The ability of regulatory T cells (Treg) to traffic to sites of inflammation supports their role in controlling immune responses. This feature supports the idea that adoptive transfer of in vitro expanded human Treg could be used for treatment of immune/inflammatory diseases. However, the migratory behavior of Treg as well as their direct influence at the site of inflammation remains poorly understood. To explore the possibility that Treg may have direct anti-inflammatory influences on tissues, independent of their well-established suppressive effects on lymphocytes, we studied the adhesive interactions between mouse Treg and endothelial cells, as well as their influence on endothelial function during acute inflammation. We show that Foxp3+ adaptive/inducible Treg (iTreg) but not naturally occurring Treg (nTreg) efficiently interact with endothelial selectins and transmigrate through endothelial monolayers in vitro. In response to activation by endothelial antigen presentation or immobilized anti-CD3ε, Foxp3+ iTreg suppressed TNFα and IL-1β mediated endothelial selectin expression and adhesiveness to effector T cells. This suppression was contact independent, rapid acting, and mediated by TGFβ-induced activin receptor like kinase [ALK]5 signaling in endothelial cells. In addition, Foxp3+ iTreg adhered to inflamed endothelium in vivo and their secretion products blocked acute inflammation in a model of peritonitis. These data support the concept that Foxp3+ iTreg help to regulate inflammation independently of their influence on effector T cells by direct suppression of endothelial activation and leukocyte recruitment.
T cells; adhesion molecules; endothelial cells; inflammation; suppression
Patients treated with vaccines based on NGlycolil gangliosides have showed benefit in progression free survival and overall survival. These molecules, which have been observed in breast cancer cells, are minimally or not expressed in normal human tissue and have been considered as antigen tumor-specific. For this reason they are very attractive to immunotherapy. A phase I/II clinical trial was carried out in metastatic breast cancer patients with the NGlycolylGM3/VSSP vaccine administered by subcutaneous route. Selecting the optimal biological doses of the vaccine in these patients was the principal objective based on the immunogenicity, efficacy and safety results. Six levels of doses of vaccine were studied. Treatment schedule consisted of five doses every two weeks and then monthly until reaching a fifteenth doses. Doses levels studied were 150, 300, 600, 900, 1200 and 1500 μg. Five patients in each level were included except at the 900 μg dose, in which ten patients were included. Immunogenicity was determined by levels of antibodies generated in patients after vaccination. The response criteria of evaluation in solid tumors (RECIST) was used to evaluate antitumoral effect. Safety was evaluated by Common Toxicity Criteria of Adverse Event (CTCAE). The vaccine administration was safe and immunogenic in all does levels. Most frequent adverse events related to vaccination were mild or moderate and were related to injection site reactions and “flu-like” symptoms. Vaccination induced specific anti-NeuGcGM3 IgM and IgG antibodies responses in all patients. Disease control (objective response or stable disease) was obtained in 72.7% of evaluated patients. Median overall survival was 15.9 months. Two patients of two different dose levels achieved overall survival values of about six years. The dose of 900 μg was selected as biological optimal dose in which overall survival was 28.5 months.
metastatic breast cancer; clinical trial; therapeutic vaccine; ganglioside; NGcGM3
Regulatory T cells (Treg) are present in atherosclerotic lesions and can modulate disease. In this study we characterized changes in Treg responses associated with prolonged hypercholesterolemia and lesion progression.
Methods and Results
Ldlr−/− mice in which Treg express green fluorescence protein (GFP) were fed a control or cholesterol-rich diet and GFP+ cells were enumerated in lymphoid tissues and in aorta. Splenic Treg numbers increased after 4, 8 and 20 weeks in cholesterol-diet fed mice, however, the number of circulating and lesional Treg peaked at 4 weeks and decreased significantly at 8 and 20 weeks, concomitant with increased numbers of CD4+ effector T cells and increased lesion size over this period. Treg expression of selectin ligands and their ability to bind to aortic endothelium decreased after prolonged hypercholesterolemia, and apoptosis of lesional Treg increased. After 4 weeks of cholesterol rich-diet, a switch to a control diet for 4 weeks reduced serum cholesterol stopped lesion growth, and the high aortic Treg content was maintained, compared to mice fed a cholesterol diet for 8 weeks. After the diet reversal, the splenic Treg retained the phenotype of Treg after 4 weeks of cholesterol diet.
