Production and proteomic characterisation of purified protein derivative from Mycobacterium avium subsp. paratuberculosis

<p>Abstract</p> <p>Background</p> <p>Effective diagnosis of Johne's disease (JD), particularly at the stage of early subclinical infection, remains one of the greatest challenges for the control of JD worldwide. The IFN-γ test of cell mediated immunity is currently one of the most suitable diagnostics for subclinical infections, however a major limitation of this test is the lack of a standardised purified protein derivative (PPD) antigen (also referred to as Johnin PPD or PPDj). While attempting to replace PPDj with more specific individual antigens is an attractive proposition, bacterial culture derived PPDj remains the most effective antigen preparation for the diagnosis of subclinical JD. It may be possible to increase the reproducibility and specificity of PPDj preparations by further characterising and standardising the PPDj production.</p> <p>Results</p> <p>Using a standardised protocol, five in-house preparations of PPDj were prepared from cultures of <it>Mycobacterium avium </it>subsp. <it>paratuberculosis </it>(MAP). Compared to PPDs obtained from other institutes/laboratories, these preparations appeared to perform similarly well in the IFN-γ test. Although the broad proteomic composition of all PPDj preparations was remarkably similar, the absolute abundance of individual proteins varied markedly between preparations. All PPDj preparations contained common immunogenic proteins which were also observed in PPD preparations from <it>Mycobacterium avium </it>subsp. <it>avium </it>(PPDa) and <it>Mycobacterium bovis </it>(PPDb). Temporal difference in protein secretion of <it>in vitro </it>cultured MAP was observed between 20 and 34 weeks suggesting that the age of MAP culture used for PPDj preparations may markedly influence PPDj composition.</p> <p>Conclusions</p> <p>This study describes a protocol for the production of PPDj and its subsequent proteomic characterisation. The broad proteomic composition of different preparations of PPDj was, for the most part, highly similar. Compositional differences between PPDj preparations were found to be a direct reflection of genetic differences between the MAP strain types used to produce these preparations and the age of MAP cultures they were derived from. A number of conserved immunogenic proteins, such as members of the cutinase-like protein family, were found to be more abundant in PPDj compared to PPDa and should be considered as possible diagnostic antigens for the future.</p

A Deep Sequencing Approach to Comparatively Analyze the Transcriptome of Lifecycle Stages of the Filarial Worm, Brugia malayi

Lymphatic filariasis, also known as elephantiasis, is a tropical disease affecting over 120 million people worldwide. More than 40 million people live with painful, disfiguring symptoms that can cause severe debilitation and social stigma. The disease is caused by infection with thread-like filarial nematodes (roundworms) that have a complex parasitic lifecycle involving both human and mosquito hosts. In the study, the authors profiled the transcriptome (the set of genes transcribed into messenger RNA rather than all of those in the genome) of the human filarial worm Brugia malayi in different lifecyle stages using deep sequencing technology. The analysis revealed major transitions in RNA expression from eggs through larval stages to adults. Using statistical approaches, the authors identified groups of genes with distinct life stage dependent transcriptional patterns, with particular emphasis on genes displaying sex-biased or germline-enriched patterns and those displaying significant changes during larval development. This study presents a first comprehensive analysis of the lifecycle transcriptome of B. malayi, providing fundamental molecular information that should help researchers better understand parasite biology and could provide clues for the development of more effective interventions

EACVI survey on investigations and imaging modalities in chronic coronary syndromes

AIMS The European Association of Cardiovascular Imaging (EACVI) Scientific Initiatives Committee performed a global survey to evaluate current practice for the assessment and management of patients with suspected and confirmed chronic coronary syndromes. METHODS AND RESULTS One-hundred and ten imaging centres from 37 countries across the world responded to the survey. Most non-invasive investigations for coronary artery disease were widely available, except cardiovascular magnetic resonance (available 40% centres). Coronary computed tomography angiography (CCTA) and nuclear scans were reported by a multi-disciplinary team in only a quarter of centres. In the initial assessment of patients presenting with chest pain, only 32% of respondents indicated that they rely on pre-test probability for selecting the optimal imaging test while 31% proceed directly to CCTA. In patients with established coronary artery disease and recurrent chest pain, respondents opted for stress echocardiography (27%) and nuclear stress perfusion scans (26%). In asymptomatic patients with coronary artery disease and an obstructive (>70%) right coronary artery stenosis, 58% of respondents were happy to pursue medical therapy without further testing or intervention. This proportion fell to 29% with left anterior descending artery stenosis and 1% with left main stem obstruction. In asymptomatic patients with evidence of moderate-to-severe myocardial ischaemia (15%), only 18% of respondents would continue medical therapy without further investigation. CONCLUSION Despite guidelines recommendations pre-test probability is used to assess patients with suspected coronary artery in a minority of centres, one-third of centres moving directly to CCTA. Clinicians remain reticent to pursue a strategy of optimal medical therapy without further investigation or intervention in patients with controlled symptoms but obstructive coronary artery stenoses or myocardial ischaemia

WHO draft guidelines on dietary saturated and trans fatty acids: time for a new approach?

