Modeling the Potential of Treg-Based Therapies for Transplant Rejection: Effect of Dose, Timing, and Accumulation Site.

Introduction: The adoptive transfer of regulatory T cells (Tregs) has emerged as a method to promote graft tolerance. Clinical trials have demonstrated the safety of adoptive transfer and are now assessing their therapeutic efficacy. Strategies that generate large numbers of antigen specific Tregs are even more efficacious. However, the combinations of factors that influence the outcome of adoptive transfer are too numerous to be tested experimentally. Here, mathematical modeling is used to predict the most impactful treatment scenarios. Methods: We adapted our mathematical model of murine heart transplant rejection to simulate Treg adoptive transfer and to correlate therapeutic efficacy with Treg dose and timing, frequency of administration, and distribution of injected cells. Results: The model predicts that Tregs directly accumulating to the graft are more protective than Tregs localizing to draining lymph nodes. Inhibiting antigen-presenting cell maturation and effector functions at the graft site was more effective at modulating rejection than inhibition of T cell activation in lymphoid tissues. These complex dynamics define non-intuitive relationships between graft survival and timing and frequency of adoptive transfer. Conclusion: This work provides the framework for better understanding the impact of Treg adoptive transfer and will guide experimental design to improve interventions.

Reversible blood-brain barrier opening utilizing the membrane active peptide melittin in vitro and in vivo.

The blood-brain barrier (BBB) tightly controls entry of molecules and cells into the brain, restricting the delivery of therapeutics. Blood-brain barrier opening (BBBO) utilizes reversible disruption of cell-cell junctions between brain microvascular endothelial cells to enable transient entry into the brain. Here, we demonstrate that melittin, a membrane active peptide present in bee venom, supports transient BBBO. From endothelial and neuronal viability studies, we first identify the accessible concentration range for BBBO. We then use a tissue-engineered model of the human BBB to optimize dosing and elucidate the mechanism of opening. Melittin and other membrane active variants transiently increase paracellular permeability via disruption of cell-cell junctions that result in transient focal leaks. To validate the results from the tissue-engineered model, we then demonstrate that transient BBBO can be reproduced in a mouse model. We identify a minimum clinically effective intra-arterial dose of 3 µM min melittin, which is reversible within one day and neurologically safe. Melittin-induced BBBO represents a novel technology for delivery of therapeutics into the brain.

A peptide for transcellular cargo delivery: Structure-function relationship and mechanism of action.

The rate of transport of small molecule drugs across biological barriers, such as the blood-brain barrier, is often a limiting factor in achieving a therapeutic dose. One proposed strategy to enhance delivery across endothelial or epithelial monolayers is conjugation to cell-penetrating peptides (CPPs); however, very little is known about the design of CPPs for efficient transcellular transport. Here, we report on transcellular transport of a CPP, designated the CL peptide, that increases the delivery of small-molecule cargoes across model epithelium approximately 10-fold. The CL peptide contains a helix-like motif and a polyarginine tail. We investigated the effect of cargo, helix-like motif sequence, polyarginine tail length, and peptide stereochemistry on cargo delivery. We showed that there is an optimal helix-like motif sequence (RLLRLLR) and polyarginine tail length (R7) for cargo delivery. Furthermore, we demonstrated that the peptide-cargo conjugate is cleaved by cells in the epithelium at the site of a two-amino acid linker. The cleavage releases the cargo with the N-terminal linker amino acid from the peptide prior to transport out of the epithelium. These studies provide new insight into the sequence requirements for developing novel CPPs for transcellular delivery of cargo.

Ebola Virus Delta Peptide Is a Viroporin.

The Ebola virus (EBOV) genome encodes a partly conserved 40-residue nonstructural polypeptide, called the delta peptide, that is produced in abundance during Ebola virus disease (EVD). The function of the delta peptide is unknown, but sequence analysis has suggested that delta peptide could be a viroporin, belonging to a diverse family of membrane-permeabilizing small polypeptides involved in replication and pathogenesis of numerous viruses. Full-length and conserved C-terminal delta peptide fragments permeabilize the plasma membranes of nucleated cells of rodent, dog, monkey, and human origin; increase ion permeability across confluent cell monolayers; and permeabilize synthetic lipid bilayers. Permeabilization activity is completely dependent on the disulfide bond between the two conserved cysteines. The conserved C-terminal portion of the peptide is biochemically stable in human serum, and most serum-stable fragments have full activity. Taken together, the evidence strongly suggests that Ebola virus delta peptide is a viroporin and that it may be a novel, targetable aspect of Ebola virus disease pathology.IMPORTANCE During the unparalleled West African outbreak of Ebola virus disease (EVD) that began in late 2013, the lack of effective countermeasures resulted in chains of serial infection and a high mortality rate among infected patients. A better understanding of disease pathology is desperately needed to develop better countermeasures. We show here that the Ebola virus delta peptide, a conserved nonstructural protein produced in large quantities by infected cells, has the characteristics of a viroporin. This information suggests a critical role for the delta peptide in Ebola virus disease pathology and as a possible target for novel countermeasures.

