The impact of time between surgery and adjuvant chemoradiotherapy in advanced oral cavity squamous cell carcinoma.

Objective: In advanced oral squamous cell carcinoma (OSCC), adjuvant therapy (AT) is an important part of the treatment to ensure extended locoregional control after primary surgical resection. The impact of the time interval between surgery and AT on the oncological prognosis remains unclear, particularly in high-risk constellations. The aim of this study is to categorize treatment delays and to determine their impact on the oncological prognosis within the context of the histopathological risk parameters of patients with advanced OSCC. Methods: In this single-institutional retrospective cohort study, all patients treated for OSCC between 2016 and 2021 and who received postoperative chemoradiation (POCRT) were included. Patients were divided into two groups: Group I: ≤ 6 weeks between surgery and POCRT; and Group II: > 6 weeks between surgery and POCRT. Results: Overall, 202 patients were included (Group I: 156 (77.2%) vs. Group II: 46 (22.8%)). There were no statistically significant differences in epidemiological aspects and histopathological risk factors between the two groups. The maximum time to initiation of POCRT was 11 weeks. Delayed POCRT initiation had no statistically significant influence on the 5-year OS (61.6% vs. 57.3%, p = 0.89), locoregional control rate (38.6% vs. 43.3%, p = 0.57), and RFS (32.3% vs. 30.4%, p = 0.21). On multivariate analysis, extracapsular spread (HR: 2.21, 95% CI: 1.21 - 4.04, p = 0.01) and incomplete surgical resection (HR: 2.01, 95% CI: 1.10 - 3.69, p = 0.02) were significantly correlated with OS. For RFS, ECS (HR: 1.82, 95% CI: 1.15 - 2.86, p = 0.01), incomplete resection (HR: 1.67, 95% CI: 1.04 - 2.71, p = 0.04), and vascular infiltration of the tumor (V-stage; HR: 2.15, 95% CI: 1.08 - 4.27, p = 0.03) were significant risk predictors. Conclusion: Delays in POCRT initiation up to 11 weeks after surgical resection for advanced OSCC were not statistically significantly associated with impaired survival. In cases of prolonged surgical treatment due to management of complications, a small delay in AT beyond the recommended time limit may be justified and AT should still be pursued.

Intracellular Dynamics of Extracellular Vesicles by Segmented Trajectory Analysis.

The analysis of nanoparticle (NP) dynamics in live cell studies by video tracking provides detailed information on their interactions and trafficking in the cells. Although the video analysis is not yet routinely used in NP studies, the equipment suitable for the experiments is already available in most laboratories. Here, we compare trajectory patterns, diffusion coefficients, and particle velocities of NPs in A549 cells with a rather simple experimental setup consisting of a fluorescence microscope and openly available trajectory analysis software. The studied NPs include commercial fluorescent polymeric particles and two subpopulations of PC-3 cell-derived extracellular vesicles (EVs). As bioderived natural nanoparticles, the fluorescence intensities of the EVs limited the recording speed. Therefore, we studied the effect of the recording frame rate and analysis parameters to the trajectory results with bright fluorescent commercial NPs. We show that the trajectory classification and the apparent particle velocities are affected by the recording frame rate, while the diffusion constants stay comparable. The NP trajectory patterns were similar for all NP types and resembled intracellular vesicular transport. Interestingly, the EV movements were faster than the commercial NPs, which contrasts with their physical sizes and may indicate a greater role of the motor proteins in their intracellular transports.

Role of Fiber Shaft Length in Tumor Targeting with Ad5/3 Vectors.