The 2018 WHO draft guidelines on dietary saturated fatty acids and trans fatty acids recommend reducing total intake of saturated fat and replacing it with polyunsaturated and monounsaturated fatty acids. The recommendations fail to take into account considerable evidence that the health effects of saturated fat varies depending on the specific fatty acid and on the specific food source. Maintaining general advice to reduce total saturated fatty acids will work against the intentions of the guidelines and weaken their effect on chronic disease incidence and mortality. A food based translation of the recommendations for saturated fat intake would avoid unnecessary reduction or exclusion of foods that are key sources of important nutrients

The NIH-NIAID Filariasis Research Reagent Resource Center

Filarial worms cause a variety of tropical diseases in humans; however, they are difficult to study because they have complex life cycles that require arthropod intermediate hosts and mammalian definitive hosts. Research efforts in industrialized countries are further complicated by the fact that some filarial nematodes that cause disease in humans are restricted in host specificity to humans alone. This potentially makes the commitment to research difficult, expensive, and restrictive. Over 40 years ago, the United States National Institutes of Health–National Institute of Allergy and Infectious Diseases (NIH-NIAID) established a resource from which investigators could obtain various filarial parasite species and life cycle stages without having to expend the effort and funds necessary to maintain the entire life cycles in their own laboratories. This centralized resource (The Filariasis Research Reagent Resource Center, or FR3) translated into cost savings to both NIH-NIAID and to principal investigators by freeing up personnel costs on grants and allowing investigators to divert more funds to targeted research goals. Many investigators, especially those new to the field of tropical medicine, are unaware of the scope of materials and support provided by the FR3. This review is intended to provide a short history of the contract, brief descriptions of the fiilarial species and molecular resources provided, and an estimate of the impact the resource has had on the research community, and describes some new additions and potential benefits the resource center might have for the ever-changing research interests of investigators

EACVI survey on investigations and imaging modalities in chronic coronary syndromes

Aims The European Association of Cardiovascular Imaging (EACVI) Scientific Initiatives Committee performed a global survey to evaluate current practice for the assessment and management of patients with suspected and confirmed chronic coronary syndromes.Methods and results One-hundred and ten imaging centres from 37 countries across the world responded to the survey. Most non-invasive investigations for coronary artery disease were widely available, except cardiovascular magnetic resonance (available 40% centres). Coronary computed tomography angiography (CCTA) and nuclear scans were reported by a multi-disciplinary team in only a quarter of centres. In the initial assessment of patients presenting with chest pain, only 32% of respondents indicated that they rely on pre-test probability for selecting the optimal imaging test while 31% proceed directly to CCTA. In patients with established coronary artery disease and recurrent chest pain, respondents opted for stress echocardiography (27%) and nuclear stress perfusion scans (26%). In asymptomatic patients with coronary artery disease and an obstructive (>70%) right coronary artery stenosis, 58% of respondents were happy to pursue medical therapy without further testing or intervention. This proportion fell to 29% with left anterior descending artery stenosis and 1% with left main stem obstruction. In asymptomatic patients with evidence of moderate-to-severe myocardial ischaemia (15%), only 18% of respondents would continue medical therapy without further investigation.Conclusion Despite guidelines recommendations pre-test probability is used to assess patients with suspected coronary artery in a minority of centres, one-third of centres moving directly to CCTA. Clinicians remain reticent to pursue a strategy of optimal medical therapy without further investigation or intervention in patients with controlled symptoms but obstructive coronary artery stenoses or myocardial ischaemia.</p

The Proteolipid Protein Promoter Drives Expression outside of the Oligodendrocyte Lineage during Embryonic and Early Postnatal Development

