Faculty Bibliography

OBJECTIVE:The aim of this study was to determine the interaction of full thickness excisional wounds and tumors in vivo. SUMMARY OF BACKGROUND DATA/BACKGROUND:Tumors have been described as wounds that do not heal due to similarities in stromal composition. On the basis of observations of slowed tumor growth after ulceration, we hypothesized that full thickness excisional wounds would inhibit tumor progression in vivo. METHODS:To determine the interaction of tumors and wounds, we developed a tumor xenograft/allograft (human head and neck squamous cell carcinoma SAS/mouse breast carcinoma 4T1) wound mouse model. We examined tumor growth with varying temporospatial placement of tumors and wounds or ischemic flap. In addition, we developed a tumor/wound parabiosis model to understand the ability of tumors and wounds to recruit circulating progenitor cells. RESULTS:Tumor growth inhibition by full thickness excisional wounds was dose-dependent, maintained by sequential wounding, and relative to distance. This effect was recapitulated by placement of an ischemic flap directly adjacent to a xenograft tumor. Using a parabiosis model, we demonstrated that a healing wound was able to recruit significantly more circulating progenitor cells than a growing tumor. Tumor inhibition by wound was unaffected by presence of an immune response in an immunocompetent model using a mammary carcinoma. Utilizing functional proteomics, we identified 100 proteins differentially expressed in tumors and wounds. CONCLUSION/CONCLUSIONS:Full thickness excisional wounds have the ability to inhibit tumor growth in vivo. Further research may provide an exact mechanism for this remarkable finding and new advances in wound healing and tumor biology.

Hepatitis B virus (HBV) remains a major global health problem with 257 million chronically infected individuals worldwide, of whom approximately 20 million are co-infected with hepatitis delta virus (HDV). Progress toward a better understanding of the complex interplay between these two viruses and the development of novel therapies have been hampered by the scarcity of suitable cell culture models that mimic the natural environment of the liver. Here, we established HBV and HBV/HDV co-infections and super-infections in self-assembling co-cultured primary human hepatocytes (SACC-PHHs) for up to 28 days in a 384-well format and highlight the suitability of this platform for high-throughput drug testing. We performed RNA sequencing at days 8 and 28 on SACC-PHHs, either HBV mono-infected or HBV/HDV co-infected. Our transcriptomic analysis demonstrates that hepatocytes in SACC-PHHs maintain a mature hepatic phenotype over time, regardless of infection condition. We confirm that HBV is a stealth virus, as it does not induce a strong innate immune response; rather, oxidative phosphorylation and extracellular matrix-receptor interactions are dysregulated to create an environment that promotes persistence. Notably, HDV co-infection also did not lead to statistically significant transcriptional changes across multiple donors and replicates. The lack of innate immune activation is not due to SACC-PHHs being impaired in their ability to induce interferon stimulated genes (ISGs). Rather, polyinosinic:polycytidylic acid exposure activates ISGs, and this stimulation significantly inhibits HBV infection, yet only minimally affects the ability of HDV to infect and persist. Conclusion: These data demonstrate that the SACC-PHH system is a versatile platform for studying HBV/HDV co-infections and holds promise for performing chemical library screens and improving our understanding of the host response to such infections.

BACKGROUND:Immunization with attenuated malaria sporozoites protects humans from experimental malaria challenge by mosquito bite. Protection in humans is strongly correlated with the production of T cells targeting a heterogeneous population of pre-erythrocyte antigen proteoforms, including liver stage antigens. Currently, few T cell epitopes derived from Plasmodium falciparum, the major aetiologic agent of malaria in humans are known. METHODS:In this study both in vitro and in vivo malaria liver stage models were used to sequence host and pathogen proteoforms. Proteoforms from these diverse models were subjected to mild acid elution (of soluble forms), multi-dimensional fractionation, tandem mass spectrometry, and top-down bioinformatics analysis to identify proteoforms in their intact state. RESULTS:These results identify a group of host and malaria liver stage proteoforms that meet a 5% false discovery rate threshold. CONCLUSIONS:This work provides proof-of-concept for the validity of this mass spectrometry/bioinformatic approach for future studies seeking to reveal malaria liver stage antigens towards vaccine development.

Axonal sorting, the controlled passage of specific cargoes from the cell soma into the axon compartment, is critical for establishing and maintaining the polarity of mature neurons. To delineate axonal sorting events, we took advantage of two neuroinvasive alpha-herpesviruses. Human herpes simplex virus 1 (HSV-1) and pseudorabies virus of swine (PRV; suid herpesvirus 1) have evolved as robust cargo of axonal sorting and transport mechanisms. For efficient axonal sorting and subsequent egress from axons and presynaptic termini, progeny capsids depend on three viral membrane proteins (Us7 (gI), Us8 (gE), and Us9), which engage axon-directed kinesin motors. We present evidence that Us7-9 of the veterinary pathogen pseudorabies virus (PRV) form a tripartite complex to recruit Kif1a, a kinesin-3 motor. Based on multi-channel super-resolution and live TIRF microscopy, complex formation and motor recruitment occurs at the trans-Golgi network. Subsequently, progeny virus particles enter axons as enveloped capsids in a transport vesicle. Artificial recruitment of Kif1a using a drug-inducible heterodimerization system was sufficient to rescue axonal sorting and anterograde spread of PRV mutants devoid of Us7-9. Importantly, biophysical evidence suggests that Us9 is able to increase the velocity of Kif1a, a previously undescribed phenomenon. In addition to elucidating mechanisms governing axonal sorting, our results provide further insight into the composition of neuronal transport systems used by alpha-herpesviruses, which will be critical for both inhibiting the spread of infection and the safety of herpesvirus-based oncolytic therapies.

Development of curative therapies for chronic hepatitis B virus (HBV) infection will likely require new animal models. Here, we evaluate HBV infection in squirrel monkeys based on the high-sequence homology of the HBV receptor, Na+/taurocholate co-transporting peptide (NTCP), between humans and squirrel monkeys. HBV PreS1 peptide was examined for binding human and squirrel monkey NTCP. Immunodeficient Fah