Melody, an ENU mutation in Caspase 3, alters the catalytic cysteine residue and causes sensorineural hearing loss in mice

Progeny from the Harwell N-ethyl-N-nitrosourea (ENU) recessive mutagenesis screen were assessed for auditory defects. A pedigree was identified with multiple progeny lacking response to a clickbox test. Auditory brainstem response (ABR) analysis showed that homozygous mutant mice were profoundly deaf and the line was named melody. We subsequently mapped this mutation to a 6-Mb region on chromosome 8 and identified a point mutation in melody that results in a C163S substitution in the catalytic site of Caspase 3, a cysteine protease involved in apoptosis. Melody fails to complement a null Caspase-3 mutant. Scanning electron microscopy (SEM) has revealed disorganised sensory hair cells and hair cell loss. Histological analysis of melody has shown degeneration of spiral ganglion cells in homozygote mice, with a gradient of severity from apical to basal turns. Melody heterozygotes also show evidence of loss of spiral ganglion neurons, suggesting that the C163S mutation may show dominant negative effects by binding and sequestering proteins at the active site. The melody line provides a new model for studying the role of Caspase 3 in deafness and a number of other pathways and systems

Initiation of V(D)J Recombination by Dβ-Associated Recombination Signal Sequences: A Critical Control Point in TCRβ Gene Assembly

T cell receptor (TCR) β gene assembly by V(D)J recombination proceeds via successive Dβ-to-Jβ and Vβ-to-DJβ rearrangements. This two-step process is enforced by a constraint, termed beyond (B)12/23, which prohibits direct Vβ-to-Jβ rearrangements. However the B12/23 restriction does not explain the order of TCRβ assembly for which the regulation remains an unresolved issue. The initiation of V(D)J recombination consists of the introduction of single-strand DNA nicks at recombination signal sequences (RSSs) containing a 12 base-pairs spacer. An RSS containing a 23 base-pairs spacer is then captured to form a 12/23 RSSs synapse leading to coupled DNA cleavage. Herein, we probed RSS nicks at the TCRβ locus and found that nicks were only detectable at Dβ-associated RSSs. This pattern implies that Dβ 12RSS and, unexpectedly, Dβ 23RSS initiate V(D)J recombination and capture their respective Vβ or Jβ RSS partner. Using both in vitro and in vivo assays, we further demonstrate that the Dβ1 23RSS impedes cleavage at the adjacent Dβ1 12RSS and consequently Vβ-to-Dβ1 rearrangement first requires the Dβ1 23RSS excision. Altogether, our results provide the molecular explanation to the B12/23 constraint and also uncover a ‘Dβ1 23RSS-mediated’ restriction operating beyond chromatin accessibility, which directs Dβ1 ordered rearrangements

Measuring Under-Five Mortality: Validation of New Low-Cost Methods

n/

Quantifying the impact of human immunodeficiency virus-1 escape from cytotoxic T-lymphocytes.

Published versio

Modelling the Evolution and Spread of HIV Immune Escape Mutants

During infection with human immunodeficiency virus (HIV), immune pressure from cytotoxic T-lymphocytes (CTLs) selects for viral mutants that confer escape from CTL recognition. These escape variants can be transmitted between individuals where, depending upon their cost to viral fitness and the CTL responses made by the recipient, they may revert. The rates of within-host evolution and their concordant impact upon the rate of spread of escape mutants at the population level are uncertain. Here we present a mathematical model of within-host evolution of escape mutants, transmission of these variants between hosts and subsequent reversion in new hosts. The model is an extension of the well-known SI model of disease transmission and includes three further parameters that describe host immunogenetic heterogeneity and rates of within host viral evolution. We use the model to explain why some escape mutants appear to have stable prevalence whilst others are spreading through the population. Further, we use it to compare diverse datasets on CTL escape, highlighting where different sources agree or disagree on within-host evolutionary rates. The several dozen CTL epitopes we survey from HIV-1 gag, RT and nef reveal a relatively sedate rate of evolution with average rates of escape measured in years and reversion in decades. For many epitopes in HIV, occasional rapid within-host evolution is not reflected in fast evolution at the population level

Progression to AIDS in South Africa Is Associated with both Reverting and Compensatory Viral Mutations

