneally at a dose of 1.3 ml/kg/day for 4 days. Controls received the vehicle in the same dose and duration. Food intake and body weight were measured daily. A regional hypothalamic NPY and galanin immunocytochemical study was also performed. Acute toluene administration in rats generated a reduction in food intake and body weight gain. A significant decrease in NPY immunostaining in the paraventricular nucleus and an increase in arcuate nucleus without changes in other hypothalamic regions were observed. Also, acute toluene administration increased galanin immunostaining in paraventricular and arcuate nuclei. These results suggest that the hypothalamic NPY arcuate-paraventricular projection is involved in the anorectic effect of acute high-dose toluene administration. Changes in regional hypothalamic galanin expression could represent a compensatory mechanism against toluene-induced anorexia.

Toluene alters p75NTR expression in the rat brainstem.

Toluene is a neurotoxic organic solvent widely used in industry. Acute toluene administration in rats induced a significant increase in the numbers of neural cells immunostained for p75NTR in several brainstem regions, such as the raphe magnus and the nucleus of the solitary tract, as well as in the lateral reticular, gigantocellular, vestibular and ventral cochlear nuclei, without any in the facial and spinal trigeminal nuclei and the dorsal horn of the spinal cord. These data suggest that p75NTR could be involved in toluene-induced neurotoxic efffects in the rat brainstem.

Dextran-protamine-solid lipid nanoparticles as a non-viral vector for gene therapy: in vitro characterization and in vivo transfection after intravenous administration to mice.

The aim of present work is to evaluate the transfection capacity of a new multicomponent system based on dextran (Dex), protamine (Prot), and solid lipid nanoparticles (SLN) after intravenous administration to mice. The vectors containing the pCMS-EGFP plasmid were characterized in terms of particle size and surface charge. In vitro transfection capacity and cell viability were studied in four cell lines, and compared with the transfection capacity of SLN without dextran and protamine. Transfection capacity was related to the endocytosis mechanism: caveolae or clathrin. The Dex-Prot-DNA-SLN vector showed a higher transfection capacity in those cells with a high ratio of activity of clathrin/caveolae-mediated endocytosis. However, the complex prepared without dextran and protamine (DNA-SLN) was more effective in those cells with a high ratio of activity of caveolae/clathrin-mediated endocytosis. The interaction with erythrocytes and the potential hemolytic effect were also checked. The Dex-Prot-DNA-SLN vector showed no agglutination of erythrocytes, probably due to the presence of dextran. After intravenous administration to BALB/c mice, the vector was able to induce the expression of the green fluorescent protein in liver, spleen and lungs, and the protein expression was maintained for at least 7 days. Although additional studies are necessary, this work reveals the promising potential of this new gene delivery system for the treatment of genetic and non-genetic diseases through gene therapy.

Adaptive response in the antioxidant defence system in the course and outcome in first-episode schizophrenia patients: a 12-months follow-up study.

Correlation of plasma antioxidant enzyme activity with the course and outcome in first-episode schizophrenia patients (n=49) was analyzed in order to assess the possible utility of peripheral markers of oxidative stress as prognostic factors. These markers were measured shortly after the onset of schizophrenia, and again 1, 6 and 12 months later. A decrease in catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and glutathione (GSH) levels and total antioxidant status (TAS), as well as an increase in thiobarbituric acid reactive substances (TBARS), were observed 1 month after (p<0.05). 6-Months later, there was a reduction in TAS, GSH, SOD and GPx, and a increase in TBARS (p<0.05), with a normalization of CAT levels, indicating a persistent alteration of the antioxidant system and the maintenance of oxidative stress. At 12-months, a considerable decrease was observed in TBARS. Additionally, while the level of GPx decreased (p<0.05) further, SOD and GSH levels and TAS were normalizing, indicating a partial regeneration of the antioxidant defence system. These results indicate the possible contribution of oxidative stress to the onset and pathophysiology of schizophrenia, suggesting the involvement of an adaptive response in the antioxidant defence system in the course and outcome in first-episode schizophrenia patients.

Cognitive impairment is related to oxidative stress and chemokine levels in first psychotic episodes.