Desmoglein 2 (DSG2) is overexpressed in many epithelial cancers and therefore represents a target receptor for oncolytic viruses, including Ad5/3-based viruses. For most Ad serotypes, the receptor-binding fiber is composed of tail, shaft, and knob domains. Here, we investigated the role of the fiber shaft in Ad5/3 tumor transduction in vitro and in human DSG2-transgenic mice carrying human DSG2high tumors. DSG2tg mice express DSG2 in a pattern similar to humans. We constructed Ad5/3L (with the "long" Ad5 shaft) and Ad5/3S (with the "short" Ad3 shaft) expressing GFP or luciferase. In in vitro studies we found that coagulation factor X, which is known to mediate undesired hepatocyte transduction of Ad5, enhances the transduction of Ad5/3(L), but not the transduction of Ad5/3(S). We therefore hypothesized that Ad5/3(S) would target DSG2high tumors while sparing the liver after intravenous injection. In vivo imaging studies for luciferase and analysis of luciferase activity in isolated organs, showed that Ad5/3(L) vectors efficiently transduced DSG2high tumors and liver but not normal epithelial tissues after intravenous injection. Ad5/3(S) showed minimal liver transduction, however it failed to transduce DSG2high tumors. Further modifications of the Ad5/3(S) capsid are required to compensate for the lower infectivity of Ad5/3(S) vectors.

Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched allogeneic CAR-T cells.

The use of T cells from healthy donors for allogeneic chimeric antigen receptor T (CAR-T) cell cancer therapy is attractive because healthy donor T cells can produce versatile off-the-shelf CAR-T treatments. To maximize safety and durability of allogeneic products, the endogenous T cell receptor and major histocompatibility complex class I molecules are often removed via knockout of T cell receptor beta constant (TRBC) (or T cell receptor alpha constant [TRAC]) and B2M, respectively. However, gene editing tools (e.g., CRISPR-Cas9) can display poor fidelity, which may result in dangerous off-target mutations. Additionally, many gene editing technologies require T cell activation, resulting in a low percentage of desirable stem cell memory T cells (TSCM). We characterize an RNA-guided endonuclease, called Cas-CLOVER, consisting of the Clo051 nuclease domain fused with catalytically dead Cas9. In primary T cells from multiple donors, we find that Cas-CLOVER is a high-fidelity site-specific nuclease, with low off-target activity. Notably, Cas-CLOVER yields efficient multiplexed gene editing in resting T cells. In conjunction with the piggyBac transposon for delivery of a CAR transgene against the B cell maturation antigen (BCMA), we produce allogeneic CAR-T cells composed of high percentages of TSCM cells and possessing potent in vivo anti-tumor cytotoxicity.

COVID-19 Vaccine Booster Strategies in Light of Emerging Viral Variants: Frequency, Timing, and Target Groups.

BACKGROUND: Vaccinations have reduced severe burden of COVID-19 and allowed for lifting of non-pharmaceutical interventions. However, with immunity waning alongside emergence of more transmissible variants of concern, vaccination strategies must be examined. METHODS: Here we apply a SARS-CoV-2 transmission model to identify preferred frequency, timing, and target groups for vaccine boosters to reduce public health burden and health systems risk. We estimated new infections and hospital admissions averted over 2 years through annual or biannual boosting of those eligible (those who received doses one and two) who are (1) most vulnerable (60+ or living with comorbidities) or (2) those 5+, at universal (98% of eligible) or lower coverage (85% of those 50+ or with comorbidities and 50% of 5-49 year olds) representing moderate vaccine fatigue and/or hesitancy. We simulated three emerging variant scenarios: (1) no new variants; (2) 25% more infectious and immune-evading Omicron-level severity variants emerge annually and become dominant; (3) emerge biannually. We further explored the impact of varying seasonality, variant immune-evading capacity, infectivity, severity, timing, and vaccine infection blocking assumptions. RESULTS: To reduce COVID-19-related hospitalisations over the next 2 years, boosters should be provided for all those eligible annually 3-4 months ahead of peak winter whether or not new variants of concern emerge. Only boosting those most vulnerable is unlikely to ensure reduced stress on health systems. Moreover, boosting all eligible better protects those most vulnerable than only boosting the vulnerable group. Conversely, while this strategy may not ensure reduced stress on health systems, as an indication of cost-effectiveness, per booster dose more hospitalisations could be averted through annual boosting of those most vulnerable versus all eligible, since those most vulnerable are more likely to seek hospital care once infected, whereas increasing to biannual boosting showed diminishing returns. Results were robust when key model parameters were varied. However, we found that the more frequently variants emerge, the less the effect boosters will have, regardless of whether administered annually or biannually. CONCLUSIONS: Delivering well-timed annual COVID-19 vaccine boosters to all those eligible, prioritising those most vulnerable, can reduce infections and hospital admissions. Findings provide model-based evidence for decision-makers to plan for administering COVID-19 boosters ahead of winter 2022-2023 to help mitigate the health burden and health system stress.