On Chip Bioelectric Impedance Spectroscopy Reveals the Effect of P-Glycoprotein Efflux Pumps on the Paracellular Impedance of Tight Junctions at the Blood-Brain Barrier.

Bioelectric impedance spectroscopy was used to elucidate the influence of P-gp efflux pumps on the kinetics of tight junction down-regulation in confluent monolayers of Madine Darby Canine Kidney Epithelial Cells (MDCK) following administration of phenylarsine oxide (PAO), a molecule that inhibits protein tyrosine phosphatases (PTP) and induces matrix metalloproteinase activity. Matrix metalloproteinases (MMPs) and phosphatase inhibitors induce modification of occludin tight junction proteins critical for the proper function of the blood-brain barrier. The addition of PAO to MDCKII cell lines resulted in a dramatic decrease in monolayer resistance. In contrast, MDCKII-MDR1 cells transfected with the MDR1 gene treated with PAO showed an initial decrease in monolayer resistance followed by a partial recovery and subsequent decrease. This resistance decay reversal was suppressed with the addition of the P-glycoprotein (P-gp) pump inhibitor elacridar, and is attributed to PAO efflux. These results illustrate impedance spectroscopy can be used to characterize the competing kinetics of efflux and down-regulation of tight junctions. In addition, the resistance decay reversal effect can be used to evaluate P-gp pump inhibitor efficacy.

Adoptive Transfer of Engineered Rhesus Simian Immunodeficiency Virus-Specific CD8+ T Cells Reduces the Number of Transmitted/Founder Viruses Established in Rhesus Macaques.

AIDS virus infections are rarely controlled by cell-mediated immunity, in part due to viral immune evasion and immunodeficiency resulting from CD4+ T-cell infection. One likely aspect of this failure is that antiviral cellular immune responses are either absent or present at low levels during the initial establishment of infection. To test whether an extensive, timely, and effective response could reduce the establishment of infection from a high-dose inoculum, we adoptively transferred large numbers of T cells that were molecularly engineered with anti-simian immunodeficiency virus (anti-SIV) activity into rhesus macaques 3 days following an intrarectal SIV inoculation. To measure in vivo antiviral activity, we assessed the number of viruses transmitted using SIVmac239X, a molecularly tagged viral stock containing 10 genotypic variants, at a dose calculated to transmit 12 founder viruses. Single-genome sequencing of plasma virus revealed that the two animals receiving T cells expressing SIV-specific T-cell receptors (TCRs) had significantly fewer viral genotypes than the two control animals receiving non-SIV-specific T cells (means of 4.0 versus 7.5 transmitted viral genotypes; P = 0.044). Accounting for the likelihood of transmission of multiple viruses of a particular genotype, the calculated means of the total number of founder viruses transmitted were 4.5 and 14.5 in the experimental and control groups, respectively (P = 0.021). Thus, a large antiviral T-cell response timed with virus exposure can limit viral transmission. The presence of strong, preexisting T-cell responses, including those induced by vaccines, might help prevent the establishment of infection at the lower-exposure doses in humans that typically transmit only a single virus. IMPORTANCE: The establishment of AIDS virus infection in an individual is essentially a race between the spreading virus and host immune defenses. Cell-mediated immune responses induced by infection or vaccination are important contributors in limiting viral replication. However, in human immunodeficiency virus (HIV)/SIV infection, the virus usually wins the race, irreversibly crippling the immune system before an effective cellular immune response is developed and active. We found that providing an accelerated response by adoptively transferring large numbers of antiviral T cells shortly after a high-dose mucosal inoculation, while not preventing infection altogether, limited the number of individual viruses transmitted. Thus, the presence of strong, preexisting T-cell responses, including those induced by vaccines, might prevent infection in humans, where the virus exposure is considerably lower.