Targeting intracellular nontuberculous mycobacteria and M. tuberculosis with a bactericidal enzymatic cocktail.

To address intracellular mycobacterial infections, we developed a cocktail of four enzymes that catalytically attack three layers of the mycobacterial envelope. This cocktail is delivered to macrophages, through a targeted liposome presented here as ENTX_001. Endolytix Cocktail 1 (EC1) leverages mycobacteriophage lysin enzymes LysA and LysB, while also including α-amylase and isoamylase for degradation of the mycobacterial envelope from outside of the cell. The LysA family of proteins from mycobacteriophages has been shown to cleave the peptidoglycan layer, whereas LysB is an esterase that hydrolyzes the linkage between arabinogalactan and mycolic acids of the mycomembrane. The challenge of gaining access to the substrates of LysA and LysB provided exogenously was addressed by adding amylase enzymes that degrade the extracellular capsule shown to be present in Mycobacterium tuberculosis. This enzybiotic approach avoids antimicrobial resistance, specific receptor-mediated binding, and intracellular DNA surveillance pathways that limit many bacteriophage applications. We show this cocktail of enzymes is bactericidal in vitro against both rapid- and slow-growing nontuberculous mycobacteria (NTM) as well as M. tuberculosis strains. The EC1 cocktail shows superior killing activity when compared to previously characterized LysB alone. EC1 is also powerfully synergistic with standard-of-care antibiotics. In addition to in vitro killing of NTM, ENTX_001 demonstrates the rescue of infected macrophages from necrotic death by Mycobacteroides abscessus and Mycobacterium avium. Here, we demonstrate shredding of mycobacterial cells by EC1 into cellular debris as a mechanism of bactericide.IMPORTANCEThe world needs entirely new forms of antibiotics as resistance to chemical antibiotics is a critical problem facing society. We addressed this need by developing a targeted enzyme therapy for a broad range of species and strains within mycobacteria and highly related genera including nontuberculous mycobacteria such as Mycobacteroides abscessus, Mycobacterium avium, Mycobacterium intracellulare, as well as Mycobacterium tuberculosis. One advantage of this approach is the ability to drive our lytic enzymes through encapsulation into macrophage-targeted liposomes resulting in attack of mycobacteria in the cells that harbor them where they hide from the adaptive immune system and grow. Furthermore, this approach shreds mycobacteria independent of cell physiology as the drug targets the mycobacterial envelope while sidestepping the host range limitations observed with phage therapy and resistance to chemical antibiotics.

Human oral lectin ZG16B acts as a cell wall polysaccharide probe to decode host-microbe interactions with oral commensals.

The oral microbiome is critical to human health and disease, yet the role that host salivary proteins play in maintaining oral health is unclear. A highly expressed gene in human salivary glands encodes the lectin zymogen granule protein 16 homolog B (ZG16B). Despite the abundance of this protein, its interaction partners in the oral microbiome are unknown. ZG16B possesses a lectin fold, but whether it binds carbohydrates is unclear. We postulated that ZG16B would bind microbial glycans to mediate recognition of oral microbes. To this end, we developed a microbial glycan analysis probe (mGAP) strategy based on conjugating the recombinant protein to fluorescent or biotin reporter functionality. Applying the ZG16B-mGAP to dental plaque isolates revealed that ZG16B predominantly binds to a limited set of oral microbes, including Streptococcus mitis, Gemella haemolysans, and, most prominently, Streptococcus vestibularis. S. vestibularis is a commensal bacterium widely distributed in healthy individuals. ZG16B binds to S. vestibularis through the cell wall polysaccharides attached to the peptidoglycan, indicating that the protein is a lectin. ZG16B slows the growth of S. vestibularis with no cytotoxicity, suggesting that it regulates S. vestibularis abundance. The mGAP probes also revealed that ZG16B interacts with the salivary mucin MUC7. Analysis of S. vestibularis and MUC7 with ZG16B using super-resolution microscopy supports ternary complex formation that can promote microbe clustering. Together, our data suggest that ZG16B influences the compositional balance of the oral microbiome by capturing commensal microbes and regulating their growth using a mucin-assisted clearance mechanism.

Radiation-Associated Primary Osteosarcoma of the Breast.