Prolonged hypercholesterolemia impairs Treg but not effector T cell (Teff) accumulation in lesions, but reversal of hypercholesterolemia can prevent loss of lesional Treg. Therefore cholesterol lowering therapies may induce dynamic and beneficial changes in Treg:Teff ratios in atherosclerotic lesions.
atherosclerosis; diet; hypercholesterolemia; T cells; regulatory T cells; CTLA-4; GITR; Ldlr; PSGL1; Treg
Smoking cessation is beneficial for our health at any point in life, both in healthy people and in people already suffering from a smoking-related disease. Any help to quit smoking can produce considerable benefits for Public Health. The purpose of the present study is to evaluate the efficacy of the CO-oximetry technique together with brief advice in smoking cessation, in terms of reduction of the number of cigarettes or in the variation of the motivation to quit smoking at month 12 compared with brief advice alone.
Randomised, parallel, single-blind clinical trial in a primary health care setting in Majorca (Spain). Smokers in contemplation or pre-contemplation phase will be included in the study. Exclusion criteria: Smokers in preparation phase, subjects with a terminal illness or whose health status does not allow them to understand the study or complete the informed consent, and pregnant or breastfeeding women. The subjects will be randomly assigned to the control group (CG) or the intervention group (IG). The CG will receive brief advice, and the IG will receive brief advice together with a measurement of exhaled CO. There will be follow-up evaluations at 6 and 12 months after inclusion. 471 subjects will be needed per group in order to detect a difference between groups ≥ 5%. Primary outcome: sustained smoking cessation (at 6 and 12 months) confirmed by urine cotinine test. Secondary outcomes: point smoking cessation at 6 and 12 months both confirmed by urine cotinine analysis and self-reported, reduction in cigarette consumption, and variation in phase of smoking cessation.
CO-oximetry is an inexpensive, non-invasive, fast technique that requires little technical training; making it a technique for risk assessment in smokers that can be easily applied in primary care and, if proven effective, could serve as a reinforcement aid in smoking cessation intervention activities.
Current Controlled Trials ISRCTN67499921
We report the first case of syngnathia with hypophyseal duplication and describe the central nervous system (CNS) and craniofacial anomalies associated with hypophyseal duplication in the reported autopsy case. We studied clinical reports, scanner images, and autopsy results of a 2-months-old female baby. The propositus had frontonasal dysmorphism, retrognathia, and bifid tongue. She also presented maxillomandibular bony fusion (syngnathia) and an intraoral hairy polyp. In the cranium, the sella turcica was broadened, with two complete hypophyses and two infundibulums. The CNS had both olfactory bulbs and corpus callosum agenesis. There are 27 previous cases of maxillomandibular fusion and seven previous autopsy cases of hypophyseal duplication associated with other frontonasal malformations. As far as the authors know, this is the first case reported in the literature that associates syngnathia with duplication of the craniofacial midline including hypophyseal duplication.
Syngnathia; alveolar ridge fusion; duplication of craniofacial midline; agenesis corpus callosum; hypophyseal duplication
Nicotine is the primary component of tobacco that maintains the smoking habit and develops addiction. The adaptive changes of nicotinic acetylcholine receptors produced by repeated exposure to nicotine play a crucial role in the establishment of dependence. However, other neurochemical systems also participate in the addictive effects of nicotine including glutamate, cannabinoids, GABA and opioids. This review will cover the involvement of these neurotransmitters in nicotine addictive properties, with a special emphasis on the endogenous opioid system. Thus, endogenous enkephalins and beta-endorphins acting on mu-opioid receptors are involved in nicotine rewarding effects, whereas opioid peptides derived from prodynorphin participate in nicotine aversive responses. An upregulation of mu-opioid receptors has been reported after chronic nicotine treatment that could counteract the development of nicotine tolerance, whereas the downregulation induced on kappa-opioid receptors seems to facilitate nicotine tolerance. Endogenous enkephalins acting on mu-opioid receptors also play a role in the development of physical dependence to nicotine. In agreement with these actions of the endogenous opioid system, the opioid antagonist naltrexone has shown to be effective for smoking cessation in certain subpopulations of smokers.
