Activation of vitamin D in the gingival epithelium and its role in gingival inflammation and alveolar bone loss.

BACKGROUND AND OBJECTIVE: Both chronic and aggressive periodontal disease are associated with vitamin D deficiency. The active form of vitamin D, 1,25(OH)2 D3 , induces the expression of the antimicrobial peptide LL-37 and innate immune mediators in cultured human gingival epithelial cells (GECs). The aim of this study was to further delineate the mechanism by which vitamin D enhances the innate defense against the development of periodontal disease (PD). MATERIALS AND METHODS: Wild-type C57Bl/6 mice were made deficient in vitamin D by dietary restriction. Cultured primary and immortalized GEC were stimulated with 1,25(OH)2 D3 , followed by infection with Porphyromonas gingivalis, and viable intracellular bacteria were quantified. Conversion of vitamin D3 to 25(OH)D3 and 1,25(OH)2 D3 was quantified by ELISA. Effect of vitamin D on basal IL-1α expression in mice was determined by topical administration to the gingiva of wild-type mice, followed by qRT-PCR. RESULTS: Dietary restriction of vitamin D led to alveolar bone loss and increased inflammation in the gingiva in the mouse model. In primary human GEC and established human cell lines, treatment of GEC with 1,25(OH)2 D3 inhibited the intracellular growth of P. gingivalis. Cultured GEC expressed two 25-hydroxylases (CYP27A1 and CYP2R1), as well as 1-α hydroxylase, enabling conversion of vitamin D to both 25(OH)D3 and 1,25(OH)2 D3 . Topical application of both vitamin D3 and 1,25(OH)2 D3 to the gingiva of mice led to rapid inhibition of IL-1α expression, a prominent pro-inflammatory cytokine associated with inflammation, which also exhibited more than a 2-fold decrease from basal levels in OKF6/TERT1 cells upon 1,25(OH)2 D3 treatment, as determined by RNA-seq. CONCLUSION: Vitamin D deficiency in mice contributes to PD, recapitulating the association seen in humans, and provides a unique model to study the development of PD. Vitamin D increases the activity of GEC against the invasion of periodontal pathogens and inhibits the inflammatory response, both in vitro and in vivo. GEC can convert inactive vitamin D to the active form in situ, supporting the hypothesis that vitamin D can be applied directly to the gingiva to prevent or treat periodontal disease.

Potent in vitro and in vivo antifungal activity of a small molecule host defense peptide mimic through a membrane-active mechanism.

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.

Identification of unstimulated constitutive immunocytes, by enzyme histochemistry, in the coenenchyme of the octocoral Swiftia exserta.

Most animals rely on circulating hemocytes as cellular effectors of immunity. These cells traditionally have been characterized by morphology, function, and cellular contents. Morphological descriptions use granule differences and cell shapes; functional descriptions rely on phagocytic ability and oxygen transport; and cellular content descriptions include cytochemical features and key enzymes. Key enzymes used to identify phagocytes in tissues include hydrolytic enzymes, peroxidase, and--in invertebrates--phenoloxidase. Cnidaria such as Swiftia exserta lack a circulatory system, thereby complicating the identification of immune effector cells. As a first step in identifying immunocytes, this study focused on basic enzymes used during phagocytosis and encapsulation; both processes have been reported in octocorals such as S. exserta. Earlier work suggested that there are two populations of phagocytic cells: a constitutive population and an induced population following a trauma-associated challenge. To identify the constitutive immune effector cells in S. exserta in a nonactivated state, we used cryosections of unstimulated animals and the following enzymes to serve as identifying proxies due to their roles in phagocytosis and encapsulation: (1) acid phosphatase, (2) alkaline phosphatase, (3) non-specific esterase, (4) ß-glucuronidase, (5) peroxidase, and (6) phenoloxidase. Our results indicate that in unstimulated animals, two distinct cell populations could function as immunocytes. These cell types were differentiated by their enzyme reactivity and their location within the mesoglea of S. exserta, and have been described as either "oblong granular cells" or "granular amoebocytes."

Plicatamide, an antimicrobial octapeptide from Styela plicata hemocytes.