INTRODUCTION: Non-epithelial primary mammary osteosarcomas are extremely rare. The differentials include metaplastic carcinoma and malignant phyllodes tumour. This is the first published case of primary breast osteosarcoma arising after local radiotherapy. CASE PRESENTATION: A 73-year-old female presented with a right-sided breast lump. The same breast had been irradiated 11 years previously for invasive ductal carcinoma. Diagnostic excision revealed a highly cellular, malignant spindle-cell lesion merged with an osteoid matrix and foci of calcification and bone formation. Immunohistochemistry and molecular studies showed no lines of differentiation. Due to the lack of epithelial/glandular differentiation, in situ carcinoma or leaf-like pattern, the diagnosis of post-irradiation osteosarcoma was made. She underwent mastectomy and is disease-free at 8 months of follow-up. CONCLUSION: Post-irradiation osteosarcoma should be considered in the differential diagnosis of breast lesions showing malignant osteoid. Extensive sampling and careful search for epithelial differentiation is required to guide management. Complete surgical excision is recommended.

Functional Comparison of Blood-Stage Plasmodium falciparum Malaria Vaccine Candidate Antigens.

The malaria genome encodes over 5,000 proteins and many of these have also been proposed to be potential vaccine candidates, although few of these have been tested clinically. RH5 is one of the leading blood-stage Plasmodium falciparum malaria vaccine antigens and Phase I/II clinical trials of vaccines containing this antigen are currently underway. Its likely mechanism of action is to elicit antibodies that can neutralize merozoites by blocking their invasion of red blood cells (RBC). However, many other antigens could also elicit neutralizing antibodies against the merozoite, and most of these have never been compared directly to RH5. The objective of this study was to compare a range of blood-stage antigens to RH5, to identify any antigens that outperform or synergize with anti-RH5 antibodies. We selected 55 gene products, covering 15 candidate antigens that have been described in the literature and 40 genes selected on the basis of bioinformatics functional prediction. We were able to make 20 protein-in-adjuvant vaccines from the original selection. Of these, S-antigen and CyRPA robustly elicited antibodies with neutralizing properties. Anti-CyRPA IgG generally showed additive GIA with anti-RH5 IgG, although high levels of anti-CyRPA-specific rabbit polyclonal IgG were required to achieve 50% GIA. Our data suggest that further vaccine antigen screening efforts are required to identify a second merozoite target with similar antibody-susceptibility to RH5.

Changes in natural killer cells and exhausted memory regulatory T Cells with corticosteroid therapy in acute autoimmune hepatitis.

Autoimmune hepatitis (AIH) is an immune-mediated liver disease currently treated by immunosuppressive medications with significant side effects. Thus, novel mechanistic treatments are greatly needed. We performed prospective deep immunophenotyping of blood immune cells in patients with acute AIH before and after corticosteroid therapy. Blood samples from 26 patients with acute AIH (United Kingdom-AIH Consortium) were phenotyped by flow cytometry at baseline and 4 months after starting corticosteroids. Pretreatment liver tissues were stained for forkhead box P3-positive (FOXP3POS) regulatory T cells (Tregs), clusters of differentiation (CD)56POS natural killer (NK) cells, and chemokine (C-X-C motif) ligand 10. Chemokine secretion by cultured primary hepatocyte and biliary epithelial cells was measured by enzyme-linked immunosorbent assay. Functional coculture assays with stimulated NK cells and Tregs were performed. CD161 ligand, lectin-like transcript-1 expression by intrahepatic immune cells was demonstrated with flow cytometry. Frequencies of NKbright cells declined with therapy (P < 0.001) and correlated with levels of alanine aminotransferase (P = 0.023). The Treg:NKbright ratio was lower pretreatment, and Tregs had an activated memory phenotype with high levels of CD39, cytotoxic T lymphocyte antigen 4, and FOXP3 but also high programmed death ligand 1, indicating exhaustion. Coculture experiments suggested the Tregs could not efficiently suppress interferon-γ secretion by NK cells. Both Tregs and NK cells had high expression of liver infiltration and T helper 17 plasticity-associated marker CD161 (P = 0.04). Pretreatment and CD161pos NK cells expressed high levels of perforin and granzyme B, consistent with an activated effector phenotype (P < 0.05). Lectin-like transcript 1, a ligand for CD161, is expressed on intrahepatic B cells, monocytes, and neutrophils. Conclusion: Activated effector NK cells, which correlate with biochemical measurements of hepatitis, and exhausted memory Tregs are increased in the blood of patients with treatment-naive AIH and decline with corticosteroid therapy. Inadequate regulation of NK cells by exhausted FOXP3pos Tregs may play a role in AIH pathogenesis and contribute to liver injury. (Hepatology Communications 2018;2:421-436).

