Advances of Recombinant Adenoviral Vectors in Preclinical and Clinical Applications.

Adenoviruses (Ad) have the potential to induce severe infections in vulnerable patient groups. Therefore, understanding Ad biology and antiviral processes is important to comprehend the signaling cascades during an infection and to initiate appropriate diagnostic and therapeutic interventions. In addition, Ad vector-based vaccines have revealed significant potential in generating robust immune protection and recombinant Ad vectors facilitate efficient gene transfer to treat genetic diseases and are used as oncolytic viruses to treat cancer. Continuous improvements in gene delivery capacity, coupled with advancements in production methods, have enabled widespread application in cancer therapy, vaccine development, and gene therapy on a large scale. This review provides a comprehensive overview of the virus biology, and several aspects of recombinant Ad vectors, as well as the development of Ad vector, are discussed. Moreover, we focus on those Ads that were used in preclinical and clinical applications including regenerative medicine, vaccine development, genome engineering, treatment of genetic diseases, and virotherapy in tumor treatment.

Transforming Growth Factor ß1 and Gap Junction Protein Alpha 4 Gene Heterogeneity in Relation to the Severity of Clinical Disease in Cystic Fibrosis.

BACKGROUND: High TGFß1-producing variants cause severe clinical disease in F508del homozygous patients. Lately, we showed that a single nucleotide polymorphism (SNP), rs41266431, in the GJA4 gene modifies the disease severity of cystic fibrosis (CF). Our aim was to investigate whether the clinical phenotype associated with GJA4 variants was independent of TGFß1 variants. METHODS: Homozygous F508del patients (n = 115, mean age 27.2 years, m/f (65/50)) were included in this study. A deep sequence analysis was performed for GJA4 and TGBß1, and disease severity was assessed over 3 years using lung function tests (LFTs), body mass index, diabetes mellitus, colonization with Pseudomonas aeruginosa, survival to end-stage lung disease (ESLD), as well as distinct inflammatory biomarkers. RESULTS: The analyses revealed that one SNP (rs41266431) in GJA4 may be clinically relevant. Carriers homozygous for the G variant (n = 84; 73%) presented with worse LFTs (forced vital capacity (FVC) % predicted: mean 80/86.6, p < 0.035) and a lower survival to ESLD (p < 0.029). For the TGBß1 variant: 509 carriers of the C variant (CT + CC genotype, n = 105, 91.3%) had better LFTs (Forced expiratory flow at 75% of the FVC (FEF75% predicted: median 40/29.5, p < 0.015), although a similar outcome to ESLD. A gene-gene interaction was not observed between TGBß1 and GJA4 variants for any clinical measure. CONCLUSIONS: GJA4 variants are independent of TGBß1 variants. Both variants had an impact on the LFTs, although only GJA4 variants were associated with an improved outcome for ESLD. CLINICAL TRIAL REGISTRATION: The study was registered with ClinicalTrials.gov, number NCT04242420, retrospectively on January 24th, 2020.

Uncovering the gastrointestinal passage, intestinal epithelial cellular uptake, and AGO2 loading of milk miRNAs in neonates using xenomiRs as tracers.

BACKGROUND: Human breast milk has a high microRNA (miRNA) content. It remains unknown whether and how milk miRNAs might affect intestinal gene regulation and homeostasis of the developing microbiome after initiating enteral nutrition. However, this requires that relevant milk miRNA amounts survive the gastrointestinal (GI) passage, are taken up by cells, and become available to the RNA interference machinery. It seems important to dissect the fate of these miRNAs after oral ingestion and GI passage. OBJECTIVES: Our goal was to analyze the potential transmissibility of milk miRNAs via the gastrointestinal system in neonate humans and a porcine model in vivo to contribute to the discussion of whether milk miRNAs could influence gene regulation in neonates and thus might vertically transmit developmental relevant signals. METHODS: We performed cross-species profiling of miRNAs via deep sequencing and utilized dietary xenobiotic taxon-specific milk miRNA (xenomiRs) as tracers in human and porcine neonates, followed by functional studies in primary human fetal intestinal epithelial cells using adenovirus-type 5-mediated miRNA gene transfer. RESULTS: Mammals share many milk miRNAs yet exhibit taxon-specific miRNA fingerprints. We traced bovine-specific miRNAs from formula nutrition in human preterm stool and 9 d after the onset of enteral feeding in intestinal cells (ICs) of preterm piglets. Thereafter, several xenomiRs accumulated in the ICs. Moreover, a few hours after introducing enteral feeding in preterm piglets with supplemented reporter miRNAs (cel-miR-39-5p/-3p), we observed their enrichment in blood serum and in argonaute RISC catalytic component 2 (AGO2)-immunocomplexes from intestinal biopsies. CONCLUSIONS: Milk-derived miRNAs survived GI passage in human and porcine neonates. Bovine-specific miRNAs accumulated in ICs of preterm piglets after enteral feeding with bovine colostrum/formula. In piglets, colostrum supplementation with cel-miR-39-5p/-3p resulted in increased blood concentrations of cel-miR-39-3p and argonaute RISC catalytic component 2 (AGO2) loading in ICs. This suggests the possibility of vertical transmission of miRNA signaling from milk through the neonatal digestive tract.