addiction; enkephalin; dynorphin; beta-endorphin; tolerance; withdrawal; naltrexone; opioid receptor
Even though technological progress has provided us with more and more sophisticated equipment for making goniometric measurements, the most commonly used clinical tools are still the universal goniometer and, to a lesser extent, the inclinometer. There is, however, no published study so far that uses an inclinometer for measurements in children with cerebral palsy (CP). The objective of this study was two-fold: to independently assess the intra and inter-examiner reliability for measuring the hip abduction range of motion in children with CP using two different instruments, the universal two-axis goniometer and electronic inclinometer. A pool of 5 examiners with different levels of experience as paediatric physiotherapists participated. The study did not compare both instruments because the measurement procedure and the hip position were different for each.
A prospective, observational study of goniometery was carried out with 14 lower extremities of 7 children with spastic CP. The inclinometer study was carried out with 8 lower extremities of 4 children with spastic CP. This study was divided into two independent parts: a study of the reliability of the hip abduction range of motion measured with a universal goniometer (hip at 0° flexion) and with an electronic inclinometer (hip at 90° flexion). The Intraclass Correlation Coefficient (ICC) was calculated to analyse intra and inter-examiner agreement for each instrument.
For the goniometer, the intra-examiner reliability was excellent (>0.80), while the inter-examiner reliability was low (0.375 and 0.475). For the inclinometer, both the intra-examiner (0.850-0.975) and inter-examiner reliability were excellent (0.965 and 0.979).
The inter-examiner reliability for goniometric measurement of hip abduction in children with CP was low, in keeping with other results found in previous publications. The inclinometer has proved to be a highly reliable tool for measuring the hip abduction range of motion in children with CP, which opens up new possibilities in this field, despite having some measurement limitations.
There is an enormous gap between the antiproliferative and in vivo antitumor efficacy of gemcitabine in cell line-based models and its clinical efficacy. This may be due to insensitiveness of the precursor, cancer stem cell (CSC) compartment to cytotoxic agents. The hedgehog pathway is associated with CSC signaling and control. We used a direct xenograft model of pancreatic cancer and a two-stage approach was used to test the hypotheses that targeting CSC could increase the efficacy of gemcitabine. Tumors from a gemcitabine-sensitive xenograft were treated with gemcitabine first, and randomized, after tumor regression to continuing treatment with gemcitabine, a hedgehog inhibitor alone or in combination with gemcitabine. We tested markers described as associated with CSC such as CD24, CD44, ALDH, nestin, and the hedgehog pathway. After induction with gemcitabine, treated tumor showed an enrichment in CSC markers such as ALDH and CD24. Subsequently, a release from gemcitabine prompted a repopulation of proliferating cells and a decrease in such markers to equilibrate from pretreatment levels. Combined treatment with gemcitabine and cyclopamine induced tumor regression and decrease in CSC markers and hedgehog signaling. Cytoplasmic CD24 and ALDH were inversely and strongly associated with growth and were expressed in a minority of cells that we propose constitute the CSC compartment. Hedgehog inhibitors as part of a dual compartment therapeutic approach were able to further reduce tumor growth and decreased both static and dynamic markers of CSC. Direct tumor xenografts are a valid platform to test multicompartment therapeutic approaches in pancreatic cancer.
Lowering of blood pressure by antihypertensive drugs reduces the risks of cardiovascular events, stroke, and total mortality. However, poor adherence to antihypertensive medications reduces their effectiveness and increases the risk of adverse events. In terms of relative risk reduction, an improvement in medication adherence could be as effective as the development of a new drug.
The proposed randomized controlled trial will include patients with a low adherence to medication and uncontrolled blood pressure. The intervention group will receive a multifactorial intervention during the first, third, and ninth months, to improve adherence. This intervention will include motivational interviews, pill reminders, family support, blood pressure self-recording, and simplification of the dosing regimen.
The primary outcome is systolic blood pressure. The secondary outcomes are diastolic blood pressure, proportion of patients with adequately controlled blood pressure, and total cost.
The trial will evaluate the impact of a multifactorial adherence intervention in routine clinical practice. Ethical approval was given by the Ethical Committee on Human Research of Balearic islands, Spain (approval number IB 969/08 PI).