Regulatory T Cell Metabolism in the Hepatic Microenvironment.

Thymic-derived naturally occurring regulatory T cells (tTreg) are crucial for maintaining peripheral immune homeostasis. They play a crucial role in preventing autoimmunity and maintaining organ transplant without requiring immunosuppression. Cellular metabolism has recently emerged as an important regulator of adaptive immune cell balance between Treg and effector T cells. While the metabolic requirements of conventional T cells are increasingly understood, the role of Treg cellular metabolism is less clear. The continuous exposure of metabolites and nutrients to the human liver via the portal blood flow influences the lineage fitness, function, proliferation, migration, and survival of Treg cells. As cellular metabolism has an impact on its function, it is crucial to understand the metabolic pathways wiring in regulatory T cells. Currently, there are ongoing early phase clinical trials with polyclonal and antigen-specific good manufacturing practice (GMP) Treg therapy to treat autoimmune diseases and organ transplantation. Thus, enhancing immunometabolic pathways of Treg by translational approach with existing or new drugs would utilize Treg cells to their full potential for effective cellular therapy.

Time use, role participation and life satisfaction of older people: impact of driving status.

BACKGROUND/AIM: Driving cessation has been recognised as affecting the health and wellbeing of older people. Further exploration of the impact of driving status on time use, role participation and life satisfaction was required. METHODS: A cross-sectional survey of 234 older people (current drivers, retired drivers and people who have never driven) was employed. Time use in the previous week, role participation and life satisfaction were measured and compared between the groups, while controlling for sociodemographic variables (health status, activities of daily living and instrumental activities of daily living status, gender, age and living situation). RESULTS: When compared to current drivers, retired drivers had significantly lower life satisfaction (P = 0.01), fewer present roles (P < 0.0001) and were less likely to participate in volunteer (P = 0.005) and family member roles (P = 0.009). Retired drivers spent less time on social leisure (P = 0.002) and away from home (P = 0.0001), and more time in solitary leisure (P= 0.0001). Comparing the participation of retired drivers with those who had never driven indicated that retired drivers spent significantly less time in volunteer work (P = 0.009). CONCLUSIONS: The findings indicate that older non-drivers may require support for participation and wellbeing.

Silver Memories: implementation and evaluation of a unique radio program for older people.

OBJECTIVES: A unique radio program, Silver Memories, specifically designed to address social isolation and loneliness in older people by broadcasting music (primarily), serials and other programs relevant to the period when older people grew up--the 1920-1950s--first aired in Brisbane, Australia, in April 2008. The impact of the program upon older listeners' mood, quality of life (QOL) and self-reported loneliness was independently evaluated. METHOD: One hundred and thirteen community-dwelling persons and residents of residential care facilities, aged 60 years and older participated in a three month evaluation of Silver Memories. They were asked to listen to the program daily and baseline and follow-up measures of depression, QOL and loneliness were obtained. Participants were also asked for their opinions regarding the program's quality and appeal. RESULTS: The results showed a statistically significant improvement in measures of depression and QOL from baseline to follow-up but there was no change on the measure of loneliness. The results did not vary by living situation (community vs. residential care), whether the participant was lonely or not lonely, socially isolated or not isolated, or whether there had been any important changes in the participant's health or social circumstances throughout the evaluation. CONCLUSION: It was concluded that listening to Silver Memories appears to improve the QOL and mood of older people and is an inexpensive intervention that is flexible and readily implemented.

Rhetoric to action: a study of stakeholder perceptions of aging well in two local communities.

This qualitative study of local perceptions of policy goals and action in relation to aging reports 31 stakeholder interviews within 2 Australian communities exploring (a) the meaning of aging well; and (b) preferred policy actions to achieve positive aging outcomes. Findings suggest that community perceptions of aging well are broadly consistent with the goals of national and international policy frameworks in focusing on 3 dimensions--health, social engagement, and security. Further, participants believe that achievement of positive aging outcomes requires a mix of self-help, community action, and government intervention--particularly government support and encouragement for aging well initiatives.

