Biology subjects

Fairfax, K.

Publications and source records attributed to Fairfax, K..

5 recordsLinked to original sources

bioRxiv2025

Development of a point-of-care field diagnostic test for DFT1 and DFT2

The Tasmanian devil (Sarcophilus harrisii) population has undergone a major decline in the wild due to the epidemics of two transmissible cancers known as devil facial tumours 1 (DFT1) and devil facial tumour 2 (DFT2). Understanding the distribution and prevalence of DFT1 and DFT2 is challenging as they can only be detected in field conditions when tumours are large enough to be visually observed. This impedes our ability to understand how the devil population is evolving in response to the tumours. PCR-based diagnostics are available, but the technique is not field-applicable. To overcome these hurdles, we developed an ultrasensitive and field-deployable rapid diagnostic tool. We used a technique called Specific High-Sensitivity Enzymatic Reporter Unlocking (SHERLOCK), which combines an isothermal nucleic acid amplification step with CRISPR-based recognition and activation of reporter probes to indicate target sequence detection. This system requires only portable laboratory equipment, making it more amenable to field conditions. The results showed that our test can detect DFT1 and DFT2 DNA within the attomolar range. We then confirmed that the test can detect DFT1 in the field using non-invasive swab samples. The SHERLOCK test can be used for more active monitoring and management approaches that are not currently possible.

molecular biology↗

bioRxiv2025

Exploring Prime Editing strategies for genetic correction of a Ceroid Neuronal Lipofuscinosis type 2 disease causing variant

Neuronal ceroid lipofuscinosis type 2 (CLN2) is a rare, fatal paediatric neurodegenerative genetic disease that causes progressive psychomotor decline, epilepsy, speech impairment, vision loss, and premature death by late childhood or early adolescence. It is caused by mutations in the TPP1 gene, which encodes the tripeptidyl peptidase 1 enzyme. To date, enzyme replacement therapy (ERT) with recombinant human TPP1 is the only approved treatment available and it does not appear to have a curative effect on disease progression or rescuing complex motor functions. This underscores the importance of developing more effective treatment strategies for CLN2 disease. In the quest for an alternative therapy, we here seek to utilise CRISPR based prime editing (PE) to correct one of the most frequent TPP1 mutations of c.622 C>T (p. Arg208Ter). In cultured mammalian HEK293a cells we screened 16 different PE and pegRNA combinations, achieving on-target editing efficiencies up to 27.36 %. To enable efficient in vivo gene editing, we packaged the most promising PE and pegRNA combination using engineered virus-like particles (eVLPs) which resulted in 12.6 % on-target editing. Given the robust on-target editing and reduced risk of bystander editing, PE is deemed suitable for in vivo testing in the pre-clinical models for CLN2 disease. Overall, our findings establish the proof of concept for CRISPR-based prime editing to correct pathogenic human TPP1 nonsense mutation of c.622 C>T (p. R208X) and provide a basis for further investigations of PE as a genetic treatment for CLN2 disease.

molecular biology↗

bioRxiv2023

ARF6-dependent endocytic trafficking of the Interferon-γ receptor drives adaptive immune resistance in cancer.

Adaptive immune resistance (AIR) is a protective process used by cancer to escape elimination by CD8+ T cells. Inhibition of immune checkpoints PD-1 and CTLA-4 specifically target Interferon-gamma (IFN{gamma})-driven AIR. AIR begins at the plasma membrane where tumor cell-intrinsic cytokine signaling is initiated. Thus, plasma membrane remodeling by endomembrane trafficking could regulate AIR. Herein we report that the trafficking protein ADP-Ribosylation Factor 6 (ARF6) is critical for IFN{gamma}-driven AIR. ARF6 prevents transport of the receptor to the lysosome, augmenting IFN{gamma}R expression, tumor intrinsic IFN{gamma} signaling and downstream expression of immunosuppressive genes. In murine melanoma, loss of ARF6 causes resistance to immune checkpoint blockade (ICB). Likewise, low expression of ARF6 in patient tumors correlates with inferior outcomes with ICB. Our data provide new mechanistic insights into tumor immune escape, defined by ARF6-dependent AIR, and support that ARF6-dependent endomembrane trafficking of the IFN{gamma} receptor influences outcomes of ICB.

cancer biology↗

bioRxiv2020

A novel subset of follicular helper-like MAIT cells has capacity for B cell help and antibody production in the mucosa.

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that aid in protection against bacterial pathogens at mucosal surfaces via release of inflammatory cytokines and cytotoxic molecules. Recent evidence suggests MAIT cells are capable of providing B cell help. In this study, we describe a previously unreported population of CXCR5+ T follicular helper (Tfh)-like MAIT cells, MAITfh, that have the capacity to provide B cell help within mucosal lymphoid organs. MAITfh cells are preferentially located near germinal centers in human tonsils and express the classical Tfh-associated transcription factor, B-cell lymphoma 6 (BCL-6), co-stimulatory markers, inducible T cell costimulatory (ICOS) and programmed death receptor 1 (PD-1), and cytokines, interleukin (IL)-21. Furthermore, we demonstrate the ability of MAIT cells to provide B cell help in vivo following mucosal challenge with Vibrio cholerae. Specifically, we show that adoptive transfer of MAIT cells into {beta} T cell-deficient mice promoted B cell differentiation and increased serum V. cholerae-specific IgA responses. Our data demonstrate the capacity of MAIT cells to participate in adaptive immune responses, and suggest that MAIT cells may be potential targets for mucosal vaccines. One Sentence SummaryWe identified and characterized a novel subset of T follicular helper-like MAIT (MAITfh) cells that has the capacity to provide B cell help, and show the sufficiency of MAIT cells to promote production of pathogen-specific IgA antibodies and B cell differentiation in mucosal challenge.

immunology↗

bioRxiv2020

Schistosoma mansoni infection induces long-lived sex-specific metabolic reprogramming of the myeloid lineage

Despite evidence that helminths protect from metabolic disease, a major gap exists in understanding the underlying mechanism(s). Here we demonstrate that bone marrow derived macrophages (BMDM) from S. mansoni infected male ApoE-/- mice have dramatically increased mitochondrial respiration compared to those from uninfected mice. This change associates with increased glucose and palmitate shuttling into TCA cycle intermediates and decreased accumulation of cellular cholesterol esters. Moreover, systemic metabolic modulation by schistosomes is a function of biological sex, where infection protects ApoE-/- male, but not female, mice from obesity and glucose intolerance. Sex-dependence extends to myeloid cells, where reprogramming leads to opposite cholesterol phenotypes in BMDM from females and males. Finally, the metabolic reprogramming of male myeloid cells is transferrable via bone marrow transplantation to an uninfected host, indicating maintenance of reprogramming in the absence of sustained antigen exposure. This work reveals that S. mansoni systemic reprograming of myeloid metabolism is sex-dependent.

immunology↗