Current controlled trials ISRCTN21229328
While some targeted agents should not be used in squamous cell carcinomas (SCCs), other agents might preferably target SCCs. In a previous microarray study, one of the top differentially expressed genes between adenocarcinomas (ACs) and SCCs is P63. It is a well-known marker of squamous differentiation, but surprisingly, its expression is not widely used for this purpose. Our goals in this study were (1) to further confirm our microarray data, (2) to analize the value of P63 immunohistochemistry (IHC) in reducing the number of large cell carcinoma (LCC) diagnoses in surgical specimens, and (3) to investigate the potential of P63 IHC to minimize the proportion of “carcinoma NOS (not otherwise specified)” in a prospective series of small tumor samples.
With these goals in mind, we studied (1) a tissue-microarray comprising 33 ACs and 99 SCCs on which we performed P63 IHC, (2) a series of 20 surgically resected LCCs studied for P63 and TTF-1 IHC, and (3) a prospective cohort of 66 small thoracic samples, including 32 carcinoma NOS, that were further classified by the result of P63 and TTF-1 IHC.
The results in the three independent cohorts were as follows: (1) P63 IHC was differentially expressed in SCCs when compared to ACs (p<0.0001); (2) half of the 20 (50%) LCCs were positive for P63 and were reclassified as SCCs; and (3) all P63 positive cases (34%) were diagnosed as SCCs.
P63 IHC is useful for the identification of lung SCCs.
Emerging evidence suggests that the hypocretinergic system is involved in addictive behavior. In this study, we investigated the role of these hypothalamic neuropeptides in anxiety-like responses of nicotine and stress-induced reinstatement of nicotine-seeking behavior. Acute nicotine (0.8 mg/kg, sc) induced anxiogenic-like effects in the elevated plus-maze and activated the paraventricular nucleus of the hypothalamus (PVN) as revealed by c-Fos expression. Pretreatment with the hypocretin receptor 1 (Hcrtr-1) antagonist SB334867 or prepro-hypocretin gene deletion blocked both nicotine effects. In the PVN, SB334867 also prevented the activation of corticotrophin releasing factor (CRF) and arginine-vasopressin (AVP) neurons, which expressed Hcrtr-1. In addition, an increase of the percentage of c-Fos positive hypocretin cells in the perifornical and dorsomedial hypothalamic (PFA/DMH) areas was found after nicotine (0.8 mg/kg, sc) administration. Intracerebroventricular (icv) infusion of hypocretin-1 (Hcrt-1) (0.75 nmol/1 μl) or footshock stress reinstated a previously extinguished nicotine-seeking behavior. The effects of Hcrt-1 were blocked by SB334867, but not by the CRF1 receptor antagonist antalarmin. Moreover, SB334867 did not block CRF-dependent footshock-induced reinstatement of nicotine-seeking while antalarmin was effective in preventing this nicotine motivational response. Therefore, the Hcrt system interacts with CRF and AVP neurons in the PVN and modulates the anxiogenic-like effects of nicotine whereas Hcrt and CRF play a different role in the reinstatement of nicotine-seeking. Indeed, Hcrt-1 reinstates nicotine-seeking through a mechanism independent of CRF activation whereas CRF mediates the reinstatement induced by stress.
nicotine; hypocretin; CRF; anxiety; reinstatement; drug-seeking
Although hippotherapy treatment has been demonstrated to have therapeutic effects on children with cerebral palsy, the samples used in research studies have been very small. In the case of hippotherapy simulators, there are no studies that either recommend or advise against their use in the treatment of children with cerebral palsy. The aim of this randomised clinical study is to analyse the therapeutic effects or the contraindications of the use of a commercial hippotherapy simulator on several important factors relating to children with cerebral palsy such as their motor development, balance control in the sitting posture, hip abduction range of motion and electromyographic activity of adductor musculature.
The study is a randomised controlled trial. It will be carried out with a sample of 37 children with cerebral palsy divided into two treatment groups. Eligible participants will be randomly allocated to receive either (a) Treatment Group with hippotherapy simulator, maintaining sitting posture, with legs in abduction and rhythmic movement of the simulator or (b) Treatment Group maintaining sitting posture, with legs in abduction and without rhythmic movement of the simulator. Data collection and analysis: all measurements will be carried out by a specially trained blind assessor. To ensure standardization quality of the assessors, an inter-examiner agreement will be worked out at the start of the study. The trial is funded by the Department of Research, Innovation and Development of the Regional Government of Aragon (Official Bulletin of Aragon 23 July 2007), project number PM059/2007.
