Biology subjects

Baker, O.

Publications and source records attributed to Baker, O..

3 recordsLinked to original sources

bioRxiv2024

Platelet-specific P2Y1 receptor deficient mice have suppressed leukocyte recruitment in response to lipopolysaccharide.

A role for the P2Y1 receptor in inflammation has been established using a pharmacological approach over an acute 4 hour time span. However, nucleotide-structure P2Y1 receptor antagonists have limited experimental use due to inadequate pharmacokinetics, and inability to decipher global versus cell specific effects in vivo. The creation of a conditional knock out (platelet) P2Y1 transgenic mouse model was designed to overcome these restrictions. A homozygous P2Y1 LoxP mouse colony was created using CRISPR/Cas9 technology, and crossed with a hemizygous P2Y1 LoxP with PF4-cre to provide offspring that are homozygous for P2Y1 LoxP flanked allele, and hemizygous for the PF4cre (platelet P2Y1-/-) and offspring homozygous for P2Y1 LoxP flanked allele, but non-carriers for PF4cre (control mice). Animals were intranasally administered LPS to induce pulmonary inflammation to assess the influence of phenotype on leukocyte recruitment. 24 hours post intranasal LPS administration; pulmonary neutrophil and platelet recruitment were significantly suppressed, despite the fact that neutrophils retained the ability to migrate to fMLP ex vivo. Circulating platelet and leukocyte numbers were not different between control and platelet P2Y1-/- animals. Tail bleeding times revealed the platelet P2Y1-/- mice had a severe bleeding phenotype. This is the first demonstration of a platelet specific P2Y1-/- mouse model to confirm the importance of platelet P2Y1 receptors in the regulation of inflammatory responses with a 60-70% inhibition of leukocyte recruitment over an extended time period compared to previous pharmacological studies. platelet P2Y1-/- mice will help further elucidate the mechanisms by which P2Y1 receptors regulate platelet activation during inflammation. Key PointsO_LIMice selectively deficient in the platelet P2Y1 have suppressed leukocyte and platelet recruitment during inflammation. C_LIO_LIWe provide a methodology to determine mechanistic relevance that is otherwise limited by pharmacological approaches. C_LI

pharmacology and toxicology↗

bioRxiv2020

Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade

While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLC{gamma}-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

immunology↗

bioRxiv2020

Reduced purine biosynthesis in humans after their divergence from Neandertals

We analyze the metabolomes of humans, chimpanzees and macaques in muscle, kidney and three different regions of the brain. Whereas several compounds in amino acid metabolism occur at either higher or lower concentrations in humans than in the other primates, metabolites in oxidative phosphorylation and purine biosynthesis are consistently present in lower concentrations in the brains of humans. In particular, metabolites downstream of adenylosuccinate lyase, which catalyzes two reactions in purine synthesis, occur at lower concentrations in humans. This enzyme carries an amino acid substitution that is present in all humans today but absent in Neandertals. By introducing the modern human substitution into the genomes of mice, as well as the ancestral, Neandertal-like substitution into the genomes of human cells, we show that this amino acid substitution is responsible for much or all of the reduction of de novo synthesis of purines in humans.

evolutionary biology↗