The proteolipid protein (Plp) gene promoter is responsible for driving expression of one of the major components of myelin – PLP and its splice variant DM-20. Both products are classically thought to express predominantly in oligodendrocytes. However, accumulating evidence suggests Plp expression is more widespread than previously thought. In an attempt to create a mouse model for inducing oligodendrocyte-specific gene deletions, we have generated transgenic mice expressing a Cre recombinase cDNA under control of the mouse Plp promoter. We demonstrate Plp promoter driven Cre expression is restricted predominantly to mature oligodendrocytes of the central nervous system (CNS) at postnatal day 28. However, crosses into the Rosa26LacZ and mT/mG reporter mouse lines reveal robust and widespread Cre activity in neuronal tissues at E15.5 and E10.5 that is not strictly oligodendrocyte lineage specific. By P28, all CNS tissues examined displayed high levels of reporter gene expression well outside of defined white matter zones. Importantly, our study reinforces the emerging idea that Plp promoter activity is not restricted to the myelinating cell lineage, but rather, has widespread activity both during embryonic and early postnatal development in the CNS. Specificity of the promoter to the oligodendrocyte cell lineage, as shown through the use of a tamoxifen inducible Plp-CreERt line, occurs only at later postnatal stages. Understanding the temporal shift in Plp driven expression is of consequence when designing experimental models to study oligodendrocyte biology

Midgut Barrier Imparts Selective Resistance to Filarial Worm Infection in Culex pipiens pipiens

Mosquitoes in the Culex pipiens complex thrive in temperate and tropical regions worldwide, and serve as efficient vectors of Bancroftian lymphatic filariasis (LF) caused by Wuchereria bancrofti in Asia, Africa, the West Indies, South America, and Micronesia. However, members of this mosquito complex do not act as natural vectors for Brugian LF caused by Brugia malayi, or for the cat parasite B. pahangi, despite their presence in South Asia where these parasites are endemic. Previous work with the Iowa strain of Culex pipiens pipiens demonstrates that it is equally susceptible to W. bancrofti as is the natural Cx. p. pipiens vector in the Nile Delta, however it is refractory to infection with Brugia spp. Here we report that the infectivity barrier for Brugia spp. in Cx. p. pipiens is the mosquito midgut, which inflicts internal and lethal damage to ingested microfilariae. Following per os Brugia exposures, the prevalence of infection is significantly lower in Cx. p. pipiens compared to susceptible mosquito controls, and differs between parasite species with <50% and <5% of Cx. p. pipiens becoming infected with B. pahangi and B. malayi, respectively. When Brugia spp. mf were inoculated intrathoracically to bypass the midgut, larvae developed equally well as in controls, indicating that, beyond the midgut, Cx. p. pipiens is physiologically compatible with Brugia spp. Mf isolated from Cx. p. pipiens midguts exhibited compromised motility, and unlike mf derived from blood or isolated from the midguts of Ae. aegypti, failed to develop when inoculated intrathoracically into susceptible mosquitoes. Together these data strongly support the role of the midgut as the primary infection barrier for Brugia spp. in Cx. p. pipiens. Examination of parasites recovered from the Cx. p. pipiens midgut by vital staining, and those exsheathed with papain, suggest that the damage inflicted by the midgut is subcuticular and disrupts internal tissues. Microscopic studies of these worms reveal compromised motility and sharp bends in the body; and ultrastructurally the presence of many fluid or carbohydrate-filled vacuoles in the hypodermis, body wall, and nuclear column. Incubation of Brugia mf with Cx. p. pipiens midgut extracts produces similar internal damage phenotypes; indicating that the Cx. p. pipiens midgut factor(s) that damage mf in vivo are soluble and stable in physiological buffer, and inflict damage on mf in vitro

Gene Therapy Restores Auditory and Vestibular Function in a Mouse Model of Usher Syndrome Type 1c

Because there are currently no biological treatments for deafness, we sought to advance gene therapy approaches to treat genetic deafness. We reasoned that gene delivery systems that target auditory and vestibular sensory cells with high efficiency would be required to restore complex auditory and balance function. We focused on Usher Syndrome, a devastating genetic disorder that causes blindness, balance disorders and profound deafness, and used a knock-in mouse model, Ush1c c.216G>A, which carries a cryptic splice site mutation found in French-Acadian patients with Usher Syndrome type IC (USH1C). Following delivery of wild-type Ush1c into the inner ears of neonatal Ush1c c.216G>A mice, we find recovery of gene and protein expression, restoration of sensory cell function, rescue of complex auditory function and recovery of hearing and balance behavior to near wild-type levels. The data represent unprecedented recovery of inner ear function and suggest that biological therapies to treat deafness may be suitable for translation to humans with genetic inner ear disorders