The Adenovirus Vector Platform: Novel Insights into Rational Vector Design and Lessons Learned from the COVID-19 Vaccine.

The adenovirus vector platform remains one of the most efficient toolboxes for generation of transfer vehicles used in gene therapy and virotherapy to treat tumors, as well as vaccines to protect from infectious diseases. The adenovirus genome and capsids can be modified using highly efficient techniques, and vectors can be produced at high titers, which facilitates their rapid adaptation to current needs and disease applications. Over recent years, the adenovirus vector platform has been in the center of attention for vaccine development against the ongoing coronavirus SARS-CoV-2/COVID-19 pandemic. The worldwide deployment of these vaccines has greatly deepened the knowledge on virus-host interactions and highlighted the need to further improve the effectiveness and safety not only of adenovirus-based vaccines but also of gene therapy and oncolytic virotherapy vectors. Based on the current evidence, we discuss here how adenoviral vectors can be further improved by intelligent molecular design. This review covers the full spectrum of state-of-the-art strategies to avoid vector-induced side effects ranging from the vectorization of non-canonical adenovirus types to novel genome engineering techniques.

The Role of Insulin Receptor Substrate Proteins in Bronchopulmonary Dysplasia and Asthma: New Potential Perspectives.

Insulin receptor substrates (IRSs) are proteins that are involved in signaling through the insulin receptor (IR) and insulin-like growth factor (IGFR). They can also interact with other receptors including growth factor receptors. Thus, they represent a critical node for the transduction and regulation of multiple signaling pathways in response to extracellular stimuli. In addition, IRSs play a central role in processes such as inflammation, growth, metabolism, and proliferation. Previous studies have highlighted the role of IRS proteins in lung diseases, in particular asthma. Further, the members of the IRS family are the common proteins of the insulin growth factor signaling cascade involved in lung development and disrupted in bronchopulmonary dysplasia (BPD). However, there is no study focusing on the relationship between IRS proteins and BPD yet. Unfortunately, there is still a significant gap in knowledge in this field. Thus, in this review, we aimed to summarize the current knowledge with the major goal of exploring the possible roles of IRS in BPD and asthma to foster new perspectives for further investigations.

An Adenoviral Vector as a Versatile Tool for Delivery and Expression of miRNAs.

Only two decades after discovering miRNAs, our understanding of the functional effects of deregulated miRNAs in the development of diseases, particularly cancer, has been rapidly evolving. These observations and functional studies provide the basis for developing miRNA-based diagnostic markers or new therapeutic strategies. Adenoviral (Ad) vectors belong to the most frequently used vector types in gene therapy and are suitable for strong short-term transgene expression in a variety of cells. Here, we report the set-up and functionality of an Ad-based miRNA vector platform that can be employed to deliver and express a high level of miRNAs efficiently. This vector platform allows fast and efficient vector production to high titers and the expression of pri-miRNA precursors under the control of a polymerase II promoter. In contrast to non-viral miRNA delivery systems, this Ad-based miRNA vector platform allows accurate dosing of the delivered miRNAs. Using a two-vector model, we showed that Ad-driven miRNA expression was sufficient in down-regulating the expression of an overexpressed and highly stable protein. Additional data corroborated the downregulation of multiple endogenous target RNAs using the system presented here. Additionally, we report some unanticipated synergistic effects on the transduction efficiencies in vitro when cells were consecutively transduced with two different Ad-vectors. This effect might be taken into consideration for protocols using two or more different Ad vectors simultaneously.

