A Phase I Double Blind, Placebo-Controlled, Randomized Study of a Multigenic HIV-1 Adenovirus Subtype 35 Vector Vaccine in Healthy Uninfected Adults

<div><h3>Background</h3><p>We conducted a phase I, randomized, double-blind, placebo-controlled trial to assess the safety and immunogenicity of escalating doses of two recombinant replication defective adenovirus serotype 35 (Ad35) vectors containing gag, reverse transcriptase, integrase and nef (Ad35-GRIN) and env (Ad35-ENV), both derived from HIV-1 subtype A isolates. The trial enrolled 56 healthy HIV-uninfected adults.</p> <h3>Methods</h3><p>Ad35-GRIN/ENV (Ad35-GRIN and Ad35-ENV mixed in the same vial in equal proportions) or Ad35-GRIN was administered intramuscularly at 0 and 6 months. Participants were randomized to receive either vaccine or placebo (10/4 per group, respectively) within one of four dosage groups: Ad35-GRIN/ENV 2×10⁹ (A), 2×10¹⁰ (B), 2×10¹¹ (C), or Ad35-GRIN 1×10¹⁰ (D) viral particles.</p> <h3>Results</h3><p>No vaccine-related serious adverse event was reported. Reactogenicity events reported were dose-dependent, mostly mild or moderate, some severe in Group C volunteers, all transient and resolving spontaneously. IFN-γ ELISPOT responses to any vaccine antigen were detected in 50, 56, 70 and 90% after the first vaccination, and in 75, 100, 88 and 86% of Groups A–D vaccine recipients after the second vaccination, respectively. The median spot forming cells (SFC) per 10⁶ PBMC to any antigen was 78–139 across Groups A–C and 158–174 in Group D, after each of the vaccinations with a maximum of 2991 SFC. Four to five HIV proteins were commonly recognized across all the groups and over multiple timepoints. CD4+ and CD8+ T-cell responses were polyfunctional. Env antibodies were detected in all Group A–C vaccinees and Gag antibodies in most vaccinees after the second immunization. Ad35 neutralizing titers remained low after the second vaccination.</p> <h3>Conclusion/Significance</h3><p>Ad35-GRIN/ENV reactogenicity was dose-related. HIV-specific cellular and humoral responses were seen in the majority of volunteers immunized with Ad35-GRIN/ENV or Ad35-GRIN and increased after the second vaccination. T-cell responses were broad and polyfunctional.</p> <h3>Trial Registration</h3><p>ClinicalTrials.gov <a href="http://clinicaltrials.gov/ct2/results?term=NCT00851383">NCT00851383</a></p> </div

The Inner Centromere Protein (INCENP) Coil Is a Single α-Helix (SAH) Domain That Binds Directly to Microtubules and Is Important for Chromosome Passenger Complex (CPC) Localization and Function in Mitosis

The chromosome passenger complex (CPC) is a master regulator of mitosis. INCENP acts as a scaffold regulating CPC localisation and activity. During early mitosis the N-terminal region of INCENP forms a three-helix bundle with Survivin and Borealin, directing the CPC to the inner centromere where it plays essential roles in chromosome alignment and the spindle assembly checkpoint. The C-terminal IN-box region of INCENP is responsible for binding and activating Aurora B kinase. The central region of INCENP has been proposed to comprise a coiled-coil domain acting as a spacer between the N and C terminal domains that is involved in microtubule binding and regulation of the spindle checkpoint. Here we show that the central region (213 residues) of chicken INCENP is not a coiled coil but a ~32 nm long single alpha helical (SAH) domain. The N-terminal half of this domain directly binds to microtubules in vitro. By analogy with previous studies of myosin 10, our data suggest that the INCENP SAH might stretch up to ~80 nm under physiological forces. Thus, the INCENP SAH could act as a flexible dog-leash allowing Aurora B to phosphorylate dynamic substrates localized in the outer kinetochore while at the same time being stably anchored to the stable chromatin of the inner centromere. Furthermore, by achieving this flexibility via a SAH domain, the CPC avoids a need for dimerization (required for coiled-coil formation), which would greatly complicate regulation of the proximity-induced trans-phosphorylation that is critical for Aurora B activation

A Mouse Model for Osseous Heteroplasia

GNAS/Gnas encodes Gsa that is mainly biallelically expressed but shows imprinted expression in some tissues. In Albright Hereditary Osteodystrophy (AHO) heterozygous loss of function mutations of GNAS can result in ectopic ossification that tends to be superficial and attributable to haploinsufficiency of biallelically expressed Gsa. Oed-Sml is a point missense mutation in exon 6 of the orthologous mouse locus Gnas. We report here both the late onset ossification and occurrence of benign cutaneous fibroepithelial polyps in Oed-Sml. These phenotypes are seen on both maternal and paternal inheritance of the mutant allele and are therefore due to an effect on biallelically expressed Gsa. The ossification is confined to subcutaneous tissues and so resembles the ossification observed with AHO. Our mouse model is the first with both subcutaneous ossification and fibroepithelial polyps related to Gsa deficiency. It is also the first mouse model described with a clinically relevant phenotype associated with a point mutation in Gsa and may be useful in investigations of the mechanisms of heterotopic bone formation. Together with earlier results, our findings indicate that Gsa signalling pathways play a vital role in repressing ectopic bone formation

