Showing papers by "Richard W. Titball published in 2018"

Standardization of G. mellonella Larvae to Provide Reliable and Reproducible Results in the Study of Fungal Pathogens

Abstract: In the past decade, Galleria mellonella (wax moth) larvae have become widely used as a non-mammalian infection model However, the full potential of this infection model has yet to be realised, limited by the variable quality of larvae used and the lack of standardised procedures Here, we review larvae suitable for research, protocols for dosing larvae, and methods for scoring illness in larvae infected with fungal pathogens The development of standardised protocols for carrying out our experimental work will allow high throughput screens to be developed, changing the way in which we evaluate panels of mutants and strains It will also enable the in vivo screening of potential antimicrobials at an earlier stage in the research and development cycle

Galleria mellonella as an infection model to investigate virulence of Vibrio parahaemolyticus.

Abstract: Non-toxigenic V. parahaemolyticus isolates (tdh−/trh−/T3SS2−) have recently been isolated from patients with gastroenteritis. In this study we report that the larvae of the wax moth (Galleria mello...

The Culture Environment Influences Both Gene Regulation and Phenotypic Heterogeneity in Escherichia coli.

Abstract: Microorganisms shape the composition of the medium they are growing in, which in turn has profound consequences on the reprogramming of the population gene-expression profile. In this paper, we investigate the progressive changes in pH and sugar availability in the medium of a growing Escherichia coli (E. coli) culture. We show how these changes have an effect on both the cellular heterogeneity within the microbial community and the gene-expression profile of the microbial population. We measure the changes in gene-expression as E. coli moves from lag, to exponential, and finally into stationary phase. We found that pathways linked to the changes in the medium composition such as ribosomal, tricarboxylic acid cycle (TCA), transport, and metabolism pathways are strongly regulated during the different growth phases. In order to quantify the corresponding temporal changes in the population heterogeneity, we measure the fraction of E. coli persisters surviving different antibiotic treatments during the various phases of growth. We show that the composition of the medium in which β-lactams or quinolones, but not aminoglycosides, are dissolved strongly affects the measured phenotypic heterogeneity within the culture. Our findings contribute to a better understanding on how the composition of the culture medium influences both the reprogramming in the population gene-expression and the emergence of phenotypic variants.

Variant proteins stimulate more IgM+ GC B-cells revealing a mechanism of cross-reactive recognition by antibody memory

Abstract: Many devastating infectious diseases are caused by viruses that change over time. When a vaccine exists, it usually protects against a particular strain of virus, but often fails to defend against new versions of the microbe. This is why the flu vaccine has to be ‘updated’ every year, for example. Vaccines rely on the memory of our immune system. When a virus enters the body, a group of immune cells known as B cells gets activated. Certain B cells can recognise the invader and produce specific proteins, the antibodies, which can target and kill the invader. During the infection some of these B cells become ‘memory B cells’, having gone through a maturation process that hones their ability to specifically recognize this particular microbe. If the same virus enters the organism again, the memory B cells rapidly identify it and produce a quicker and more efficient immune response than during the first attack. This is how vaccines work. However memory B cells may not be able to recognize a previous intruder if it has changed too much. The memory B cell population is diverse. Some cells are fully mature and can quickly recognize the original virus. But others have not finished their maturation process: these cells are less focussed, and cannot target the original microbe with the same exact precision shown by mature memory cells. For almost forty years it was thought that this reduced focus might make the immature cells better at identifying new versions of the original attacker, but up until now, it was not clear what these memory cells could do. Here Burton, Tennant et al. injected a group of mice with proteins from the Dengue virus, which prompted an immune reaction. After several weeks, the animals received either the same proteins again, or proteins that were different. Compared to the fully mature cells, the immature memory B cells were much better at recognizing the variants of the proteins, and these cells then multiplied and mounted an immune response. Without the original protein injection, the response without the immature memory B cells was not as efficient. The body therefore has a pool of memory B cells that can recognise a wider range of virus protein variants than the ones that caused the first immune reaction. Understanding the role of immature memory B cells in immunity could help design vaccines that protect against several strains or fast-evolving viruses. This could have the potential to reduce the severity of diseases that affect hundreds of millions of people every year.

Interactions Between Pseudomonas Immunotoxins and the Plasma Membrane: Implications for CAT-8015 Immunotoxin Therapy.

Abstract: Acute Lymphoblastic Leukaemia (ALL) remains the most frequent cause of cancer-related mortality in children and novel therapies are needed for the treatment of relapsed/refractory childhood ALL. One approach is the targeting of ALL blasts with the Pseudomonas immunotoxin CAT-8015. Although CAT-8015 has potent anti-leukaemia activity, with a 32 % objective response rate in a phase 1 study of childhood ALL, haemolytic-uremic syndrome (HUS) and vascular leak syndrome (VLS), major dose-limiting toxicities, have limited the use of this therapeutic approach in children. Investigations into the pathogenesis of CAT-8015-induced HUS/VLS are hindered by the lack of an adequate model system that replicates clinical manifestations, but damage to vascular endothelial cells (ECs) and blood cells are believed to be major initiating factors in both syndromes. Since there is little evidence that murine models replicate human HUS/VLS, and CAT-8015-induced HUS/VLS predominantly affects children, we developed human models and used novel methodologies to investigate CAT-8015 interactions with red blood cells (RBCs) from paediatric ALL patients and ECs of excised human mesenteric arteries. We provide evidence that CAT-8015 directly interacts with RBCs, mediated by Pseudomonas toxin. We also show correlation between the electrical properties of the RBC membrane and RBC susceptibility to CAT-8015-induced lysis, which may have clinical implication. Finally, we provide evidence that CAT-8015 is directly cytototoxic to ECs of excised human mesenteric arteries. In conclusion, the human models we developed constitutes the first, and very important, step in understanding the origins of HUS/VLS in immunotoxin therapy and will allow further investigations of HUS/VLS pathogenesis.

Genome Sequence of Staphylococcus aureus Ex1, Isolated from a Patient with Spinal Osteomyelitis.

Abstract: Here, we present the genome sequence of Staphylococcus aureus Ex1, isolated in 2015 from a patient with spinal osteomyelitis at the Royal Devon and Exeter Hospital in the United Kingdom. The availability of the Ex1 genome sequence provides a resource for studying the basis for spinal infection and horizontal gene transfer in S. aureus.