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Customer Spotlight: Using Multiplex IHC in Infectious Disease Research

In a recent paper published in the American Journal of Pathology, researchers from Boston University School of Medicine and collaborators studied temporospatial disease progression in Ebola virus disease (EVD). They used multiplexed fluorescent immunohistochemistry (mIHC) and in situ hybridization to quantify viral and host factors related to Zaire ebolavirus (EBOV) pathogenesis in the liver of rhesus macaques.

With the rising interest in infectious disease research, we took the opportunity to speak with the principal investigator of this study, Nick Crossland, DVM, DACVP, to understand his team’s findings as well as to learn how mIHC can characterize progression of infectious disease. Dr. Crossland is an Assistant Professor in Pathology & Laboratory Medicine at Boston University School of Medicine.

nick crossland

Dr. Nick Crossland

What sparked your interest in studying infectious disease in animals?

My veterinary anatomic pathology residency training at Louisiana State University is where my passion for studying infectious diseases originates. The hot and humid climate of the South proved to quite literally represent the perfect storm for some of the craziest and fulminant infectious diseases I could have ever imagined. Many of my cases had never been described in the literature, and I was able to publish 11 manuscripts, a testament to our unique case load at LSU. It wasn’t uncommon for me to be orchestrating a team of senior veterinary students to perform ten necropsies, all on different species of animals simultaneously! I wouldn’t trade those days for anything – we had such a blast learning together, and it really taught me invaluable lessons on communicating and working together as a team to achieve a common goal.

After residency, I knew I wanted to pursue a career in research so I could use my skills to help understand why the host so often loses the battle to infectious diseases. In order to do this, I knew I needed to add additional skills to my tool belt, so I completed a postdoctoral fellowship at Tulane National Primate Research Center, where I helped characterize a rhesus monkey model of chronic Lyme Disease.

The rest is history, as I have never looked back and took my first professional job as an Assistant Professor at Boston University in the Fall of 2017. I work at the National Emerging Infectious Diseases Laboratories (NEIDL), where I serve both as an Investigator and Director of the NEIDL’s Comparative Pathology Laboratory (NCPL), offering pathology support across all levels of biocontainment (BSL-2/3/4).

I know that’s a lot of backstory, but I think it’s important to understand my career trajectory. Ultimately, I had to come to terms with how I could marry my passions, while simultaneously utilizing my expertise in veterinary pathology to best serve mankind. In my current role, my knowledge and skillset are best suited in characterizing the value and limitations of translational animal models of emerging infectious diseases, which ultimately serve to evaluate the safety and efficacy of our most promising medical counter measures to alleviate and prevent human suffering.

Tell us about your recent paper on the quantification of Zaire Ebolavirus biomarkers in rhesus macaques. What were your findings?

Fortunately, upon arriving at BU, I was quickly immersed into a rich collaborative network at the NEIDL and nationally with our biocontainment partners. I’m especially grateful to Dr. John Connor at BU and our NIH NIAID partners at Rocky Mountain Laboratory and Frederick National Laboratory that helped jumpstart my research program. This manuscript reflects that early work and was my first project serving as the lead PI. My first graduate student, Ali Greenberg, who is now in medical school, serves as the first author.

Our findings offer unique insight into Ebola virus disease (EVD) progression in the liver of rhesus monkeys, which faithfully recapitulate human disease. Many of the clinical signs affiliated with EVD can be attributed to liver disease and reflect why we chose to focus solely on this organ to begin with. We were able to characterize statistically significant differences in molecular signatures of disease across early time points that were not feasible with routine ordinal histomorphological characterization. We also achieved temporospatial context to important differentially expressed genes previously described in transcriptomics analysis. More specifically, we characterized gene expression of the pro-inflammatory cytokine (IL-6) and interferon stimulating gene- 15 (ISG-15), as the former has been affiliated with poor clinical outcomes, and induction of the latter has been shown to be inhibited by the Ebola viral protein VP35. These findings were supported in our work, where ISG-15 was upregulated near infected cells, but not within them. IL-6 expression was originally restricted to sinusoidal macrophages but progressed to involve the fibrovascular compartment surrounding blood vessels in later and terminal timepoints, clearly illustrating a temporospatial evolution of pro-inflammatory gene expression.

liver rhesus macaque

Legend: Gray – DAPI nuclear stain; Green – Ebola Zaire VP35 viral protein; Red – Tissue Factor, CD142 – extrinsic coagulation, Blue – CD68 monocyte/macrophage marker. Image courtesy of Dr. Nick Crossland.

