Centenary Institute

Over the last century, the awareness of air pollution awareness and its harm to public health have resulted in the implementation of new protective measures to try and limit exposure. Bushfires generate waves of air pollution with orders of magnitudes higher than normal background pollution, necessitating studies to understand the physiological impact. Previous work has strongly linked bushfire smoke to respiratory disease (chronic obstructive pulmonary disease COPD, asthma, lung cancer) cardiovascular disease (myocardial infarction, stroke), and increased susceptibility to infection (COVID-19). Nevertheless, the underlying mechanisms of pathogenesis remain unclear. There is little consensus on what in vitro and in vivo techniques are best used to examine disease mechanisms. Individual investigations are useful but a lack of standard methods creates variability and makes comparisons difficult. Developing adaptable in vitro and in vivo models that are replicable, physiologically relevant, and affordable may reduce variability enabling comparisons. These models should also be capable of integrating multiple types of pollutants or reference materials to develop standards that other studies can follow, facilitating comparisons. Here, we discuss advance in vitro and in vivo experimental models to study the impact of bushfire smoke exposure induced pathophysiology. The goal is to improve comparison and translation across studies, and to lay the groundwork for future research into the mechanisms that underpin bushfire smoke-induced pathogenesis to enable the development of preventative measures and effective therapies.

Abstract Human Leukocyte Antigen (HLA)-G is selectively expressed by fetal trophoblast cells that invade maternal tissue and encounter maternal natural killer (NK) cells early in pregnancy. In NK cells, the endosomal receptor KIR2DL4 responds to soluble HLA-G by inducing a broad transcriptional program to support placental development. Structural features of KIR2DL4 that control ligand binding and endocytosis are unknown. Random mutagenesis revealed that three cysteines in the first immunoglobulin domain of KIR2DL4 regulated endocytosis and uptake of HLA-G. We found that the Cys10-Cys28 bond visible in the KIR2DL4 crystal structure is an allosteric disulfide with potential to switch to a Cys28-Cys74 bond. Mass spectrometry analysis showed that KIR2DL4 in human cells exists in both disulfide-bonded states. The Cys10-Cys28 bond in purified KIR2DL4 was reduced by protein disulfide isomerase (PDI) in vitro. Inhibition of PDI caused retention of KIR2DL4 at the plasma membrane and prevented HLA-G uptake. Mutants in the Cys10-Cys28 configuration endocytosed spontaneously but did not bind HLA-G. Conversely, KIR2DL4 with a Cys28-Cys74 bond was at the plasma membrane and endocytosed HLA-G. A structural change predicted by AlphaFold upon disulfide switching to the Cys28-Cys74 form reorients the D0 domain into a conformation that binds HLA-G. Thus, conversion of KIR2DL4 from an inactive state to an HLA-G binding form can regulate NK cell function to promote fetal development.

