Transfer of central nervous system autoantigens and presentation in secondary lymphoid organs

Dendritic cells are thought to regulate tolerance induction vs immunization by transferring Ags and peripheral signals to draining lymph nodes (LN). However, whether myelin Ag transfer and presentation in LN occurs during demyelinating brain disease is unknown. In this study, we demonstrate redistribution of autoantigens from brain lesions to cervical LN in monkey experimental autoimmune encephalomyelitis (EAE) and in multiple sclerosis (MS). Immunohistochemical analysis revealed significantly more cells containing myelin Ags in cervical LN of monkeys with EAE compared with those of healthy control monkeys. Myelin Ags were observed in cells expressing dendritic cell/macrophage-specific markers, MHC class II, and costimulatory molecules. Moreover, these cells were directly juxtaposed to T cells, suggesting that cognate interactions between myelin-containing APC and T cells are taking place in brain-draining LN. Indeed, myelin Ag-reactive T cells were observed in cervical LN from marmosets and rhesus monkeys. Importantly, these findings were paralleled by our findings in human tissue. We observed significantly more myelin Ag-containing cells in LN of individuals with MS compared with those of control individuals. These cells expressed APC markers, as observed in marmosets and rhesus monkeys. These findings suggest that during MS and EAE, modulation of T cell reactivity against brain-derived Ags also takes place in cervical LN and not necessarily inside the brain. A major implication is that novel therapeutic strategies may be targeted to peripheral events, thereby circumventing the blood-brain barrier.     

Neighbourhood Deprivation and Outcomes of Stop Smoking Support – An Observational Study

Background

Rates of smoking and smoking cessation vary with socio-economic status. The objectives were to assess the association between neighbourhood deprivation, completion of treatment to support quit attempts and success of quit attempts—while taking into account other predictors of outcome.

Methods

555,744 quit attempts supported by English Stop Smoking Services in 2009–2012 were linked to the Index of Multiple Deprivation (IMD) 2010 ranks for the clients’ neighbourhood and split into deciles relative to the national IMD. Logistic regressions tested the association between neighbourhood deprivation and completion (4-week follow-up) of treatment and biochemically validated success (expired-air carbon monoxide <10ppm) while adjusting for demographics and intervention characteristics. Sensitivity analyses assessed subsamples: first supported attempts (n = 364,397), those with recorded cigarette dependence (n = 98,659) and completed treatment (n = 416,436).

Results

Higher neighbourhood deprivation was associated with reduced completion (OR_adj = 0.949, 95% CI: 0.947 to 0.951) and success (OR_adj = 0.957, 95% CI: 0.955 to 0.959). Results of sensitivity analyses were consistent with those of the main analysis.

Conclusions

Neighbourhood deprivation was associated with small but consistent reductions in completion and success of evidence-based interventions. These associations were not explained by intervention characteristics, demographics or dependence and reduced completion did not fully account for reduced success.     

A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish

Piwi proteins specify an animal-specific subclass of the Argonaute family that, in vertebrates, is specifically expressed in germ cells. We demonstrate that zebrafish Piwi (Ziwi) is expressed in both the male and the female gonad and is a component of a germline-specifying structure called nuage. Loss of Ziwi function results in a progressive loss of germ cells due to apoptosis during larval development. In animals that have reduced Ziwi function, germ cells are maintained but display abnormal levels of apoptosis in adults. In mammals, Piwi proteins associate with approximately 29-nucleotide-long, testis-specific RNA molecules called piRNAs. Here we show that zebrafish piRNAs are present in both ovary and testis. Many of these are derived from transposons, implicating a role for piRNAs in the silencing of repetitive elements in vertebrates. Furthermore, we show that piRNAs are Dicer independent and that their 3' end likely carries a 2'O-Methyl modification.     

An inventory of shedding data from clinical gene therapy trials

Viruses are the most commonly used vectors for clinical gene therapy. The risk of dissemination of a viral vector into the environment via excreta from the treated patient, a phenomenon called shedding, is a major safety concern for the environment. Despite the significant number of clinical gene therapy trials that have been conducted worldwide, there is currently no overview of actual shedding data available. In this article, an inventory of shedding data obtained from a total of 100 publications on clinical gene therapy trials using retroviral, adenoviral, adeno-associated viral and pox viral vectors is presented. In addition, the experimental set-up for shedding analysis including the assays used and biological materials tested is summarized. The collected data based on the analysis of 1619 patients in total demonstrate that shedding of these vectors occurs in practice, mainly determined by the type of vector and the route of vector administration. Due to the use of non-quantitative assays, the lack of information on assay sensitivity in most publications, and the fact that assay sensitivity is expressed in various ways, general conclusions cannot be made as to the level of vector shedding. The evaluation of the potential impact and consequences of the observations is complicated by the high degree of variety in the experimental design of shedding analysis between trials. This inventory can be supportive to clinical gene therapy investigators for the establishment of an evidence-based risk assessment to be included in a clinical protocol application, as well as to national regulatory authorities for the ongoing development of regulatory guidelines regarding gene therapy.     

