Toll-like receptor-dependent activation of several human blood cell types by protamine-condensed mRNA

We reported that RNA condensed on protamine is protected from RNase-mediated degradation and can be used for vaccination. Here, we show that such complexes are also danger signals that activate mouse cells through a MyD88-dependent pathway. Moreover, mRNA-protamine complexes stimulate human blood cells. They strongly activate DC and monocytes, leading to TNF-alpha and IFN-alpha secretion. In addition, protamine-RNA complexes directly activate B cells, NK cells and granulocytes. The detailed analysis of the activated cell types, the study of the cytokines released from PBMC cultured with protamine-RNA complexes and recently published results suggest that TLR-7 and TLR-8 may be involved in the recognition of protamine-stabilized RNA. Our data indicate that protamine-stabilized RNA, which may be similar to RNA condensed in the nucleocapsids of RNA viruses, is a strong danger signal. Thus, similarly to plasmid DNA, protamine-RNA combines antigen production and non-specific immunostimulation. The studies presented here explain the capacity of protamine-RNA to act as a vaccine, and pave the way towards the development of safe and efficient mRNA-based immunotherapies.     

Can Women Provide Reliable Information about Their Children''s Fathers? Cross-informant Agreement about Men''s Lifetime Antisocial Behaviour

It is difficult to study the contribution of fathers' antisocial behaviour to children's development because fathers with behavioural problems are often absent or reluctant to participate in research. This study examines whether mothers' reports about their children's fathers' antisocial behaviour can be substituted for interviews with fathers. Both members of 67 couples (N = 134) were interviewed separately and independently about the men's lifetime antisocial behaviour. There was strong relative agreement: the women's reports about men's antisocial behaviour and the men's self-reports about the same behaviour were highly correlated. However, there was poor agreement about absolute level: compared to men's self-reports, women reported fewer of the men's antisocial behaviours. Women's reports provide a reliable index of men's relative standing in a distribution and can be used in research about their children's fathers, but should not be used to make diagnostic decisions about men's antisocial disorders.     

Familial hypercholesterolaemia: what’s new?

Autosomal Dominant Familial Hypercholesterolaemia (FH) is the commonest inherited disorder of lipoprotein metabolism. Untreated monogenic FH caused by mutations in the LDLR, APOB or PCSK9 genes result in early onset cardiovascular death (below the age of 60 years). In the UK the prevalence of heterozygous FH is 1 in 270 and homozygous FH is 160,000 approximately.The introduction of statins nearly three decades ago has altered the natural history of FH, with a significant reduction of cardiovascular related morbidity and mortality. There is increasing evidence that early childhood interventions such as lifestyle choices, healthy eating and commencing statins by the age of 10 years would potentially prevent early onset cardiovascular disease and mortality in monogenic FH. The medium term safety of statins in children has been demonstrated. The UK paediatric FH register data has shown that children with FH are less obese than the normal population and the register aims to monitor the longer-term safety of statins in children with FH. Child-parent screening would potentially benefit the child and enables identifying a parent with FH, before the onset of a life threatening cardiovascular event. In addition, genetic cascade testing of relatives of an affected individual has been shown to be highly cost effective.We review the current literature with brief updates on genetics, the UK paediatric FH register data, published recommendations for the management of homozygous and heterozygous FH, lipid lowering therapies in children and screening for FH in childhood.     

Scanning of the dopamine D1 and D5 receptor genes by REF in neuropsychiatric patients reveals a novel missense change at a highly conserved amino acid

In previous analyses of schizophrenic patients, multiple missense changes and one nonsense change were identified in the D5 dopamine receptor (DRD5) gene, but no sequence changes of likely functional significance were identified in the D1 dopamine receptor (DRD1) gene. In the present study, we examined these genes in patients with certain other neuropsychiatric disorders that may be related to dopaminergic dysregulation. The coding regions of the DRD1 and DRD5 genes were examined in 25 and 25 autistic patients, 25 and 28 attention deficit hyperactivity disorder patients, and 51 and 43 alcoholic patients, respectively. In addition, the DRD5 gene was examined in 75 schizophrenic patients to search for additional variants affecting protein structure or expression (VAPSEs). These patients were analyzed with REF (restriction endonuclease fingerprinting), a hybrid between SSCP and restriction endonuclease digestion, which allows the entire coding sequence to be screened in one lane of a gel. Approximately 800 kb of genomic sequence were examined. No sequence changes were identified in the DRD1 gene among the 101 patient samples analyzed. Two sequence changes were identified in the DRD5 gene among the 171 patient samples. These included one previously identified silent polymorphism at base pair 978 (P326P). The change was identified in patients from all disease categories and from different ethnic backgrounds. One novel missense change, L88F, occurred in transmembrane domain II at a highly conserved amino acid in all dopamine receptors as well as in α1- and β-adrenergic receptors. The mutation was identified in a Caucasian male patient with autism. Further analysis is necessary to determine if this missense change is associated with a particular neuropsychiatric phenotype. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81:172–178, 1998. © 1998 Wiley-Liss, Inc.     

Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha

Telomere proteins protect the chromosomal terminus from nucleolytic degradation and end-to-end fusion, and they may contribute to telomere length control and the regulation of telomerase. The current studies investigate the effect of Oxytricha single-stranded telomere DNA-binding protein subunits α and β on telomerase elongation of telomeric DNA. A native agarose gel system was used to evaluate telomere DNA-binding protein complex composition, and the ability of telomerase to use these complexes as substrates was characterized. Efficient elongation occurred in the presence of the α subunit. Moreover, the α–DNA cross-linked complex was a substrate for telomerase. At higher α concentrations, two α subunits bound to the 16-nucleotide single-stranded DNA substrate and rendered it inaccessible to telomerase. The formation of this α·DNA·α complex may contribute to regulation of telomere length. The α·β·DNA ternary complex was not a substrate for telomerase. Even when telomerase was prebound to telomeric DNA, the addition of α and β inhibited elongation, suggesting that these telomere protein subunits have a greater affinity for the DNA and are able to displace telomerase. In addition, the ternary complex was not a substrate for terminal deoxynucleotidyltransferase. We conclude that the telomere protein inhibits telomerase by rendering the telomeric DNA inaccessible, thereby helping to maintain telomere length.     

Inhibition of Salmonella typhimurium Invasion by Host Cell Expression of Secreted Bacterial Invasion Proteins

Pathogenic Salmonella species initiate infection of a host by inducing their own uptake into intestinal epithelial cells. An invasive phenotype is conferred to this pathogen by a number of proteins that are components of a type III secretion system. During the invasion process, the bacteria utilize this secretion system to release proteins that enter the host cell and apparently interact with unknown host cell components that induce alterations in the actin cytoskeleton. To investigate the role of secreted proteins as direct modulators of invasion, we have evaluated the ability of Salmonella typhimurium to enter mammalian cells that express portions of the Salmonella invasion proteins SipB and SipC. Plasma membrane localization of SipB and SipC was achieved by fusing carboxyl- and amino-terminal portions of each invasion protein to the intracellular carboxyl-terminal tail of a membrane-bound eukaryotic receptor. Expression of receptor chimeras possessing the carboxyl terminus of SipB or the amino terminus of SipC blocked Salmonella invasion, whereas expression of their chimeric counterparts had no effect on invasion. The effect on invasion was specific for Salmonella since the perturbation of uptake was not extended to other invasive bacterial species. These results suggest that Salmonella invasion can be competitively inhibited by preventing the intracellular effects of SipB or SipC. In addition, these experiments provide a model for examining interactions between bacterial invasion proteins and their host cell targets.     

Dynamics of host blood effects inGlossina morsitans sspp. infected withTrypanosoma congolense andT. brucei

The pattern of infection inGlossina morsitans morsitans andG. m. centralis membrane-fed on eland, buffalo or goat blood mixed withTrypanosoma congolense orT. brucei was studied from day 1 to day 10. Tsetse were initially permissive vectors, with most flies harbouring infections of 10⁴–10⁵ parasites on day 3. However, after a second blood meal on day 3, flies cleared many infections withG. m. morsitans clearing more infections thanG. m. centralis. Infective feeds of goat blood consistently increased final infection rates by limiting the number of infections lost between days 3 and 6. In further experiments withG. m. morsitans only, this effect was replicated by feeding flies on erythrocytes but not on serum. These results suggest that compounds from some mammalian erythrocytes match the target specificity ofG. m. morsitans midgut lectins and, hence, have a protective effect on trypanosome establishment in the fly.     

Models for epilepsy and epileptogenesis: report from the NIH workshop,Bethesda, Maryland

PURPOSE: The workshop explored the current problems, needs, and potential usefulness of existing methods of discovery of new therapies to treat epilepsy patients. Resistance to medical therapy (pharmacoresistance) and the development of epilepsy (epileptogenesis) are recognized as two of the major problems in epilepsy treatment today. At the same time, there is growing awareness that the development of new therapies has slowed, a trend that has economic and scientific roots. To move toward new and more effective therapies, novel approaches to therapy discovery are needed. METHODS: A workshop was held in March 2001 with the charge to develop a plan to move the exploration and discovery process forward. Participants from academia, government, and industry reviewed the current status of epilepsy therapy and explored the identification of potential new therapies. RESULTS: At the end of the 2-day meeting, the panel made a series of recommendations. The two major recommendations were (a) to establish a means for continuing the examination of new approaches to therapy discovery, and (b) to identify models and approaches to therapy discovery that may identify treatments that are more successful than those available. Further recommendations were made to support the development of technology (miniaturization, computerization, video monitoring, etc.) to facilitate the use of the new models and to identify the mechanisms of therapy success and failure. CONCLUSIONS: Understanding the epidemiology of therapy resistance and providing support for new approaches to therapy development were identified as key issues for introduction of new and more effective treatments.