real-time-imaging-quantification-fungal-biofilm-development-using-two

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

University at Buffalo

Salivary gland cell differentiation has been a recurring challenge for researchers as primary salivary cells show a loss of phenotype in culture. Particularly, parotid cells show a marked decrease in amylase expression, the loss of tight junction organization and proper cell function. Previously, Matrigel has been used successfully as an extracellular matrix; however, it is not practical for in vivo applications as it is tumorigenic. An alternative method could rely on the use of fibrin hydrogel (FH), which has been used extensively in biomedical engineering applications ranging from cardiovascular tissue engineering to wound-healing experiments. Although several groups have examined the effects of a three-dimensional (3D) environment on salivary cell cultures, little is known about the effects of FH on salivary cell cultures. The current study developed a 3D cell culture model to support parotid gland cell differentiation using a combination of FH and growth factor-reduced Matrigel (GFR-MG). Furthermore, FH polymerized with a combination of EGF and IGF-1 induced formation of 3D spheroids capable of amylase expression and an agonist-induced increase in the intracellular Ca(2+) concentration ([Ca(2+)]i) in salivary cells. These studies represent an initial step toward the construction of an artificial salivary gland to restore salivary gland dysfunction. This is necessary to reduce xerostomia in patients with compromised salivary function.

Growth factors polymerized within fibrin hydrogel promote amylase production in parotid cells.

Sjögren's syndrome (SS) is an autoimmune disorder characterized by chronic inflammation and destruction of salivary and lacrimal glands, leading to dry mouth, dry eyes, and the presence of anti-nuclear antibodies. Despite modern advances, the current therapies for SS have no permanent benefit. A potential treatment could involve the use of resolvins, which are highly potent endogenous lipid mediators that are synthesized during the resolution of inflammation to restore tissue homeostasis. Our previous studies indicate that ALX/FPR2, the receptor for RvD1, is expressed and active in the rat parotid cell line Par-C10. Specifically, activation of ALX/FPR2 with RvD1 blocked inflammatory signals caused by TNF-α and enhanced salivary epithelial integrity. The goal of this study was to investigate RvD1 receptor expression and signaling pathways in primary salivary cells. Additionally, we determined the role of the aspirin-triggered 17R analog (AT-RvD1, a more chemically stable RvD1 epimeric form) in prevention of TNF-α-mediated salivary inflammation in mouse submandibular glands (mSMG). Our results indicate that ALX/FPR2 is expressed in mSMG and is able to elicit intracellular Ca2+ responses and phosphorylation of Erk1/2, as well as Akt. Given that these signaling pathways are linked to cell survival, we investigated whether AT-RvD1 was able to prevent programmed cell death in mSMG. Specifically, we determined that AT-RvD1 prevented TNF-α-mediated caspase-3 activation. Finally, we show that ALX/FPR2 is expressed in human minor salivary glands with and without SS, indicating the potential therapeutic use of AT-RvD1 for this condition.

ALX/FPR2 receptor for RvD1 is expressed and functional in salivary glands.

Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disorder that causes secretory dysfunction of the salivary glands leading to dry mouth. Previous studies reported that tight junction (TJ) proteins are down-regulated and lose polarity in human minor salivary glands with SS, suggesting that TJ structure is compromised in SS patients. In this paper, we utilized the NOD/ShiLtJ mouse with the main goal of evaluating this model for future TJ research. We found that the organization of apical proteins in areas proximal and distal to lymphocytic infiltration remained intact in mouse and human salivary glands with SS. These areas looked comparable to control glands (i.e., with no lymphocytic infiltration). TJ staining was absent in areas of lymphocytic infiltration coinciding with the loss of salivary epithelium. Gene expression studies show that most TJs are not significantly altered in 20-week-old NOD/ShiLtJ mice as compared with age-matched C57BL/6 controls. Protein expression studies revealed that the TJ proteins, zonula occludens-1 (ZO-1), occludin, claudin-12, as well as E-cadherin, do not significantly change in NOD/ShiLtJ mice. Our results suggest that ZO-1, occludin and E-cadherin are not altered in areas without lymphocytic infiltration. However, future studies will be necessary to test the functional aspect of these results.

Zonula occludens-1, occludin and E-cadherin expression and organization in salivary glands with Sjögren's syndrome.

 is a newly identified species causing invasive candidemia and candidiasis. It has broad multidrug resistance (MDR) not observed for other pathogenic  species. Histatin 5 (Hst 5) is a well-studied salivary cationic peptide with significant antifungal activity against  and is an attractive candidate for treating MDR fungi, since antimicrobial peptides induce minimal drug resistance. We investigated the susceptibility of  to Hst 5 and neutrophils, two first-line innate defenses in the human host. The majority of  clinical isolates, including fluconazole-resistant strains, were highly sensitive to Hst 5: 55 to 90% of cells were killed by use of 7.5 μM Hst 5. Hst 5 was translocated to the cytosol and vacuole in  cells; such translocation is required for the killing of  by Hst 5. The inverse relationship between fluconazole resistance and Hst 5 killing suggests different cellular targets for Hst 5 than for fluconazole.  showed higher tolerance to oxidative stress than , and higher survival within neutrophils, which correlated with resistance to oxidative stress  Thus, resistance to reactive oxygen species (ROS) is likely one, though not the only, important factor in the killing of  by neutrophils. Hst 5 has broad and potent candidacidal activity, enabling it to combat MDR  strains effectively.

Fluconazole-Resistant Candida auris Is Susceptible to Salivary Histatin 5 Killing and to Intrinsic Host Defenses.

The ability of  to form biofilms is a virulence factor that allows tissue attachment and subsequent infection of host tissues. Fungal biofilms have been particularly well studied, however the vast majority of these studies have been conducted under static conditions. Oral biofilms form in the presence of salivary flow, therefore we developed a novel flow system used for real-time imaging of fungal biofilm development.  wild-type (WT) cells readily attached to the substrate surface during the 2 h attachment phase, then formed heterogeneous biofilms after 18 h flow. Quantitative values for biomass, rates of attachment and detachment, and cell-cell adhesion events were obtained for  WT cells and for a hyperfilamentous mutant Δ. Attachment rates of  WT cells were nearly 2-fold higher than  Δ cells, although Δ cells formed 4-fold higher biomass. The reduced normalized detachment rate was the primary factor responsible for the increased biomass of Δ biofilm, showing that cell detachment rates are an important predictor for ultimate biofilm mass under flow. Unlike static biofilms,  cells under constant laminar flow undergo continuous detachment and seeding that may be more representative of the development of in vivo biofilms.

Andrew D. McCall

Department of Oral Biology

University at Buffalo

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

Andrew D. McCall

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Oral Biology

University at Buffalo

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

Department of Oral Biology