Rapid Transepithelial Transport of Prions following Inhalation

Prion infection and pathogenesis are dependent on the agent crossing an epithelial barrier to gain access to the recipient nervous system. Several routes of infection have been identified, but the mechanism(s) and timing of in vivo prion transport across an epithelium have not been determined. The hamster model of nasal cavity infection was used to determine the temporal and spatial parameters of prion-infected brain homogenate uptake following inhalation and to test the hypothesis that prions cross the nasal mucosa via M cells. A small drop of infected or uninfected brain homogenate was placed below each nostril, where it was immediately inhaled into the nasal cavity. Regularly spaced tissue sections through the entire extent of the nasal cavity were processed immunohistochemically to identify brain homogenate and the disease-associated isoform of the prion protein (PrP^d). Infected or uninfected brain homogenate was identified adhering to M cells, passing between cells of the nasal mucosa, and within lymphatic vessels of the nasal cavity at all time points examined. PrP^d was identified within a limited number of M cells 15 to 180 min following inoculation, but not in the adjacent nasal mucosa-associated lymphoid tissue (NALT). While these results support M cell transport of prions, larger amounts of infected brain homogenate were transported paracellularly across the respiratory, olfactory, and follicle-associated epithelia of the nasal cavity. These results indicate that prions can immediately cross the nasal mucosa via multiple routes and quickly enter lymphatics, where they can spread systemically via lymph draining the nasal cavity.     

Polymorphic Variation in TIRAP Is Not Associated with Susceptibility to Childhood TB but May Determine Susceptibility to TBM in Some Ethnic Groups

Host recognition of mycobacterial surface molecules occurs through toll like receptors (TLR) 2 and 6. The adaptor protein TIRAP mediates down stream signalling of TLR2 and 4, and polymorphisms in the TIRAP gene (TIRAP) have been associated with susceptibility and resistance to tuberculosis (TB) in adults. In order to investigate the role of polymorphic variation in TIRAP in childhood TB in South Africa, which has one of the highest TB incidence rates in the world, we screened the entire open reading frame of TIRAP for sequence variation in two cohorts of childhood TB from different ethnic groups (Xhosa and mixed ancestry). We identified 13 SNPs, including seven previously unreported, in the two cohorts, and found significant differences in frequency of the variants between the two ethnic groups. No differences in frequency between individual SNPs or combinations were found between TB cases and controls in either cohort. However the 558C→T SNP previously associated with TB meningitis (TBM) in a Vietnamese population was found to be associated with TBM in the mixed ancestry group. Polymorphisms in TIRAP do not appear to be involved in childhood TB susceptibility in South Africa, but may play a role in determining occurrence of TBM.     

Rapid Screening of Gene Function by Systemic Delivery of Morpholino Oligonucleotides to Live Mouse Embryos

Traditional gene targeting methods in mice are complex and time consuming, especially when conditional deletion methods are required. Here, we describe a novel technique for assessing gene function by injection of modified antisense morpholino oligonucleotides (MOs) into the heart of mid-gestation mouse embryos. After allowing MOs to circulate through the embryonic vasculature, target tissues were explanted, cultured and analysed for expression of key markers. We established proof-of-principle by partially phenocopying known gene knockout phenotypes in the fetal gonads (Stra8, Sox9) and pancreas (Sox9). We also generated a novel double knockdown of Gli1 and Gli2, revealing defects in Leydig cell differentiation in the fetal testis. Finally, we gained insight into the roles of Adamts19 and Ctrb1, genes of unknown function in sex determination and gonadal development. These studies reveal the utility of this method as a means of first-pass analysis of gene function during organogenesis before committing to detailed genetic analysis.     

Preadolescents’ relationships with pet dogs: Relationship continuity and associations with adjustment

Research on human–animal interaction in children has been studied in isolation rather than integrated with core theories of children’s relationships. This study is one of the first to examine how children’s relationships with pet dogs are related to their human relationships (parent–child attachments, friendships) and to child adjustment, and to include observational assessment of children’s interactions with their pet dog. Children (9 to 11 years old, n = 99) completed questionnaires regarding relationships with pet dogs, parents, and friends. Half the children were observed interacting with their pet dog. Children and teachers reported children’s adjustment. Children who felt closer to their dogs were more securely attached to mothers and fathers and reported more positive qualities and less conflict with friends. Children with more secure attachments to mothers, and greater companionship with dogs, interacted more with their dogs. Parental attachment and friendship quality, but not the pet dog relationship, were related to child adjustment.     

Diminished Reovirus Capsid Stability Alters Disease Pathogenesis and Littermate Transmission

Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.     

Automatic Detection of Attention Shifts in Infancy: Eye Tracking in the Fixation Shift Paradigm

This study measured changes in switches of attention between 1 and 9 months of age in 67 typically developing infants. Remote eye-tracking (Tobii X120) was used to measure saccadic latencies, related to switches of fixation, as a measure of shifts of attention, from a central stimulus to a peripheral visual target, measured in the Fixation Shift Paradigm. Fixation shifts occur later if the central fixation stimulus stays visible when the peripheral target appears (competition condition), than if the central stimulus disappears as the peripheral target appears (non-competition condition). This difference decreases with age. Our results show significantly faster disengagement in infants over 4 months than in the younger group, and provide more precise measures of fixation shifts, than behavioural observation with the same paradigm. Reduced saccadic latencies in the course of a test session indicate a novel learning effect. The Fixation Shift Paradigm combined with remote eye-tracking measures showed improved temporal and spatial accuracy compared to direct observation by a trained observer, and allowed an increased number of trials in a short testing time. This makes it an infant-friendly non-invasive procedure, involving minimal observational training, suitable for use in future studies of clinical populations to detect early attentional abnormalities in the first few months of life.