Erratum: Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched allogeneic CAR-T cells.

[This corrects the article DOI: 10.1016/j.omtn.2022.06.003.].

Modified McLaughlin's Dynamic Muscle Support in the 21st Century? A Retrospective Study With a Prospective Follow-Up Analysis for Patients With Long Standing Facial Paralysis.

INTRODUCTION: Facial nerve paralysis can dramatically affect the life of a patient as it leads to significant alterations of the facial symmetry and functional limitations. Various methods exist including free neuromuscular flaps to reanimate patients suffering from uni- or even bilateral facial nerve paralysis. The more than 60-year-old technique described by McLaughlin continues to offer an alternative with distinct advantages for the individual patient. The present study aimed to evaluate clinical outcome and satisfaction of patients treated with a modified McLaughlin's Dynamic Muscle Support. MATERIALS AND METHODS: A total of 13 patients (mean age of 58.4 years) who received a modified McLaughlin's Dynamic Muscle Support due to uni- or bilateral long-standing facial paralysis were included. Medical records were reviewed retrospectively, and patients were contacted for additional follow-up. Patients who agreed to participate in the follow-up study were asked to answer a self-developed questionnaire. RESULTS: In all patients, a rehabilitation of facial symmetry with an improvement of the mimic expression could be achieved. Mean length of inpatient stay was 6.5 days and average duration of surgery was 121 minutes. No surgical site infection occurred. Mean follow-up was 23 months. Most of the patients were fully satisfied with the result and could experience functional and esthetic improvement.Patients who participated in the prospective follow-up study were very satisfied with the esthetic result and functional outcome. CONCLUSIONS: Even in times of advanced microsurgical techniques, McLaughlin's Dynamic Muscle Support appears to be a good alternative for the successful treatment of long-standing facial paralysis.

The Prognostic Significance of p16 and its Role as a Surrogate Marker for Human Papilloma Virus in Oral Squamous Cell Carcinoma: An Analysis of 281 Cases.

BACKGROUND/AIM: This study analyzed the expression of p16 in a large cohort of patients suffering from oral squamous cell carcinoma (OSCC) who received initial surgical therapy in order to evaluate the prognostic significance of p16 expression and to analyze its value as a surrogate marker to determine human papilloma virus (HPV) status. MATERIALS AND METHODS: Immunohistochemical staining of p16 was performed on tissue microarrays. Different expression levels of p16 (>25%; >50%; ≥70%) with a moderate to strong intensity were correlated with the clinical outcome. HPV DNA was analyzed by polymerase chain reaction (PCR). RESULTS: A total of 281 patients were included in this study. The p16 expression obtained using the abovementioned three different cutoffs did not significantly influence 5-year overall survival (OS) (p=0.23; p=0.45; p=0.23) nor recurrence-free survival (RFS) (p=0.79; p=0.45; p=0.142). In univariate Cox regression analysis, the p16 expression level was not a risk factor for OS (HR=0.637; 95%CI=0.271-1.5; p=0.300) and RFS (HR=0.74; 95%CI=0.339-1.61; p=0.449). A total of 17 patients (6.0%) were p16 positive with a cutoff ≥70%. HPV DNA was found in 4/11 of these cases by PCR, resulting in a positive predictive value of 0.36. In patients receiving adjuvant radio(chemo)therapy, a significantly (p=0.042) longer OS was observed in patients with p16 expression greater than 25% vs. ≤25%. CONCLUSION: In comparison with OPSCC, (strong) p16 positivity is rare in OSCC; however, in patients receiving primary surgery with adjuvant radio(chemo)therapy, p16 expression is associated with a higher survival rate. In conjunction with prior studies, p16 does not seem to be a reliable surrogate marker for HPV infection in OSCC.