Primary glioblastoma cultures: can profiling of stem cell markers predict radiotherapy sensitivity?

Human glioblastomas may be hierarchically organized. Within this hierarchy, glioblastoma‐initiating cells have been proposed to be more resistant to radiochemotherapy and responsible for recurrence. Here, established stem cell markers and stem cell attributed characteristics such as self‐renewal capacity and tumorigenicity have been profiled in primary glioblastoma cultures to predict radiosensitivity. Furthermore, the sensitivity to radiotherapy of different subpopulations within a single primary glioblastoma culture was analyzed by a flow cytometric approach using Nestin, SRY (sex‐determining region Y)‐box 2 (SOX2) and glial fibrillary acidic protein. The protein expression of Nestin and SOX2 as well as the mRNA levels of Musashi1, L1 cell adhesion molecule, CD133, Nestin, and pleiomorphic adenoma gene‐like 2 inversely correlated with radioresistance in regard to the clonogenic potential. Only CD44 protein expression correlated positively with radioresistance. In terms of proliferation, Nestin protein expression and Musashi1, pleiomorphic adenoma gene‐like 2, and CD133 mRNA levels are inversely correlated with radioresistance. Higher expression of stem cell markers does not correlate with resistance to radiochemotherapy in the cancer genome atlas glioblastoma collective. SOX2 expressing subpopulations exist within single primary glioblastoma cultures. These subpopulations predominantly form the proliferative pool of the primary cultures and are sensitive to irradiation. Thus, profiling of established stem cell markers revealed a surprising result. Except CD44, the tested stem cell markers showed an inverse correlation between expression and radioresistance.

     

Mouse models for methylmalonic aciduria

Methylmalonic aciduria (MMA) is a disorder of organic acid metabolism resulting from a functional defect of methylmalonyl-CoA mutase (MCM). MMA is associated with significant morbidity and mortality, thus therapies are necessary to help improve quality of life and prevent renal and neurological complications. Transgenic mice carrying an intact human MCM locus have been produced. Four separate transgenic lines were established and characterised as carrying two, four, five or six copies of the transgene in a single integration site. Transgenic mice from the 2-copy line were crossed with heterozygous knockout MCM mice to generate mice hemizygous for the human transgene on a homozygous knockout background. Partial rescue of the uniform neonatal lethality seen in homozygous knockout mice was observed. These rescued mice were significantly smaller than control littermates (mice with mouse MCM gene). Biochemically, these partial rescue mice exhibited elevated methylmalonic acid levels in urine, plasma, kidney, liver and brain tissue. Acylcarnitine analysis of blood spots revealed elevated propionylcarnitine levels. Analysis of mRNA expression confirms the human transgene is expressed at higher levels than observed for the wild type, with highest expression in the kidney followed closely by brain and liver. Partial rescue mouse fibroblast cultures had only 20% of the wild type MCM enzyme activity. It is anticipated that this humanised partial rescue mouse model of MMA will enable evaluation of long-term pathophysiological effects of elevated methylmalonic acid levels and be a valuable model for the investigation of therapeutic strategies, such as cell transplantation.     

Long-lasting hippocampal synaptic protein loss in a mouse model of posttraumatic stress disorder

Despite intensive research efforts, the molecular pathogenesis of posttraumatic stress disorder (PTSD) and especially of the hippocampal volume loss found in the majority of patients suffering from this anxiety disease still remains elusive. We demonstrated before that trauma-induced hippocampal shrinkage can also be observed in mice exhibiting a PTSD-like syndrome. Aiming to decipher the molecular correlates of these trans-species posttraumatic hippocampal alterations, we compared the expression levels of a set of neurostructural marker proteins between traumatized and control mice at different time points after their subjection to either an electric footshock or mock treatment which was followed by stressful re-exposure in several experimental groups. To our knowledge, this is the first systematic in vivo study analyzing the long-term neuromolecular sequelae of acute traumatic stress combined with re-exposure. We show here that a PTSD-like syndrome in mice is accompanied by a long-lasting reduction of hippocampal synaptic proteins which interestingly correlates with the strength of the generalized and conditioned fear response but not with the intensity of hyperarousal symptoms. Furthermore, we demonstrate that treatment with the serotonin reuptake inhibitor (SSRI) fluoxetine is able to counteract both the PTSD-like syndrome and the posttraumatic synaptic protein loss. Taken together, this study demonstrates for the first time that a loss of hippocampal synaptic proteins is associated with a PTSD-like syndrome in mice. Further studies will have to reveal whether these findings are transferable to PTSD patients.