Choosing to run: a history of gender and athletics in Kenya, c. 1940s - 1980s

Choosing to Run: A History of Athletics and Gender in Kenya, c. 1940s – 1980s explores the history of gender and athletics in Kenya, with focus on the Rift Valley Province, from the onset of late colonial rule in the 1940s through the professionalisation of the sport during the last decades of the twentieth century. The first two empirical chapters provide a history of athletics during the colonial period. The first highlights the continuity of ideas about sport and masculinity that were developed in nineteenth century Britain and were subsequently perpetuated by the men in charge of colonial sport in Kenya. The next chapter considers how pre-colonial divisions of labour and power within Rift Valley communities informed local peoples' cultures of running. The absence of women’s running was not only the result of sexism translated from the British metropole to its Kenyan colony but also of pre-existing divisions of responsibilities of indigenous Kenyan men and women into separate, gendered domains. The second half of the thesis considers the impact of social change within women’s athletics internationally and of marriage, childbirth and education locally on female runners in the Rift Valley during the post-colonial period. Most women abandoned athletics once they reached maturity. Those who sought to do otherwise, as the final chapter argues, found that they could only do so by replicating the prototype of masculine runners that had already been established. Later, after the professionalisation of running allowed women to become wealthy, female patrons took this a step further by providing resources to those in their community in need, setting themselves up as 'Big (Wo)men'. This thesis uses athletics to reveal how gender relations and gender norms have evolved and the benefits and challenges that the sport has brought both to individual Kenyan women and their communities.This thesis is not currently available in ORA

Urethral Obstruction by Seminal Coagulum is Associated with Medetomidine–Ketamine Anesthesia in Male Mice on C57BL/6J and Mixed Genetic Backgrounds

Male and female mice were anesthetized by intraperitoneal injection with a mixture delivering 0.5 mg/kg medetomidine and 50 mg/kg ketamine to achieve immobilization for whole-body radiographs and bone densitometry, as part of a phenotypic screen for bone and mineral disorders in mice carrying genetic modifications induced through mutagenesis with N′-ethyl-N′-nitrosourea. Morbidity and mortality occurred in 19 of 628 (3%) of male mice 24 to 72 h after a seemingly uneventful recovery from anesthesia. No morbidity or mortality occurred in 1564 female mice that were similar in age to the affected male mice and that underwent the same procedure. Of the 7 male mice that underwent postmortem examinations, 5 had urinary bladders grossly distended with urine and 1 had ascites. In addition, the pelvic or penile urethra in 5 of the examined male mice was obstructed with seminal coagulum associated with varying degrees of erosion of the urothelial lining and inflammation of the urethra. In 2 of these animals, from which plasma samples were recovered, azotemia, hyperphosphatemia, and hyperkalemia were present. The predilection for delayed morbidity and mortality in males after anesthesia suggests that anesthesia with 0.5 mg/kg medetomidine and 50 mg/kg ketamine is a potential risk factor for obstructive uropathy due to release of seminal coagulum. This adverse effect did not recur when we altered our anesthesia protocol to 10 mg/kg xylazine and 100 mg/kg ketamine

The Pseudosubstrate domain of Protein Kinase C-epsilon directs translocation to phagosomes via binding to phosphomonoinositides (135.46)

Abstract Protein kinase C-epsilon (PKC-ε) translocation is required for efficient IgG-mediated phagocytosis. We have published that the C1B region, binding to DAG generated by PLCγ1, is necessary for translocation (Cheeseman et al, MCB, 2006). However, C1B is not sufficient for translocation. Deletion and mutational analysis, as well as the use of PKC-ε/PKC-δ chimeras, indicate that the pseudosubstrate domain (εPS) is necessary for PKC-ε translocation. A PKC-ε fragment containing only the C1 and PS regions, as well as a chimera of PKC-δ containing εPS and εC1B, accumulated, demonstrating that these two regions are necessary and sufficient for targeting PKC-ε to phagosomes. Protein-lipid overlays of εPS-GST indicate that εPS preferentially associates with phosphomonoinositides (PI5P, PI3P&amp;gt;PI4P&amp;gt;&amp;gt;&amp;gt;polyphospho-inositides). Of the PI3 Kinase inhibitors, 100 nM wortmannin, but not 50 µM LY294002 inhibits PKC-ε translocation to phagosomes. That both drugs block the formation of PI3P as indicated by 2XFYVE staining argues against PI3P as the docking lipid for PKC-ε. Similar to PKC-ε, wortmannin, but not LY294002, blocked accumulation of PLCδPH (a PI4,5P2 reporter) at forming phagosomes. Together, these data suggest that εPS binding to PI4P and/or PI5P is necessary for PKC-ε translocation to phagosomes. Efforts are underway to identify the specific lipid binding partner for PKC-ε. Funded by NIH R01 AI05821 (MRL).</jats:p