Sek, K. | bioRxiv Science

bioRxiv2026

Accurate quantification of canine mitochondrial DNA copy number and its evaluation as a biomarker of brain injury

Acute brain injury is challenging to manage in veterinary medicine, with limited validated means of prognostication currently available. The brain is rich in mitochondrial content and contains thousands of copies of mitochondrial DNA (mtDNA) per cell. We hypothesized that brain cell loss following acute brain injury may result in release of mtDNA into the systemic circulation. To investigate this, mtDNA-CN was measured in blood (n=4-6/group) and cerebral cortex (n=1/group) samples from dogs with and without brain injury using absolute quantification by real-time qPCR. By filtering out regions with homology to nuclear mitochondrial insertion sequences (NumtS) and repetitive regions, oligonucleotide primers were designed to the canine mitochondrial and nuclear genomes. In controls, blood mtDNA-CN ranged from 98 to 288 (mean 193{+/-}72), in cases of brain injury, there was a non-significant trend for higher mtDNA-CN, ranging from 163-453 (mean 244{+/-}106). As expected, cerebral cortex contained higher mtDNA-CN than the blood. In a single case with serial sampling, mtDNA-CN increased five days post-injury. We present for the first time an assay to accurately quantify mtDNA-CN in canine samples, with potential as a biomarker for acute brain injury in veterinary practice. Simple SummaryThis work describes a novel assay to accurately measure absolute levels of canine mitochondrial DNA copy number (mtDNA-CN) in biological samples. We describe the range of mtDNA-CN in canine blood and show pilot data suggesting that blood mtDNA-CN should be evaluated as a potential biomarker of acute brain injury.

molecular biology↗

bioRxiv2021

PTP1B is an intracellular checkpoint that limits T cell and CAR T cell anti-tumor immunity

Immunotherapies aimed at alleviating the inhibitory constraints on T cells have revolutionised cancer management. To date, these have focused on the blockade of cell surface checkpoints such as PD-1. Herein we identify protein-tyrosine-phosphatase 1B (PTP1B) as an intracellular checkpoint that is upregulated in T cells in tumors. We show that the increased PTP1B limits T cell expansion and cytotoxicity to contribute to tumor growth. T cell-specific PTP1B deletion increased STAT-5 signaling and this enhanced the antigen-induced expansion and cytotoxicity of CD8+ T cells to suppress tumor growth. The pharmacological inhibition of PTP1B recapitulated the T cell-mediated repression of tumor growth and enhanced the response to PD-1 blockade. Furthermore, the deletion or inhibition of PTP1B enhanced the efficacy of adoptively-transferred chimeric-antigen-receptor (CAR) T cells against solid tumors. Our findings identify PTP1B as an intracellular checkpoint whose inhibition can alleviate the inhibitory constraints on T cells and CAR T cells to combat cancer. STATEMENT OF SIGNIFICANCETumors subvert anti-tumor immunity by engaging checkpoints that promote T-cell exhaustion. Here we identify PTP1B as an intracellular checkpoint and therapeutic target. We show that PTP1B is upregulated in intra-tumoral T-cells and that its deletion or inhibition enhances T-cell anti-tumor activity and increases CAR T-cell effectiveness against solid tumors.

cancer biology↗