Reflexivity and minimization of the impact of age-cohort differences between researcher and research participants.

Reflexivity in research can be defined as (a) the acknowledgment and identification of one's place and presence in the research, and (b) the process of using these insights to critically examine the entire research process. Many authors implore qualitative researchers to be reflexive. Very few, however, specify how to do this in practice. Furthermore, in discussions of the presence and place of the researcher, the tendency has been to focus on such factors as gender and race or ethnicity with very little attention being given to age or cohort. In this article we seek to redress this deficiency by examining how reflexivity was practiced in a context in which there was a marked difference in age and cohort membership between researcher and research participants. Specifically, we describe the methodological challenges faced by a younger researcher conducting research with older study participants on the lived experience of the body, and how reflexivity was used to adapt the methodology employed so it became more appropriate and productive within this context.

Ethical concerns in the community about technologies to extend human life span.

Debates about the ethical and social implications of research that aims to extend human longevity by intervening in the ageing process have paid little attention to the attitudes of members of the general public. In the absence of empirical evidence, conflicting assumptions have been made about likely public attitudes towards life-extension. In light of recent calls for greater public involvement in such discussions, this target article presents findings from focus groups and individual interviews which investigated whether members of the general public identify ethical issues surrounding life-extension, and if so, what these ethical issues are? In this study, while some participants were concerned primarily with the likely personal consequences of life-extension, for others the question of whether or not to pursue interventions to extend longevity, and how they should be implemented, clearly raised important ethical issues, many of which have been prominent in debates among bioethicists.

Ethical, social, and personal implications of extended human lifespan identified by members of the public.

There are a number of ethical, social, and personal implications generated by the potential development and use of technologies that may extend human longevity by intervening in aging. Despite speculations about likely public attitudes toward life extension, to date there have been few attempts to empirically examine the public's perspective of these issues. Using open-ended survey questions via telephone interviews, this study explored the attitudes of 605 members of the Australian public toward the implications of life extension. Participants were asked to briefly describe in their own words what they believed would be the beneficial, as well as negative, implications arising from life extension (if there were any), both for themselves personally and for society as a whole. Participants were also asked to describe any ethical concerns they had about life extension, if they had any at all. All open-ended responses were collated and then underwent a thematic analysis to uncover commonly cited issues regarding personal benefits/negatives, societal benefits/negatives, and ethical concerns. A considerable number of participants envisioned at least some beneficial as well as negative implications for themselves and for society, and many claimed to have at least some ethical concerns. Some novel issues were raised as well as a number of those discussed within the bioethical literature. The results should encourage researchers, bioethicists, and policy makers to engage with members of the public about the goals of research surrounding life extension, the expected outcomes of such research, and the likely implications for individuals and society.

What makes a community age-friendly: A review of international literature.

This paper undertakes a comprehensive review of the growing international literature on age-friendly communities. It examines a range of approaches and identifies the key attributes associated with creating a sustainable environment for seniors. The authors critically evaluate emerging policy trends and models and suggest directions for future research attention. The discussion provides important information and insights for the development of ageing policy and planning in Australia.

The impact of ageing-in-place policies on structural change in residential aged care.

OBJECTIVES: This paper reports findings from a study to investigate the impact of ageing-in-place policies on resident dependency levels and to explore the relationship between structural variables and outcome ratings. METHODS: The study involved a time series data collection of high- and low-care places and accreditation ratings from both the first and the second accreditation cycles in Queensland (N = 482 and 499). Paired-samples t-tests were also conducted on a sample of homes providing both high- and low-level care to assess changing dependency levels over the two time periods. RESULTS: A statistically significant increase (0.005) was found in the proportion of residents classified as high care living in low-care homes between the first and the second accreditation cycles. CONCLUSIONS: The findings suggest that the ageing-in-place regulatory intervention achieved the intended policy goal and homes have effectively implemented staffing and quality control strategies to accommodate the changes.

Involving older people in community-based research: Developing a guiding framework for researchers and community organisations.