Properties of Adenovirus Vectors with Increased Affinity to DSG2 and the Potential Benefits of Oncolytic Approaches and Gene Therapy.

Carcinomas are characterized by a widespread upregulation of intercellular junctions that create a barrier to immune response and drug therapy. Desmoglein 2 (DSG2) represents such a junction protein and serves as one adenovirus receptor. Importantly, the interaction between human adenovirus type 3 (Ad3) and DSG2 leads to the shedding of the binding domain followed by a decrease in the junction protein expression and transient tight junction opening. Junction opener 4 (JO-4), a small recombinant protein derived from the Ad3 fiber knob, was previously developed with a higher affinity to DSG2. JO-4 protein has been proven to enhance the effects of antibody therapy and chemotherapy and is now considered for clinical trials. However, the effect of the JO4 mutation in the context of a virus remains insufficiently studied. Therefore, we introduced the JO4 mutation to various adenoviral vectors to explore their infection properties. In the current experimental settings and investigated cell lines, the JO4-containing vectors showed no enhanced transduction compared with their parental vectors in DSG2-high cell lines. Moreover, in DSG2-low cell lines, the JO4 vectors presented a rather weakened effect. Interestingly, DSG2-negative cell line MIA PaCa-2 even showed resistance to JO4 vector infection, possibly due to the negative effect of JO4 mutation on the usage of another Ad3 receptor: CD46. Together, our observations suggest that the JO4 vectors may have an advantage to prevent CD46-mediated sequestration, thereby achieving DSG2-specific transduction.

Mannose-Binding Lectin (MBL) and Gap Junction Protein Alpha 4 (GJA4) Gene Heterogeneity in Relation to Severity of Clinical Disease in Cystic Fibrosis.

BACKGROUND: Recently, we provided evidence that a single nucleotide polymorphism (SNP), rs41266431, on the gap junction protein alpha 4 (GJA4) gene, acts as a modifier for clinical disease severity in patients with cystic fibrosis (CF). These features are very similar to those of variants of the mannose-binding lectin (MBL). This study aimed to clarify whether the clinical disease phenotype associated with GJA4 variants is independent of MBL variants. METHODS: One hundred and twelve patients with homozygous F508del (mean age, 27.6 years; m/f, 61/51) were recruited from the CF centers of Bonn, Frankfurt, and Amsterdam. A sequence analysis was performed for GJA4 and MBL. The clinical phenotype was assessed over three years using pulmonary function tests, body mass index, Pseudomonas aeruginosa colonization, diabetes mellitus, survival to end-stage lung disease, and inflammatory markers. RESULTS: A clinically relevant SNP of GJA4 was identified by sequence analysis. Pulmonary function (FVC% pred, mean 78/85; p < 0.055) and survival to end-stage lung disease were lower (p < 0.043) for this variant (rs41266431) in carriers homozygous for the G variant (n = 82/112; 73%) than in other carriers. Serum MBL (820/372 ng/mL, p < 0.001) was significantly higher in "MBL-sufficient" genotypes (n = 79/112; 71%) than in "MBL-insufficient" genotypes, and a trend for a significant difference in BMI percentiles (35.2/23.8; p < 0.059) was observed. For the MBL-sufficient genotype (median age at death, 38/26 years), there was a trend for better survival (p < 0.076). There was no augmentation by gene-gene interaction between MBL and GJA4 variants for any outcome parameter. CONCLUSIONS: The clinical disease phenotype associated with GJA4 variants is independent of MBL variants. MBL-sufficient variants were associated with superior BMI and a trend for better survival than MBL insufficient variants.

The Communication between Ocular Surface and Nasal Epithelia in 3D Cell Culture Technology for Translational Research: A Narrative Review.