Why did you decide to incorporate mIHC using the Vectra Polaris in your study? What is the value of multiplex IHC in infectious disease research?

Quite honestly, I got tired of reading about how multiplexed IHC was being utilized to generate quantitative temporospatial outputs that could be used to predict patient outcomes to immunomodulatory treatments. Why should the oncopathologist have all the fun, I thought to myself. I also became frustrated by the limitations of routine pathologic approaches and knew I had more to offer the research teams I work with by adapting novel technologies into my workflow. I realized that the complete absence of quantitative pathology in the infectious disease realm represented a great opportunity to define the value through my own lens. I’d like to think the value of these approaches are endless, with the primary restriction being our imaginations.

I also became frustrated by the lack of capacity for correlating pathology datasets with other study modalities. Generation of quantitative continuous outputs solved this problem and allows for very powerful statistical modeling and establishment of clinicopathological correlates of disease.


The Vectra Polaris Quantitative Pathology Imaging System

I’ve used pretty much every slide scanner on the market. There are a small handful I really like, with the Vectra Polaris falling into that department. The biggest advantage they offer, which no one else has been able to match, is their integrated multispectral unmixing workflow. This technology does wonders in enhancing the sensitivity, and thus biological accuracy, of signal while simultaneously removing tissue autofluorescence. This plays a critical role in enhancing the ease of subsequent image analysis that cannot be overstated enough. Furthermore, Akoya’s arsenal of compatible Opal fluorophores and unique filter sets allows for rapid acquisition of high plex assays, ensuring we harvest every ounce of biologically relevant data we can from a single tissue section.

How has the COVID-19 pandemic affected your lab? Are you planning any research around COVID-19?

The last few months have been inspirational from a research standpoint. Across the commonwealth, we’re fostering new collaborations at an accelerated pace driven by our shared motivation to alleviate the ongoing crisis. A great example is the establishment of the Massachusetts Consortium on Pathogen Readiness (MassCPR). Their mission is to develop and invest in both the research process and supporting infrastructure to address the current global COVID-19 pandemic, and better position the Consortium for potential future outbreaks. I serve as a member of the pathogenesis working group within the consortium, where my collaborators continue to expand and grow with each coming week. Given the novelty of the SARS-CoV-2 virus, there is so much that remains unknown, and this collaborative approach is our best chance to make meaningful differences in a timely fashion.

Some of the more immediate projects in my laboratory are the development of tools, such as multiplex IHC and ISH assays, to best characterize a broad array of animal models and evaluate their translational relevance and value in developing medical counter measures, including therapeutics and vaccines. I also have ongoing collaborations to characterize COVID-19 disease in human autopsy cases through my MD departmental partners.

I am grateful to have access to a Vectra Polaris Quantitative Pathology Imaging System through a collaborator. I’m actively writing a shared instrument grant to acquire a Vectra Polaris for the Boston University Medical Campus which would be integrated into a core facility with a complimentary dedicated computer laboratory armed with inFORM licenses. I’m grateful for the opportunity to serve as a leader in establishing the value computational pathology, or “pathomics”, serves in research discovery.

Our thanks to Nick for taking the time to speak with us about his research. The paper he discussed is available online:
Quantification of Viral and Host Biomarkers in the Liver of Rhesus Macaques: a Longitudinal Study of Zaire Ebolavirus strain Kikwit (EBOV/Kik)

Interested in learning more about the Vectra Polaris imaging system? Click here.

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