Upregulated epithelial junction expression represents a novel parameter of the epithelial radiation response to fractionated irradiation in oral mucosa.

PURPOSE: During head and neck cancer treatment, the radiation response of the oral mucosa represents a frequent early side effect. Besides radiation-induced inhibition of proliferation, various other cellular responses occur. The radiation response of adherens and tight junction proteins was so far mostly investigated with large single-dose irradiation protocols, in vivo and in vitro. Therefore, the current study was initiated to investigate the impact of daily fractionated irradiation on the expression of adherens and tight junction proteins in vivo. MATERIALS AND METHODS: Fractionation with 5â€¯× 3 Gy/week (days 0-4, 7-11) was given to the snouts of mice. Groups of 5 animals per day were euthanized every second day between day 0 (unirradiated controls) and day 14, and their tongues subjected to histological processing. Adherens junction marker (ß-catenin and E­cadherin) and tight junction marker (claudin-1 and occludin) expression was analysed in the oral mucosa of unirradiated controls and during two weeks of fractionated irradiation. RESULTS: Adherens as well as tight junction marker proteins were rapidly and consistently upregulated in both the germinal as well as the functional layer of the oral mucosa. This represents a previously unknown parameter of the epithelial radiation response to clinically relevant fractionation protocols. CONCLUSION: Fractionated irradiation significantly enhanced the expression of all proteins investigated. This study revealed a new parameter of the epithelial radiation response to fractionated irradiation.

Radioprotective Effects of Dermatan Sulfate in a Preclinical Model of Oral Mucositis-Targeting Inflammation, Hypoxia and Junction Proteins without Stimulating Proliferation.

Oral mucositis is the most frequently occurring early side effect of head-and-neck cancer radiotherapy. Systemic dermatan sulfate (DS) treatment revealed a significant radioprotective potential in a preclinical model of oral mucositis. This study was initiated to elucidate the mechanistic effects of DS in the same model. Irradiation comprised daily fractionated irradiation (5 × 3 Gy/week) over two weeks, either alone (IR) or in combination with daily dermatan sulfate treatment of 4 mg/kg (IR + DS). Groups of mice (n = 5) were sacrificed every second day over the course of 14 days in both experimental arms, their tongues excised and evaluated. The response to irradiation with and without DS was analyzed on a morphological (cell numbers, epithelial thickness) as well as on a functional (proliferation and expression of inflammation, hypoxia and epithelial junction markers) level. The mucoprotective activity of DS can be attributed to a combination of various effects, comprising increased expression of epithelial junctions, reduced inflammation and reduced hypoxia. No DS-mediated effect on proliferation was observed. DS demonstrated a significant mucositis-ameliorating activity and could provide a promising strategy for mucositis treatment, based on targeting specific, radiation-induced, mucositis-associated signaling without stimulating proliferation.

Growth defect of domain III glycoprotein B mutants of human cytomegalovirus reverted by compensatory mutations co-localizing in post-fusion conformation.

Cell entry is a crucial step for a virus to infect a host cell. Human cytomegalovirus utilizes glycoprotein B (gB) to fuse the viral and host cell membranes upon receptor binding of gH/gL-containing complexes. Fusion is mediated by major conformational changes of gB from a metastable pre-fusion to a stable post-fusion state whereby the central trimeric coiled-coils, formed by domain (Dom)III α helices, remain structurally nearly unchanged. To better understand the role of the stable core, we individually introduced three potentially helix-breaking or one disulfide bond-breaking mutation in the DIII α3 to study different aspects of the viral behavior upon long-term culturing. Two of the three helix-breaking mutations, gB_Y494P and gB_I495P, were lethal for the virus in either fibroblasts or epithelial cells. The third substitution, gB_G493P, on the other hand, displayed a delayed replication and spread, which was more pronounced in epithelial cells, hinting at an impaired fusion. Interestingly, the disulfide bond-breaker mutation, gB_C507S, performed strikingly differently in the two cell types - lethal in epithelial cells and an atypical phenotype in fibroblasts, respectively. Replication curve analyses paired with the infection efficiency, the spread morphology, and the cell-cell fusogenicity suggest a dysregulated fusion process, which could be reverted by second-site mutations mapping predominantly to gB DomV. Our findings underline the functional importance of a stable DomIII core for a well-regulated DomV rearrangement during fusion.IMPORTANCEHuman cytomegalovirus (HCMV) can establish a lifelong infection. In most people, the infection follows an asymptomatic course; however, it is a major cause of morbidity and mortality in immunocompromised patients or neonates. HCMV has a very broad cell tropism, ranging from fibroblasts to epi- and endothelial cells. The virus uses different entry pathways utilizing the core fusion machinery consisting of glycoprotein complexes gH/gL and glycoprotein B (gB). The fusion protein gB undergoes fundamental rearrangements from a metastable pre-fusion to a stable post-fusion conformation. Here, we characterized the viral behavior after the introduction of four single-point mutations in the gB central core. These led to various cell type-specific atypical phenotypes and the emergence of compensatory mutations, demonstrating an important interaction between domains III and V. We provide a new basis for the development of a structurally and functionally altered gB, which can further serve as a tool for drug and vaccine development.