Plicatamide (Phe-Phe-His-Leu-His-Phe-His-dc Delta DOPA), where dc Delta DOPA represents decarboxy-(E)-alpha,beta-dehydro-3,4-dihydroxyphenylalanine, is a potently antimicrobial octapeptide from the blood cells of the solitary tunicate, Styela plicata. Wild type and methicillin-resistant Staphylococcus aureus (MRSA) responded to plicatamide exposure with a massive potassium efflux that began within seconds. Soon thereafter, treated bacteria largely ceased consuming oxygen, and most became nonviable. Native plicatamide also formed cation-selective channels in model lipid bilayers composed of bacterial lipids. Methicillin-resistant S. aureus treated with plicatamide for 5 min contained prominent mesosomes as well as multiple, small dome-shaped surface protrusions that suggested the involvement of osmotic forces in its antimicrobial effects. To ascertain the contribution of the C-terminal dc Delta DOPA residue to antimicrobial activity, we synthesized several analogues of plicatamide that lacked it. One of these peptides, PL-101 (Phe-Phe-His-Leu-His-Phe-His-Tyr-amide), closely resembled native plicatamide in its antimicrobial activity and its ability to induce potassium efflux. Plicatamide was potently hemolytic for human red blood cells but did not lyse ovine erythrocytes. The small size, rapid action, and potent anti-staphylococcal activity of plicatamide and PL-101 make them intriguing subjects for future antimicrobial peptide design.

Natural Peptide antibiotics from tunicates: structures, functions and potential uses.

Because tunicates rely on innate immunity, their hemocytes are important contributors to host defense. Styela clava, a solitary ascidian, have eight hemocyte subtypes. Extracts of their total hemocyte population contained multiple small (2-4 kDa) antimicrobial peptides. When purified, these fell into two distinct families that were named styelins and clavanins.Styelins A-E are phenylalanine-rich, 32 residue peptides with activity against marine bacteria and human pathogens. They show considerable sequence homology to pleurocidins, antimicrobial peptides of the flounder, Pseudopleuronectes americanus. Styelin D, one of the five styelins identified by peptide isolation and cDNA cloning, was remarkable in containing 12 post-translationally modified residues, including a 6-bromotryptophan, two monohydroxylysines, four 3,4-dihydroxyphenylalanines (DOPA), four dihydroxylysines and one dihydroxyarginine. These modifications enhanced Styelin D's bactericidal ability at acidic pH and high salinity. A novel histochemical stain for DOPA suggested that Styelin D was restricted to granulocytes.Clavanins A-E are histidine-rich, 23 residue peptides that are C-terminally amidated and most effective at acidic pH. Clavaspirin is a newly described family member that also has potent cytotoxic properties. By immunocytochemistry, clavanins were identified in the granules of five eosinophilic granulocyte subtypes and in macrophage cytoplasm.Transmission and scanning electron micrographs of methicillin-resistant Staphylococcus aureus (MRSA) and E. coli that had been treated with Styelin D and clavaspirin suggested that both peptides induced osmotic disregulation. Treated bacteria manifested cytoplasmic swelling and extrusion of cytoplasmic contents through their peptidoglycan cell wall. The diverse array of antimicrobial peptides in S. clava hemocytes constitutes an effective host defense mechanism.

Immunolocalization of clavanins in Styela clava hemocytes.

Antimicrobial peptides play an important role in innate host defenses against infection. Clavanins are histidine-rich, amidated, 23-residue alpha-helical antimicrobial peptides that were isolated from a mixed population of Styela clava hemocytes. To learn which types of hemocytes contained clavanins, we raised a polyclonal antibody that recognized five different clavanins, and used it to localize these peptides by light and electron microscopy. Clavanins were present in the cytoplasmic granules and/or cytoplasm of five different types of granulocytes and they also occurred throughout the cytoplasm of macrophages. The orange G component of Mallory's trichrome stain had a high affinity for clavanins, and for the cytoplasmic granules of S. clava's hemocytes. Semiquantitative analysis of acid urea-PAGE gels suggested that clavanins and styelins comprised between 10 and 20% of the total cellular protein of eosinophilic granulocytes. Orange G and the century-old trichrome stain may provide simple screening tools for identifying cells that contain large amounts of antimicrobial peptides in mixed hemocyte populations.