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Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which often leads to protein misfolding and no CFTR surface localization. This then leads to chronic airway infections, inflammation, and tissue damage. Although vitamin D has been explored as a therapy to treat CF due to its antimicrobial-inducing and anti-inflammatory properties, the effect of 1,25-dihydroxyvitamin D 3 (1α,25(OH) 2 D 3 ) on CFTR directly has not been studied. We treated cultured healthy and diseased bronchial epithelial cells (BEC) with 10nM 1α,25(OH) 2 D 3 for 6 and 24h and found that 1α,25(OH) 2 D 3 increases both mRNA and protein CFTR levels using RT-qPCR, flow cytometry and fluorescence immunohistochemistry. Treatment of CF cells with 10nM 1α,25(OH) 2 D 3 led to an increase in both total and surface CFTR expression, suggesting 1α,25(OH) 2 D 3 could be used to increase properly localized CFTR in airway cells. To determine if BEC could convert the more clinically relevant cholecalciferol to 25OHD 3 , cultured non-CF and CF BECs were treated with a range of cholecalciferol concentrations, and 25OHD 3 levels were quantified by ELISA. We found that 25OHD 3 levels increased in a concentration-dependent manner. Treatment of BEC with 10μM cholecalciferol led to increases in both CYP24A1 and CFTR mRNA levels, even when added to the apical surface of cells grown in an air-liquid interface, suggesting that topical administration of vitamin D could be used therapeutically. To demonstrate this in vivo, we intranasally delivered 1μM 1α,25(OH) 2 D 3 into mice. After 6h, we observed induction of both Cyp24A1 and CFTR expression in the tracheas of treated mice. The major findings of this study are that vitamin D can be converted to the active form when topically administered to the airway, and this could be used to increase CFTR levels in patients with CF. This could potentially be useful as an adjunctive therapy, together with newly developed CF treatments., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lethal systemic fungal infections of Candida species are increasingly common, especially in immune compromised patients. By in vitro screening of small molecule mimics of naturally occurring host defense peptides (HDP), we have identified several active antifungal molecules, which also exhibited potent activity in two mouse models of oral candidiasis. Here we show that one such compound, C4, exhibits a mechanism of action that is similar to the parent HDP upon which it was designed. Specifically, its initial interaction with the anionic microbial membrane is electrostatic, as its fungicidal activity is inhibited by cations. We observed rapid membrane permeabilization to propidium iodide and ATP efflux in response to C4. Unlike the antifungal peptide histatin 5, it did not require energy-dependent transport across the membrane. Rapid membrane disruption was observed by both fluorescence and electron microscopy. The compound was highly active in vitro against numerous fluconazole-resistant clinical isolates of C. albicans and non-albicans species, and it exhibited potent, dose-dependent activity in a mouse model of invasive candidiasis, reducing kidney burden by three logs after 24 hours, and preventing mortality for up to 17 days. Together the results support the development of this class of antifungal drug to treat invasive candidiasis.

Leukotoxin (LtxA) is a protein secreted from the oral bacterium Aggregatibacter actinomycetemcomitans. LtxA binds to the β2 integrin lymphocyte-associated function antigen-1 (LFA-1) on human white blood cells (WBCs), resulting in cell death. LtxA is currently under investigation as a novel therapy (Leukothera(®)) for treating hematologic malignancies and autoimmune diseases. We show here that LtxA has potent in vivo anti-lymphoma activity in mice. LtxA caused complete regression of B-cell tumors and promoted long-term survival of mice. The mechanism of LtxA-mediated killing of malignant lymphocytes was further examined. We found that LtxA kills malignant lymphocytes by a novel mechanism requiring the death receptor Fas and caspase-8, but not Fas ligand (FasL) or caspase-9. We also determined that LFA-1 and Fas are closely associated on the cell surface and this proximity of LFA-1 and Fas could explain how signaling through an integrin can lead to cell death. In addition to LFA-1, this work reveals a second surface protein, Fas, that is critical for LtxA-mediated cell death. Knowledge of the mechanism of cell death induced by LtxA will facilitate the development and understanding of this potent experimental therapeutic agent., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Leukotoxin is a protein that is secreted by Aggregatibacter actinomycetemcomitans and that primarily targets the active form of leukocyte function associated antigen 1 (LFA1) on WBC. Because of its specificity for WBC, leukotoxin is being developed as a novel biologic treatment for hematologic malignancies and autoimmune-inflammatory diseases. Early studies indicated that leukotoxin is specific for WBC from humans and Old World primates. In the current study, we used in vivo and in vitro assays to show that leukotoxin has a wider host range than previously believed and can kill rodent WBC. Administration of leukotoxin to rats and mice resulted in a rapid drop in WBC number but had no effect on RBC or platelet counts. Using LFA1-knockout mice, we showed that leukotoxin-mediated depletion of WBC is dependent on LFA1. In addition, similar to its effect on human monocytes, leukotoxin kills murine myeloid leukemia via a lysosome-mediated pathway that is dependent on cathepsin D. This newly described broader host range of leukotoxin enables the biology of the protein to be studied in rodent species and offers the possibility of using rodent models for evaluating the therapeutic efficacy of leukotoxin in various diseases.

Leukotoxin (LtxA) is a protein toxin that is secreted from the oral bacterium, Aggregatibacter actinomycetemcomitans. LtxA targets specifically the β(2) integrin, leukocyte function antigen-1 (LFA-1) on white blood cells (WBCs) and causes cell death. LtxA preferentially targets activated WBCs and is being developed as a therapeutic agent for the treatment of WBC diseases such as hematologic malignancies and autoimmune/inflammatory diseases. However, the mechanism by which interaction between LtxA and LFA-1 results in cell death is not well understood. Furthermore, how LtxA preferentially recognizes activated WBCs is not known. We show here that LtxA interacts specifically with LFA-1 in the active (exposed) conformation. In THP-1 monocytes, LtxA caused rapid activation of caspases, but LtxA could overcome the inhibition of caspases and still intoxicate. In contrast, inhibiting the vesicular trafficking pathway or cathepsin D release from the lysosome resulted in significant inhibition of LtxA-mediated cytotoxicity, indicating a more potent, lysosomal mediated cell death pathway. LtxA caused rapid disruption of the lysosomal membrane and release of lysosomal contents into the cytosol. Binding of LtxA to LFA-1 resulted in the internalization of both LtxA and LFA-1, with LtxA localizing specifically to the lysosomal compartment. To our knowledge, LtxA represents the first bacterial toxin shown to localize to the lysosome where it induces rapid cell death.