SUMMARY Here we describe a multiplexed immunohistochemical platform, with computational image processing workflows including image cytometry, enabling simultaneous evaluation of 12 biomarkers in one formalin-fixed paraffin-embedded tissue section. To validate this platform, we used tissue microarrays containing 38 archival head and neck squamous cell carcinomas, and revealed differential immune profiles based on lymphoid and myeloid cell densities, correlating with human papilloma virus status and prognosis. Based on these results, we investigated 24 pancreatic ductal adenocarcinomas from patients who received neoadjuvant GVAX vaccination, and revealed that response to therapy correlated with degree of mono-myelocytic cell density, and percentages of CD8+ T cells expressing T cell exhaustion markers. These data highlight the utility of in situ immune monitoring for patient stratification, and provide digital image processing pipelines (https://github.com/multiplexIHC/cppipe) to the community for examining immune complexity in precious tissue sections, where phenotype and tissue architecture are preserved to thus improve biomarker discovery and assessment.

P450 eicosanoids are important regulators of the cerebral microcirculation, but their role in cerebral small vessel disease is unclear. We tested the hypothesis that vascular cognitive impairment (VCI) is linked to reduced cerebral microvascular eicosanoid signaling. We analyzed human brain tissue from individuals formerly enrolled in the Oregon Brain Aging Study, who had a history of cognitive impairment histopathological evidence of microvascular disease. VCI subjects had significantly higher lesion burden both on premortem MRI and postmortem histopathology compared to age- and sex-matched controls. Mass spectrometry-based eicosanoid analysis revealed that 14,15-dihydroxyeicosatrienoic acid (DHET) was elevated in cortical brain tissue from VCI subjects. Immunoreactivity of soluble epoxide hydrolase (sEH), the enzyme responsible for 14,15-DHET formation, was localized to cerebral microvascular endothelium, and was enhanced in microvessels of affected tissue. Finally, we evaluated the genotype frequency of two functional single nucleotide polymorphisms of sEH gene EPHX2 in VCI and control groups. Our findings support a role for sEH and a potential benefit from sEH inhibitors in age-related VCI.

Background Patients recovering from aneurysmal subarachnoid hemorrhage (SAH) are at risk for developing delayed cerebral ischemia (DCI). Experimental and human studies implicate the vasoconstrictor P450 eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE) in the pathogenesis of DCI. To date, no studies have evaluated the role of vasodilator epoxyeicosatrienoic acids (EETs) in DCI. Methods Using mass spectrometry, we measured P450 eicosanoids in cerebrospinal fluid (CSF) from 34 SAH patients from 1 to 14 days after admission. CSF eicosanoid levels were compared in patients who experienced DCI versus those who did not. We then studied the effect of EETs in a model of SAH using mice lacking the enzyme soluble epoxide hydrolase, which catabolizes EETs into their inactive diol. To assess changes in vessel morphology and cortical perfusion in the mouse brain we used optical microangiography, a non-invasive coherence based imaging technique. Results Along with increases in 20-HETE, we found that CSF levels of 14, 15-EET were elevated in SAH patients compared to control CSF, and levels were significantly higher in patients who experienced DCI compared to those who did not. Mice lacking sEH had elevated 14, 15-EET and were protected from the delayed decrease in microvascular cortical perfusion after SAH, compared to wild type mice. Conclusions Our findings suggest that P450 eicosanoids play an important role in the pathogenesis of DCI. While 20-HETE may contribute to the development of DCI, 14, 15-EET may afford protection against DCI. Strategies to enhance 14, 15-EET, including sEH inhibition, should be considered as part of a comprehensive approach to preventing DCI.

Object Patients with aneurysmal subarachnoid hemorrhage (SAH) are at high risk for delayed cerebral ischemia (DCI) and stroke. Epoxyeicosatrienoic acids (EETs) play an important role in cerebral blood flow regulation and neuroprotection after brain injury. Polymorphisms in the gene for the enzyme soluble epoxide hydrolase (sEH), which inactivates EETs, are associated with ischemic stroke risk and neuronal survival after ischemia. In this prospective observational study of patients with SAH we compare vital and neurologic outcomes based on functional polymorphisms of sEH. Methods Allelic discrimination based on quantitative real-time PCR was used to differentiate wild type (WT) sEH from K55R heterozygotes (predictive of increased sEH activity and reduced EETs) and R287Q heterozygotes (predictive of decreased sEH activity and increased EETs). The primary outcome was new stroke after SAH. Secondary outcomes were mortality, Glasgow outcome scale (GOS) score and neurologic deterioration attributable to delayed cerebral ischemia (DCI). Results Multivariable logistic regression models adjusted for admission age and Glasgow coma scale revealed an increase in the odds of new stroke (OR 5.48 (1.51–19.91) and mortality (OR 7.62 (1.19–48.7) in the K55R group, but no change in the odds of new stroke 0.56 (0.16–1.96) or death 3.09 (0.51–18.52) in patients with R287Q genotype, compared to wild-type sEH. R287Q genotype was associated with reduced odds of having a GOS ≤ 3 (0.23 (0.06–0.82)). There were no significant differences in the odds of neurologic deterioration due to DCI. Conclusions Genetic polymorphisms of sEH are associated with neurologic and vital outcomes after aneurysmal subarachnoid hemorrhage.

BackgroundThere has been a dramatic increase in T cell receptor (TCR) sequencing spurred, in part, by the widespread adoption of this technology across academic medical centers and by the rapid commercialization of TCR sequencing. While the raw TCR sequencing data has increased, there has been little in the way of approaches to parse the data in a biologically meaningful fashion. The ability to parse this new type of 'big data' quickly and efficiently to understand the T cell repertoire in a structurally relevant manner has the potential to open the way to new discoveries about how the immune system is able to respond to insults such as cancer and infectious diseases.