Anatomic risk factors for the occurrence of medial talar osteochondral lesions: a case-control study.

OBJECTIVE: This study aimed to determine the anatomical risk factors that may play a role in the etiology of medial-sided osteochondral lesions of the talus (OLT) using morphological parameters in magnetic resonance imaging (MRI). SUBJECTS AND METHODS: One hundred twenty-four patients with medial-sided OLT and age- and sex-matched 124 controls were included in this retrospective study. Two examiners conducted independent OLT classification and measurements of five MRI parameters: tibial axis-medial malleolus angle (TMM), the anterior opening angle of the talus (AOT), talus position (TalPos), the ratio of the distal tibial articular surface to the length of the trochlea tali arc (TAS/TAL), depth of the incisura fibularis (IncDep). Statistical analysis included intraclass correlation coefficients, independent t-tests, receiver-operating characteristic (ROC) analysis, area under the curve (AUC) calculation, and logistic regression analysis. A p-value < 0.05 was considered statistically significant. RESULTS: TTM, AOT, TalPos, and TAL values were significantly higher and the TAS/TAL ratio was significantly lower in the case group than in the control group (p < 0.001). Cut-off and AUC values for TMM were 15.15° (AUC 0.763), AOT 13.05° (AUC 0.826), TalPos 0.75 mm (AUC 0.887), TAL 35.45 mm (AUC 0.642), and TAS/TAL ratio 0.82 (AUC 0.784), p < 0.001. Multivariate logistic regression analysis results were odds ratio (OR) = 6.1 for TMM ≥ 15.15°, OR = 8.9 for AOT ≥ 13.05°, OR = 36.1 for TalPos ≥ 0.75 mm, and OR = 6.7 for TAS/TAL ratio ≤ 0.82. CONCLUSION: Ankle morphology might have an influence on OLT development. The talus position (TalPos) and anterior opening angle of the talus (AOT) seemed to be the strongest predisposing factors.

Alterations in magnetic resonance imaging characteristics of bioabsorbable magnesium screws over time in humans: a retrospective single center study.

OBJECTIVES: This study aimed to examine the alterations in magnetic resonance imaging (MRI) characteristics of bioabsorbable magnesium (Mg) screws over time in a single center study in humans. METHODS: Seventeen patients who underwent medial malleolar (MM) fracture or osteotomy fixation using bioabsorbable Mg screws and had at least one postoperative MRI were included in this retrospective study. Six of them had more than one MRI in the postoperative period and were subject of the artifact reduction measurements. 1.5T or 3T MRI scans were acquired in different periods in each patient. The size and extent of the artifact were assessed independently by two experienced radiologists both quantitatively (distance measurement) and qualitatively (Likert scale). RESULTS: In the quantitative measurements of the six follow-up patients the screw's signal loss artifact extent significantly decreased over the time, regardless of the MRI field strength (p<0.001). The mean artifact reduction was 0.06 mm (95% confidence interval [CI]: 0.05-0.07) for proton density weighted [PDw] and 0.04 mm (95% CI: 0.03-0.05) for T1 weighted (T1w) sequences per week. The qualitative assessments similarly showed significant artifact reduction in all MRI sequences. Different imaging findings, like bone marrow edema (BME), liquid collections, and gas formation were reported. The overall inter-reader agreement was high (κ=0.88, p<0.001). CONCLUSIONS: The time-dependent artifact reduction of Mg screws in postoperative controls might indicate the expected self-degradation of the Mg implants. In addition, different MRI findings were reported, which are characteristic of Mg implants. Further MRI studies are required to get a better understanding of Mg imaging properties.

Enhancing the Stabilization Potential of Lyophilization for Extracellular Vesicles.