Interest in this project is due to the following factors: Clinical originality (there are no previous studies analysing the effect of simulators on the population group of children with CP, nor any studies using as many variables as this project); Clinical impact (infantile cerebral palsy is a chronic multisystemic condition that affects not only the patient but also the patient's family and their close circle of friends); Practical benefits (the development of an effective treatment is very important for introducing this element into the rehabilitation of these children).
Current Controlled Trials ISRCTN03663478.
Recent reports suggest dyslipidemia impairs dendritic cell (DC) function and adaptive immunity. This study aimed to characterize the effect of hypercholesterolemia on antigen-presenting cell function of DCs and DC-dependent CD4+ T-cell responses. DCs incubated in vitro with acetylated low-density lipoprotein cholesterol with or without an acyl-coenzyme A:cholesterol acyl-transferase inhibitor maintained their ability to prime CD4+ T cells. Analysis of T-cell proliferation and interferon-γ and tumor necrosis factor-α production after ex vivo coculture of naïve CD4+ T cells with splenic, inguinal, or iliac DCs from low-density lipoprotein receptor–deficient (LDLR−/−) or apolipoprotein E–deficient (ApoE−/−) mice fed an atherogenic diet highlighted DC efficacy in effector T-cell generation under hypercholesterolemic conditions. Adoptive transfer of carboxyfluorescein diacetate, succinimidyl ester (CFSE)-labeled naïve CD4+ T cells in LDLR−/− recipients and subsequent immunization demonstrated effective priming of naïve T cells in hypercholesterolemic mice. CFSE dilution analyses revealed that hypercholesterolemic DCs were equipotent in naïve CD4+ T-cell priming efficacy with normocholesterolemic DCs. Quantitative real-time PCR and flow cytometric analyses demonstrated that DC expression of multiple molecules involved in antigen processing, presentation, and T-cell stimulation remained unaltered by dyslipidemia. Finally, endogenous antigen-primed CD4+ T cells responded equivalently to a secondary ex vivo antigenic challenge, regardless of whether they were primed in vivo under hypercholesterolemic or control conditions, demonstrating that all essential steps in CD4+ T-cell responses remain intact under atherogenic conditions. This study affirms that the adaptive immune response prevails under the hypercholesterolemic conditions present in atherosclerosis. In particular, DCs remain functional antigen-presenting cells and maintain their ability to prime CD4+ T cells even when cholesterol-loaded.
dendritic cells; hypercholesterolemia; atherosclerosis; adaptive immunity
Amplification of the human epidermal growth factor receptor 2 (HER2) gene has been reported in gastric carcinoma (GC). Accordingly, trastuzumab plus chemotherapy has recently become the new standard treatment for HER2-positive advanced GCs. The aim was to compare the alleged gold standard for hybridization [fluorescence in-situ hybridization (FISH)] with a novel, fully automated brightfield dual-colour silver-enhanced in-situ hybridization (SISH) method.
Methods and results:
The studies series was comprised of 166 GC samples. Additionally, tumours with discordant results obtained by FISH and SISH were analysed by real-time quantitative polymerase chain reaction (PCR) with the LightMix kit HER-2/neu. Of the samples, 17.5% and 21% were amplified by FISH and SISH, respectively. Heterogeneity was identified in up to 52% of cases. In 96.4% of cases, FISH showed the same results as SISH. All six discordant cases were positive by SISH and negative by FISH. On review of the FISH slides, all contradictory cases were polysomic and were confirmed to be negative for amplification by real-time PCR. Interestingly, all ratios in this latter group were between 2.06 and 2.50, so setting the cut-off for amplification at ≥3 resulted in perfect concordance.
Dual-colour SISH represents a novel method for the determination of HER2 status in GC.
dual-colour hybridization; fluorescence in-situ hybridization; gastric carcinoma; human epidermal growth factor receptor 2; silver-enhanced in-situ hybridization