The purpose of this paper is to outline the development of a guiding framework for both researchers and community organisations seeking to involve older people in research. Such a framework is needed to facilitate good quality, multidisciplinary research that can be used to inform policy and practice responses to the challenges of ageing. There is increased recognition that involving older people in the research process can lead to increased benefits for all involved. The guidelines outlined below put forward the following six principles: (i) acknowledge research as a process; (ii) clarify roles and levels of involvement; (iii) communicate effectively; (iv) recognise different expectations; (v) recognise difference; and (vi) ensure representativeness and diversity. These guidelines are now being promoted through the ARC/NHMRC Research Network in Ageing Well and collaborating stakeholder organisations that will be working together to implement these principles in future research involving older people.

Professional and personal attitudes of researchers in ageing towards life extension.

It is often assumed that there is broad public support for strong life extension research (i.e. research aimed at the dramatic extension of human life beyond the current maximum), and that there would be a near universal interest in using any life extending technologies that this research may produce. In this paper we report the opinions of researchers in ageing on the controversial promise of life extension, and compare these views. This paper describes the professional attitudes, personal interest and concerns expressed by Australian and international researchers in ageing (n = 14) as expressed during semi-structured, in-depth interviews. Researchers held varying opinions about the possibility of significantly extending human life. Some saw a limit to the extension of human life, while others did not. Some felt that research into the fundamental ageing process was a priority; others did not. Researchers tended to weigh up the potential risks and benefits of life extension with most expressing a personal interest in life extension that was contingent on the technology providing a good quality of life. Some participants were not interested in the prospect of life extension for personal reasons, because they felt the potential risks outweighed the potential benefits, or because life extension raised issues of justice and equity.

Community perceptions on the significant extension of life: an exploratory study among urban adults in Brisbane, Australia.

Some researchers in the field of ageing claim that significant extension of the human lifespan will be possible in the near future. While many of these researchers have assumed that the community will welcome this technology, there has been very little research on community attitudes to life extension. This paper presents the results of an in-depth qualitative study of community attitudes to life extension across age groups and religious boundaries. There were 57 individual interviews, and 8 focus groups (totalling 72 focus group participants) conducted with community members in Brisbane, Australia. Community attitudes to life extension were more varied and complex than have been assumed by some biogerontologists and bioethicists. While some participants would welcome the opportunity to extend their lives others would not even entertain the possibility. This paper details these differences of opinion and reveals contrasting positions that reflect individualism or social concern among community members. The findings also highlight the relationship between Christianity, in particular belief in an afterlife, and attitudes to life extension technology. Overall, the study raises questions about the relationship between interest in life extension, the medicalisation of ageing and the increasing acceptability of enhancement technologies that need to be addressed in more representative samples of the community.

Dog-assisted therapy for older people with dementia: a review.

This review summarises and critiques the published literature regarding dog therapy for older people with dementia living in residential aged care facilities. Nine studies were identified for inclusion and although the methodological variability of studies makes it difficult to draw firm conclusions, research suggests that dog therapy is beneficial for people with dementia. The most frequently reported findings were an increase in social behaviour and a decrease in agitated behaviour during dog contact. Improvement in social behaviour was found to be unrelated to the severity of dementia. Various improvements on measures of global function were also reported. No study adopted a randomised controlled trial design and a number of potentially important factors were not controlled for, including halo effects of animals on caregivers that may bias caregivers' responses when acting as proxies for their relatives or residents. The premorbid relationship with dogs may be an important variable influencing outcomes.

'I went in feeling like a student and came out feeling like a researcher'. An evaluation of the first Australian Masterclass for Emerging Researchers in Ageing.

OBJECTIVE: To assess how well the 2008 Masterclass for Emerging Researchers in Ageing (ERA) increased capacity for building careers in ageing research in Australia. METHODS: Twenty-six delegates rated 13 career-development activities for (i) the importance of each to their future careers and (ii) their perceived competence in each, before and after the Masterclass. Further qualitative feedback was collected at programme conclusion. RESULTS: Publishing research and working on large-scale, collaborative research projects were rated significantly more important after the Masterclass. Delegates' competence in 11 of the 13 activities increased significantly, as did averaged overall competence. Qualitative data analysis indicated the Masterclass was particularly valued for networking opportunities and for providing access to senior people in the field. CONCLUSIONS: This inaugural ERA Masterclass assisted emerging researchers in ageing to develop capacity in career development activities. These outcomes have important implications for building research capacity in ageing in Australia.