Extracellular vesicles (EV) are an emerging technology as immune therapeutics and drug delivery vehicles. However, EVs are usually stored at -80 °C which limits potential clinical applicability. Freeze-drying of EVs striving for long-term stable formulations is therefore studied. The most appropriate formulation parameters are identified in freeze-thawing studies with two different EV types. After a freeze-drying feasibility study, four lyophilized EV formulations are tested for storage stability for up to 6 months. Freeze-thawing studies revealed improved colloidal EV stability in presence of sucrose or potassium phosphate buffer instead of sodium phosphate buffer or phosphate-buffered saline. Less aggregation and/or vesicle fusion occurred at neutral pH compared to slightly acidic or alkaline pH. EVs colloidal stability can be most effectively preserved by addition of low amounts of poloxamer 188. Polyvinyl pyrrolidone failed to preserve EVs upon freeze-drying. Particle size and concentration of EVs are retained over 6 months at 40 °C in lyophilizates containing 10 mm K- or Na-phosphate buffer, 0.02% poloxamer 188, and 5% sucrose. The biological activity of associated beta-glucuronidase is maintained for 1 month, but decreased after 6 months. Here optimized parameters for lyophilization of EVs that contribute to generate long-term stable EV formulations are presented.

German oncology certification system for colorectal cancer - relative survival rates of a single certified centre vs. national and international registry data.

OBJECTIVES: To evaluate how the certification of specialised Oncology Centres in Germany affects the relative survival of patients with colorectal cancer (CRC) by means of national and international comparison. METHODS: Between 2007 and 2013, 675 patients with colorectal cancer, treated at the Hildesheim Hospital, an academic teaching hospital of the Hannover Medical School (MHH), were included. A follow-up of the entire patient group was performed until 2014. To obtain international data, a SEER-database search was done. The relative survival of 148,957 patients was compared to our data after 12, 36 and 60 months. For national survival data, we compared our rates with 41,988 patients of the Munich Cancer Registry (MCR). RESULTS: Relative survival at our institution tends to be higher in advanced tumour stages compared to national and international cancer registry data. Nationally we found only little variation in survival rates for low stages CRC (UICC I and II), colon, and rectal cancer. There were notable variations regarding relative survival rates for advanced CRC tumour stages (UICC IV). These variations were even more distinct for rectal cancer after 12, 36 and 60 months (Hildesheim Hospital: 89.9, 40.3, 30.1%; Munich Cancer Registry (MCR): 65.4, 28.7, 16.6%). The international comparison of CRC showed significantly higher relative survival rates for patients with advanced tumour stages after 12 months at our institution (77 vs. 54.9% for UICC IV; raw p<0.001). CONCLUSIONS: Our findings suggest that patients with advanced tumour stages of CRC and especially rectal cancer benefit most from a multidisciplinary and guidelines-oriented treatment at Certified Oncology Centres. For a better evaluation of cancer treatment and improved national and international comparison, the creation of a centralised national cancer registry is necessary.

Approaches to surface engineering of extracellular vesicles.

Extracellular vesicles (EVs) are cell-derived nanoparticles that are important mediators in intercellular communication. This function makes them auspicious candidates for therapeutic and drug-delivery applications. Among EVs, mammalian cell derived EVs and outer membrane vesicles (OMVs) produced by gram-negative bacteria are the most investigated candidates for pharmaceutical applications. To further optimize their performance and to utilize their natural abilities, researchers have strived to equip EVs with new moieties on their surface while preserving the integrity of the vesicles. The aim of this review is to give a comprehensive overview of techniques that can be used to introduce these moieties to the vesicle surface. Approaches can be classified in regards to whether they take place before or after the isolation of EVs. The producing cells can be subjected to genetic manipulation or metabolic engineering to produce surface modified vesicles or EVs are engineered after their isolation by physical or chemical means. Here, the advantages and disadvantages of these processes and their applicability for the development of EVs as therapeutic agents are discussed.

Author Correction: Extracellular vesicles protect glucuronidase model enzymes during freeze-drying.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evaluation of the Storage Stability of Extracellular Vesicles.