Abstract Aims Gestational diabetes mellitus (GDM) is the most common pregnancy-related medical complication. GDM is linked to aberrant immune responses in both mothers and offsprings, specifically, the subsequent development of inflammatory diseases. Whereas prior research has focused on specific immune cell subsets, a comprehensive overview of the impacts of GDM on maternal and fetal immune landscape is lacking. Here, we aim to comprehensively decipher how GDM modulates various immune cell populations in mothers and offsprings. Methods A prospective, longitudinal case-control study was carried out. Maternal blood from GDM-affected (GDM, n=18) and non-GDM-affected (Ctrl, n=21) mothers were collected at ante-(36-38 weeks of gestation) and post-partum (6-8 weeks post-partum) timepoints. Cord blood from GDM (n=7) and Ctrl (n=11) pregnancies were collected upon C-section. They were analyzed with the state-of-the-art cytometry by time of flight (CyTOF) with a 40-marker panel. Additionally, a publicly available RNA-seq dataset for cord blood mononuclear cells was re-analyzed to confirm results from CyTOF experiments. Results Compared to Ctrl, GDM was associated with more activated maternal T cell subsets ante-partum, including increased CD45RO + and Ki67 + CD4 + T cell populations, which reverted post-partum. GDM-affected maternal innate lymphoid cell (ILC) also exhibited increased granzyme B production ante-partum. On the other hand, in GDM-impacted cord blood, fetal T and B cells were more activated, displaying less naïve and more effector phenotypes, further supported by RNA-seq analyses. Conclusions Our comprehensive analyses revealed that GDM is linked to profound changes in the immune landscapes of the mothers (ante-/post-partum) and foetuses (at birth), casting novel insights towards GDM pathophysiology. Longitudinal immune profiling might be warranted for early detection and stratification of risk, and development of targeted interventions to prevent inflammatory disorders in GDM mothers and their offspring. Research in context What is already known about this subject? The maternal and intrauterine environments are important contributors to long-term health outcomes of mothers and offsprings . Some maternal and fetal immunity changes have been observed in gestational diabetes mellitus (GDM)-affected pregnancies . GDM is associated with increased risk of later-life metabolic and inflammatory diseases in mothers as well as offsprings . What is the key question? What are the longitudinal alterations in maternal and fetal immune landscapes in GDM-affected pregnancies? What are the new findings? High-dimensional immune profiling provided the most comprehensive overview of alterations in maternal and fetal immune landscapes associated with GDM . GDM is associated with skewing of maternal CD4 + T cell and ILC towards activated phenotypes ante-partum . GDM is linked to more activated fetal T and B cell profiles . How might this impact on clinical practice in the foreseeable future? Understanding the complex alterations in the maternal and fetal immune landscape in GDM-affected pregnancy provides insights into the long-term impacts of GDM on the mother and offspring .

Pancreatic cancer (PC) is an aggressive malignancy and remains one of the leading causes of cancer-related mortality worldwide. Although imaging modalities remain central to clinical practice, they rarely detect disease at the stage when curative intervention is possible. Recently, extracellular vesicles (EVs) have been increasingly recognized for their potential in biomedical applications, particularly as biomarkers for liquid biopsies. However, conventional bulk analyzes average signals across heterogeneous vesicle populations, obscuring the contribution of rare tumor-derived EVs that could serve as critical indicators of the early stages of cancer. Single EV analysis (sEVA) technologies have emerged to address this challenge by enabling high-resolution characterization of individual vesicles, enhancing analytical sensitivity, and offering strong potential for clinical translation. In this review, we focus on the emerging role of sEVA technologies in reshaping liquid biopsy-based early detection and clinical management of PC. By integrating recent advances in EV biomarker development and current sEVA technologies, this review outlines a translational strategy for integrating sEVA into PC early detection frameworks and the key barriers to clinical implementation.

Inhalable liquid formulation of endolysins represents a promising alternative to conventional antibiotics. Dry powder formulations offer improved stability for endolysin pulmonary delivery. This study aimed to evaluate the efficacy of an inhalable dry powder or liquid formulation of endolysin Cpl-1 alone and to compare it with liquid combinations of Cpl-1 with either gentamicin or endolysin Pal in a murine model of S. pneumoniae lung infection. A dry powder formulation of Cpl-1 was produced via spray drying, while liquid formulations were prepared by dissolving Cpl-1, or in combination with gentamicin or endolysin Pal in liquid. The droplet size distribution of aerosolized formulations was also characterized. Mice were intratracheally infected with S. pneumoniae and treated with either powder or liquid formulations. The bacterial load in respiratory system was assessed 26h post-infection. The stability and activity of Cpl-1 in BALF were also evaluated ex vivo. A single dose of Cpl-1 powder formulation or Cpl-1 liquid formulation (40µg/mouse) reduced pulmonary bacterial load by approximately 1 log10. Importantly, the combination of Cpl-1 and Pal in liquid form resulted in a synergistic 2.0 log10 reduction, significantly greater than either endolysin alone, while combining Cpl-1 with gentamicin did not enhance antibacterial activity. Ex vivo assays confirmed that Cpl-1 retained full enzymatic activity after incubation in BALF. This proof-of-principle study demonstrated that inhalable endolysin liquid and powder formulations could potentially be used to treat bacterial lung infections. Moreover, the combination of multiple endolysins could increase antimicrobial activity over endolysin monotherapy.

