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Keratinocytes express many pattern recognition receptors that enhance the skin's adaptive immune response to epicutaneous antigens. We have shown that these pattern recognition receptors are expressed below tight junctions (TJ), strongly implicating TJ disruption as a critical step in antigen responsiveness. To disrupt TJs, we designed peptides inspired by the first extracellular loop of the TJ transmembrane protein CLDN1. These peptides transiently disrupted TJs in the human lung epithelial cell line 16HBE and delayed TJ formation in primary human keratinocytes. Building on these observations, we tested whether vaccinating mice with an epicutaneous influenza patch containing TJ-disrupting peptides was an effective strategy to elicit an immunogenic response. Application of a TJ-disrupting peptide patch resulted in barrier disruption as measured by increased transepithelial water loss. We observed a significant increase in antigen-specific antibodies when we applied patches with TJ-disrupting peptide plus antigen (influenza hemagglutinin) in either a patch-prime or a patch-boost model. Collectively, these observations demonstrate that our designed peptides perturb TJs in human lung as well as human and murine skin epithelium, enabling epicutaneous vaccine delivery. We anticipate that this approach could obviate currently used needle-based vaccination methods that require administration by health care workers and biohazard waste removal.

In the present study, 1 g/ml each of Barleria prionitis leaves (BPL) and B. prionitis stem (BPS) were extracted from different solvents like petroleum ether, chloroform ethyl acetate and methanol. Ethyl acetate extract of BPL showed maximum inhibition zone on Gram positive Bacillus pumilus (9.83 mm) and methanol extract of BPS showed minimum inhibition zone on Gram negative Escherichia coli (0.16 mm). Petroleum ether extract did not show inhibition except petroleum ether extract of BPS on Gram positive B. pumilus (0.46 mm). Minimum inhibition concentration (MIC) was shown by petroleum ether extract of BPL on Gram positive B. pumilus and Gram negative Pseudomonas aeruginosa (1.0 mg/ml). Leaves and stem extract of Barleria prionitis L. showed difference in antibacterial activity.

Atopic dermatitis (AD) is a skin disease characterized by an impaired epidermal barrier and increased susceptibility to cutaneous viral infections. We have previously observed that barrier disruption (caused by our tight junction disrupting peptide [TJDPÒ]), enhances vaccinia virus (VV) infection of primary human foreskin keratinocytes (PHFK). To evaluate the importance of AD-relevant tight junction proteins in epidermal viral infections, we sought to identify an epidermal cell line that we could genetically manipulate. We tested N/TERT cells (immortalized PHFK) to determine whether they faithfully recapitulated primary cells in both our barrier and VV infection models. We found that N/TERTs developed a robust barrier, as measured by transepithelial electrical resistance (TEER), with peak values of 300e400 ohms/ cm 2 observed 4-5 days after treatment with high Ca +2 (1.8mM) containing media. After exposure to our TJDPÒ, N/TERTs exhibited a substantial reduction in barrier compared to media controls (e.g. TEER reduction: 46% AE 13 and 68% AE 11 at Days 2 and 3, respectively; n¼3). We previously observed that PHFK are more susceptible to VV when treated with TJDPÒ during differentiation. With N/TERTs, we observed an increase in VV susceptibility as measured by % change in plaque number (179% AE 64 at Day 2; n¼3) and % of the monolayer infected (27% AE 24 vs 42% AE 20 at Day 2 for media compared to TJDPÒ treated samples, n¼3). These observations suggest that N/TERTs behave similarly to PHFK in both our infection and barrier experiments. Future studies will focus on determining how reduction in AD-relevant TJ barrier proteins (Claudin 1, 4, 23, occludin, and zonula occludens 1) using the CRISPR/Cas9 system impacts the susceptibility of N/TERTs to viral infections (VV and herpes simplex virus).

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