Extracellular vesicles (EVs) are promising targets in current research, to be used as drugs, drug-carriers, and biomarkers. For their clinical development, not only their pharmaceutical activity is important but also their production needs to be evaluated. In this context, research focuses on the isolation of EVs, their characterization, and their storage. The present manuscript aims at providing a facile procedure for the assessment of the effect of different storage conditions on EVs, without genetic manipulation or specific functional assays. This makes it possible to quickly get a first impression of the stability of EVs under a given storage condition, and EVs derived from different cell sources can be compared easily. The stability measurement is based on the physicochemical parameters of the EVs (size, particle concentration, and morphology) and the preservation of the activity of their cargo. The latter is assessed by the saponin-mediated encapsulation of the enzyme beta-glucuronidase into the EVs. Glucuronidase acts as a surrogate and allows for an easy quantification via the cleavage of a fluorescent reporter molecule. The present protocol could be a tool for researchers in the search for storage conditions that optimally retain EV properties to advance EV research toward clinical application.

Extracellular vesicles protect glucuronidase model enzymes during freeze-drying.

Extracellular vesicles (EVs) are natural nanoparticles that play important roles in intercellular communication and are increasingly studied for biosignalling, pathogenesis and therapy. Nevertheless, little is known about optimal conditions for their transfer and storage, and the potential impact on preserving EV-loaded cargoes. We present the first comprehensive stability assessment of different widely available types of EVs during various storage conditions including -80 °C, 4 °C, room temperature, and freeze-drying (lyophilisation). Lyophilisation of EVs would allow easy handling at room temperature and thus significantly enhance their expanded investigation. A model enzyme, ß-glucuronidase, was loaded into different types of EVs derived from mesenchymal stem cells, endothelial cells and cancer cells. Using asymmetric flow field-flow fractionation we proved that the model enzyme is indeed stably encapsulated into EVs. When assessing enzyme activity as indicator for EV stability, and in comparison to liposomes, we show that EVs are intrinsically stable during lyophilisation, an effect further enhanced by cryoprotectants. Our findings provide new insight for exploring lyophilisation as a novel storage modality and we create an important basis for standardised and advanced EV applications in biomedical research.

A Combined In Vivo HSC Transduction/Selection Approach Results in Efficient and Stable Gene Expression in Peripheral Blood Cells in Mice.

We recently reported on an in vivo hematopoietic stem cell (HSC) gene therapy approach. It involves the subcutaneous injections of G-CSF/AMD3100 to mobilize HSCs from the bone marrow into the peripheral blood stream and the intravenous injection of an integrating helper-dependent adenovirus vector system. HSCs transduced in the periphery homed back to the bone marrow, where they persisted long-term. However, high transgene marking rates found in primitive bone marrow HSCs were not reflected in peripheral blood cells. Here, we tested small-molecule drugs to achieve selective mobilization and transduction of HSCs. We found more efficient GFP marking in bone marrow HSCs but no increased marking in the peripheral blood cells. We then used an in vivo HSC chemo-selection based on a mutant of the O6-methylguanine-DNA methyltransferase (mgmtP140K) gene that confers resistance to O6-BG/BCNU and should give stably transduced HSCs a proliferation stimulus and allow for the selective survival and expansion of progeny cells. Short-term exposure of G-CSF/AMD3100-mobilized, in vivo-transduced mice to relatively low selection drug doses resulted in stable GFP expression in up to 80% of peripheral blood cells. Overall, the further improvement of our in vivo HSC transduction approach creates the basis for a simpler HSC gene therapy.

In Vivo Hematopoietic Stem Cell Transduction.

Current protocols for hematopoietic stem cell (HSC) gene therapy, involving the transplantation of ex vivo lentivirus vector-transduced HSCs into myeloablated recipients, are complex and not without risk for the patient. In vivo HSC gene therapy can be achieved by the direct modification of HSCs in the bone marrow after intraosseous injection of gene delivery vectors. A recently developed approach involves the mobilization of HSCs from the bone marrow into peripheral the blood circulation, intravenous vector injection, and re-engraftment of genetically modified HSCs in the bone marrow. We provide examples for in vivo HSC gene therapy and discuss advantages and disadvantages.