Abstract Mitophagy is an essential component of the mitochondrial quality control program, maintaining mitochondrial homeostasis in metabolic tissues such as skeletal muscle. With age, it is thought that mitochondrial quality control becomes dysregulated, leading to the progression of age-associated diseases such as sarcopenia. Exercise is known to enhance skeletal muscle mitochondrial health and may be an effective intervention to prevent sarcopenia, however the role of mitophagy in this process is unknown. Utilising mitophagy reporter mice (mito-QC), we assessed adaptations in skeletal muscle mitophagy in response to increased age (3-26 months) and following an 8-week endurance exercise training period. Immunofluorescent imaging revealed that ageing led to an accumulation of mitolysosomes in sarcopenic old mice indicative of increased mitophagy, an adaptive response that was reversed by exercise training. In parallel to reducing age-associated mitophagy, exercise training increased mitochondrial respiratory capacity and improved muscle strength, suggesting that alterations in mitochondrial quality control led to improvements in skeletal muscle function. Exercise-mediated alterations in mitophagy were accompanied by increases in BNIP3, FUNDC1 and BCL2L13 protein content post training. Collectively our data suggests that sarcopenia leads to dysregulation of mitophagy in skeletal muscle. Restoring mitophagy balance with exercise training leads to improvements in mitochondrial respiration and skeletal muscle strength, identifying a novel cellular mechanism to explain the benefits of exercise training in old age.

Currently, the only known clinically relevant hereditary risk factor for emphysema is limited to mutations within the SERPINA1 gene, encoding alpha-1 antitrypsin. Although several additional rare high-impact variants have been proposed, their role in emphysema pathophysiology is unclear. This review discusses recent cases investigating novel candidate genes that may be Mendelian causes for emphysema development. We also explore potential methods to confirm the causal relation to COPD. Identifying potential new rare high-impact genetic variants may lead to novel therapeutic targets, thus improving the personalised treatment of COPD. Several gene mutations have been implicated in emphysema development, including SERPINA1, SERPINA3, PTPN6, TERT, TR, NAF1, BICD1, ELN, FBLN, FLNA and SFTPC. Mutations of the SERPINA1 and PTPN6 genes are considered definitive causes of emphysema. Studies have ascertained rare variants in cutis laxa genes (ELN, FBLN and FLNA), which cause early-onset emphysema in infants and children via defective elastin synthesis. Telomerase pathway genes (TERT, TR, NAF1 and BICD1) have also been implicated in increased COPD risk along with another member of the serpin family (SERPINA3) and SFTPC. These probable mutations for emphysema tend to present later in life. Due to being unconfirmed, they may involve a more complex gene interaction that requires further interrogation with next-generation sequencing and molecular methods, including CRISPR (clustered regularly interspaced short palindromic repeats) screening libraries, whole-exome sequencing or whole-genome sequencing. Although multiple novel mutations have been reported to cause emphysema, further validation is needed. Next-generation sequencing offers a promising method to understand early-onset emphysema and COPD pathogenesis.

Cerebral cavernous malformation (CCM) is a cluster of abnormal blood vessels in the brain that leads to severe neurological deficits, seizures, and fatal hemorrhagic stroke. Currently, there is no available drug treatment for CCM. Most CCMs are conservatively managed by observing change in appearance (MRI), recent hemorrhage, or any clinical symptoms. Neurosurgery is the only current treatment option, but it is only effective in a few cases. Since most CCM lesions are surgically inaccessible, when left untreated they lead to severe neurological deficits, seizures, and fatal hemorrhagic stroke. Hence, new non-invasive, safe, and effective treatment strategies are urgently needed. Recent research has identified gut microbiome dysbiosis and its innate immune response as the critical stimulus in experimental CCM pathogenesis, demonstrating the importance of the gut–brain axis in CCM. Importantly, CCM patients also manifest gut microbiome dysbiosis and gut barrier health can impact CCM disease course. This review highlights the emerging involvement of the gut microbiome in CCM pathogenesis and its potential as a therapeutic target. While preclinical data suggest mechanistic links, the lack of clinical intervention studies limits current applicability and underscores the need for translational research.