INTRODUCTION: This study measures the levels of various markers of oxidative stress and inflammation in blood samples from first-episode psychosis (FEP) patients, and examines the association between these peripheral biomarkers and cognitive performance at 6 months after treatment. METHODS: Twenty-eight FEP patients and 28 healthy controls (matched by age, sex and educational level) had blood samples taken at admission for assessment of total antioxidant status, superoxide dismutase (SOD), total glutathione (GSH), catalase (CAT), glutathione peroxidase, lipid peroxidation, nitrites and the chemokine monocyte chemoattractant protein-1 (MCP-1). A battery of cognitive tests was also applied to the healthy controls and those FEP patients who were in remission at 6 months after the acute episode. RESULTS: FEP patients had significantly lower levels of total antioxidant status, catalase and glutathione peroxidase, compared with the healthy controls. Regression analyses found that MCP-1 levels were negatively associated with learning and memory (verbal and working), nitrite levels were negatively associated with executive function, and glutathione levels were positively associated with executive function. CONCLUSION: Our results suggest an association between certain peripheral markers of oxidative stress and inflammation and specific aspects of cognitive functioning in FEP patients. Further studies on the association between MCP-1 and cognition are warranted.

Solid lipid nanoparticles as potential tools for gene therapy: in vivo protein expression after intravenous administration.

Naked plasmid DNA is a powerful tool for gene therapy, but it is rapidly eliminated from the circulation after intravenous administration. Therefore, the development of optimized DNA delivery systems is necessary for its successful clinical use. Solid lipid nanoparticles (SLNs) have demonstrated transfection capacity in vitro, but their application for gene delivery has not been conveniently investigated in vivo. We aimed to evaluate the capacity of SLN-DNA vectors to transfect in vivo after intravenous administration to mice. The SLNs, composed of Precirol ATO 5, DOTAP and Tween 80 were complexed with the plasmid pCMS-EGFP which encodes the enhanced green fluorescent protein (EGFP). The resulting systems were characterized in vitro showing a mean particle size of 276 nm, superficial charge of +28 mV, the ability to protect the plasmid and transfection capacity in culture cells. The intravenous administration in mice led to transfection in hepatic tissue and spleen. Protein expression was detected from the third day after administration, and it was maintained for at least 1 week. This work shows for the first time the capacity of SLN-DNA vectors to induce the expression of a foreign protein after intravenous administration, supporting the potential of SLNs for gene therapy.

Distribution and neurochemical characterization of neurons expressing GIRK channels in the rat brain.

G-protein inwardly rectifying potassium (GIRK) channels mediate the synaptic actions of numerous neurotransmitters in the mammalian brain and play an important role in the regulation of neuronal excitability in most brain regions through activation of various G-protein-coupled receptors such as the serotonin 5-HT(1A) receptor. In this report we describe the localization of GIRK1, GIRK2, and GIRK3 subunits and 5-HT(1A) receptor in the rat brain, as assessed by immunohistochemistry and in situ hybridization. We also analyze the co-expression of GIRK subunits with the 5-HT(1A) receptor and cell markers of glutamatergic, gamma-aminobutyric acid (GABA)ergic, cholinergic, and serotonergic neurons in different brain areas by double-label in situ hybridization. The three GIRK subunits are widely distributed throughout the brain, with an overlapping expression in cerebral cortex, hippocampus, paraventricular nucleus, supraoptic nucleus, thalamic nuclei, pontine nuclei, and granular layer of the cerebellum. Double-labeling experiments show that GIRK subunits are present in most of the 5-HT(1A) receptor-expressing cells in hippocampus, cerebral cortex, septum, and dorsal raphe nucleus. Similarly, GIRK mRNA subunits are found in glutamatergic and GABAergic neurons in hippocampus, cerebral cortex, and thalamus, in cholinergic cells in the nucleus of vertical limb of the diagonal band, and in serotonergic cells in the dorsal raphe nucleus. These results provide a deeper knowledge of the distribution of GIRK channels in different cell subtypes in the rat brain and might help to elucidate their physiological roles and to evaluate their potential involvement in human diseases.