The frequent and underrecognised co-occurrence of acute exacerbated COPD and depression warrants screening: a systematic review.

Patients with acute exacerbated chronic obstructive pulmonary disease (AECOPD) and concurrent depression suffer significant psychological stress and decreased quality of life. The aim of this study was to collate data, guidelines and recommendations from publications on the screening and management of depressive mood disorders in patients hospitalised with AECOPD.We systematically searched four databases for publications reporting screening or management of depression in patients hospitalised for AECOPD. The identification of articles was based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement.Out of 1494 original articles screened, 35 met all inclusion criteria. These report a prevalence of depression in AECOPD ranging between 9.5% and 85.6%. Some studies report high postadmission mortality rates for depressive AECOPD patients, and higher readmission rates in depressive versus nondepressive AECOPD patients. Importantly, none of the 35 publications included suggestions on the screening and management of depression in AECOPD.Depression and AECOPD frequently co-occur, and this worsens outcomes. Yet we did not find recommendations on management, and few interventional studies. Patients hospitalised with AECOPD should be systematically screened for depression and treatment recommendations should be developed for these patients. Randomised studies on how to screen and treat depression in hospitalised AECOPD are necessary.

Studies on the Interaction of Tumor-Derived HD5 Alpha Defensins with Adenoviruses and Implications for Oncolytic Adenovirus Therapy.

Defensins are small antimicrobial peptides capable of neutralizing human adenovirus (HAdV) in vitro by binding capsid proteins and blocking endosomal escape of virus. In humans, the alpha defensin HD5 is produced by specialized epithelial cells of the gastrointestinal and genito-urinary tracts. Here, we demonstrate, using patient biopsy specimens, that HD5 is also expressed as an active, secreted peptide by epithelial ovarian and lung cancer cells in situ This finding prompted us to study the role of HD5 in infection and spread of replication-competent, oncolytic HAdV type 3 (HAdV3). HAdV3 produces large amounts of penton-dodecahedra (PtDd), virus-like particles, during replication. We have previously shown that PtDd are involved in opening epithelial junctions, thus facilitating lateral spread of de novo-produced virions. Here, we describe a second function of PtDd, namely, the blocking of HD5. A central tool to prove that viral PtDd neutralize HD5 and support spread of progeny virus was an HAdV3 mutant virus in which formation of PtDd was disabled (mut-Ad3GFP, where GFP is green fluorescent protein). We demonstrated that viral spread of mut-Ad3GFP was blocked by synthetic HD5 whereas that of the wild-type (wt) form (wt-Ad3GFP) was only minimally impacted. In human colon cancer Caco-2 cells, induction of cellular HD5 expression by fibroblast growth factor 9 (FGF9) significantly inhibited viral spread and progeny virus production of mut-Ad3GFP but not of wt-Ad3GFP. Finally, the ectopic expression of HD5 in tumor cells diminished the in vivo oncolytic activity of mut-Ad3GFP but not of wt-Ad3GFP. These data suggest a new mechanism of HAdV3 to overcome innate antiviral host responses. Our study has implications for oncolytic adenovirus therapy.IMPORTANCE Previously, it has been reported that human defensin HD5 inactivates specific human adenoviruses by binding to capsid proteins and blocking endosomal escape of virus. The central new findings described in our manuscript are the following: (i) the discovery of a new mechanism used by human adenovirus serotype 3 to overcome innate antiviral host responses that is based on the capacity of HAdV3 to produce subviral penton-dodecahedral particles that act as decoys for HD5, thus preventing the inactivation of virus progeny produced upon replication; (ii) the demonstration that ectopic HD5 expression in cancer cells decreases the oncolytic efficacy of a serotype 5-based adenovirus vector; and (iii) the demonstration that epithelial ovarian and lung cancers express HD5. The study improves our understanding of how adenoviruses establish infection in epithelial tissues and has implications for cancer therapy with oncolytic adenoviruses.