We lack the understanding of why HIV-infected individuals in South Africa progress to AIDS. We hypothesised that in end-stage disease there is a shifting dynamic between T cell imposed immunity and viral immune escape, which, through both compensatory and reverting viral mutations, results in increased viral fitness, elevated plasma viral loads and disease progression. We explored how T cell responses, viral adaptation and viral fitness inter-relate in South African cohorts recruited from Bloemfontein, the Free State (n = 278) and Durban, KwaZulu-Natal (n = 775). Immune responses were measured by γ-interferon ELISPOT assays. HLA-associated viral polymorphisms were determined using phylogenetically corrected techniques, and viral replication capacity (VRC) was measured by comparing the growth rate of gag-protease recombinant viruses against recombinant NL4-3 viruses. We report that in advanced disease (CD4 counts <100 cells/µl), T cell responses narrow, with a relative decline in Gag-directed responses (p<0.0001). This is associated with preserved selection pressure at specific viral amino acids (e.g., the T242N polymorphism within the HLA-B*57/5801 restricted TW10 epitope), but with reversion at other sites (e.g., the T186S polymorphism within the HLA-B*8101 restricted TL9 epitope), most notably in Gag and suggestive of “immune relaxation”. The median VRC from patients with CD4 counts <100 cells/µl was higher than from patients with CD4 counts ≥500 cells/µl (91.15% versus 85.19%, p = 0.0004), potentially explaining the rise in viral load associated with disease progression. Mutations at HIV Gag T186S and T242N reduced VRC, however, in advanced disease only the T242N mutants demonstrated increasing VRC, and were associated with compensatory mutations (p = 0.013). These data provide novel insights into the mechanisms of HIV disease progression in South Africa. Restoration of fitness correlates with loss of viral control in late disease, with evidence for both preserved and relaxed selection pressure across the HIV genome. Interventions that maintain viral fitness costs could potentially slow progression

Can Non-lytic CD8+T Cells Drive HIV-1 Escape?

The CD8+ T cell effector mechanisms that mediate control of HIV-1 and SIV infections remain poorly understood. Recent work suggests that the mechanism may be primarily non-lytic. This is in apparent conflict with the observation that SIV and HIV-1 variants that escape CD8+ T cell surveillance are frequently selected. Whilst it is clear that a variant that has escaped a lytic response can have a fitness advantage compared to the wild-type, it is less obvious that this holds in the face of non-lytic control where both wild-type and variant infected cells would be affected by soluble factors. In particular, the high motility of T cells in lymphoid tissue would be expected to rapidly destroy local effects making selection of escape variants by non-lytic responses unlikely. The observation of frequent HIV-1 and SIV escape poses a number of questions. Most importantly, is the consistent observation of viral escape proof that HIV-1- and SIV-specific CD8+ T cells lyse infected cells or can this also be the result of non-lytic control? Additionally, the rate at which a variant strain escapes a lytic CD8+ T cell response is related to the strength of the response. Is the same relationship true for a non-lytic response? Finally, the potential anti-viral control mediated by non-lytic mechanisms compared to lytic mechanisms is unknown. These questions cannot be addressed with current experimental techniques nor with the standard mathematical models. Instead we have developed a 3D cellular automaton model of HIV-1 which captures spatial and temporal dynamics. The model reproduces in vivo HIV-1 dynamics at the cellular and population level. Using this model we demonstrate that non-lytic effector mechanisms can select for escape variants but that outgrowth of the variant is slower and less frequent than from a lytic response so that non-lytic responses can potentially offer more durable control

Postpartum psychiatric disorders

Pregnancy is a complex and vulnerable period that presents a number of challenges to women, including the development of postpartum psychiatric disorders (PPDs). These disorders can include postpartum depression and anxiety, which are relatively common, and the rare but more severe postpartum psychosis. In addition, other PPDs can include obsessive–compulsive disorder, post-traumatic stress disorder and eating disorders. The aetiology of PPDs is a complex interaction of psychological, social and biological factors, in addition to genetic and environmental factors. The goals of treating postpartum mental illness are reducing maternal symptoms and supporting maternal–child and family functioning. Women and their families should receive psychoeducation about the illness, including evidence-based discussions about the risks and benefits of each treatment option. Developing effective strategies in global settings that allow the delivery of targeted therapies to women with different clinical phenotypes and severities of PPDs is essential