There is a lack of knowledge regarding the connection between the ocular and nasal epithelia. This narrative review focuses on conjunctival, corneal, ultrastructural corneal stroma, and nasal epithelia as well as an introduction into their interconnections. We describe in detail the morphology and physiology of the ocular surface, the nasolacrimal ducts, and the nasal cavity. This knowledge provides a basis for functional studies and the development of relevant cell culture models that can be used to investigate the pathogenesis of diseases related to these complex structures. Moreover, we also provide a state-of-the-art overview regarding the development of 3D culture models, which allow for addressing research questions in models resembling the in vivo situation. In particular, we give an overview of the current developments of corneal 3D and organoid models, as well as 3D cell culture models of epithelia with goblet cells (conjunctiva and nasal cavity). The benefits and shortcomings of these cell culture models are discussed. As examples for pathogens related to ocular and nasal epithelia, we discuss infections caused by adenovirus and measles virus. In addition to pathogens, also external triggers such as allergens can cause rhinoconjunctivitis. These diseases exemplify the interconnections between the ocular surface and nasal epithelia in a molecular and clinical context. With a final translational section on optical coherence tomography (OCT), we provide an overview about the applicability of this technique in basic research and clinical ophthalmology. The techniques presented herein will be instrumental in further elucidating the functional interrelations and crosstalk between ocular and nasal epithelia.

Enhanced Antiviral Function of Magnesium Chloride-Modified Heparin on a Broad Spectrum of Viruses.

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

Human Species D Adenoviruses Isolated from Diarrheal Feces Show Low Infection Rates in Primary Nasal Epithelial Cells.

The importance of adenovirus (Ad) research is significantly increasing with respect to virotherapy for vaccine development, tumor, and gene therapy. Due to the different species and subtypes of this virus, the characterization of the biological significance of especially rare Ad is necessary. Previously, rare Ad types 70, 73, and 74 were originally isolated from fecal samples of immunocompromised patients and they represent recombinants of other Ad types. Here we investigated transduction experiments of these reporter gene tagged Ad types in primary cells exemplified by subject-derived primary nasal epithelial cells (NAEPCs). To analyze the transduction rates, we performed flow cytometry, quantitative polymerase chain reaction (PCR), and cytokine analyses 25 h post-infection. We found that, in contrast to Ad type 5 (as a positive control), the transduction rates of NAEPCs with Ad types 70, 73, and 74 were interestingly low. The major Ad receptor (coxsackievirus-adenovirus receptor and CD46) expression levels showed no significant change after infection with Ad types 70, 73 and 74. Moreover, Interleukin 6 (IL-6) was not released after in vitro Ad transduction. Due to the high risk of developing life-threatening complications in immunocompromised patients by these human species D Ads, even more attention needs to be investigated into the development of diagnostic and therapeutic concepts to prevent and treat those opportunistic infections in susceptible patients.

The mystery behind the nostrils - technical clues for successful nasal epithelial cell cultivation.

OBJECTIVES: Research involving the nose reveals important information regarding the morphology and physiology of the epithelium and its molecular response to agents. The role of nasal epithelial cells and other cell subsets within the nasal epithelium play an interesting translational split between experimental and clinical research studying respiratory disorders or pathogen reactions. With an additional technical manuscript including a detailed description of important technical aspects, tips, tricks, and nuances for a successful culturing of primary, human nasal epithelial cells (NAEPCs), we here aim to improve the process of communication between experimentalists and physicians, supporting the purpose of a fruitful work for future translational projects. METHODS: Based on previous work on various complex culture models of subject-derived NAEPCs, this additional manuscript harmonizes previously published facts combined with own experiences for a trouble-free implementation in laboratories. RESULTS: A well-designed experimental question is essential prior to the establishment of different NAEPCs culture models. The correct method of cell extraction from the nasal cavity is essential and represent an important basis for successful culture work. Prior enzymatic processing of biopsy specimens, cell culture materials, collagenization procedure, culture conditions, and choice of culture medium are some important practical notes that increase the quality of the culture. Moreover, protocols on imaging techniques including histologic and electron microscopy must be adapted for NAEPC culture. Adapted flow cytometric protocols and transepithelial electrical resistance measurements can add valuable information. OUTLOOK: A successful culturing of NAEPCs can provide an important basis for genetic studies and the implementation of omics-science, which is increasingly receiving broad attention in the scientific community. The common aim of in vitro 'mini-noses' will be a breakthrough in laboratories aiming to perform research under in vivo conditions. Here, organoid models are interesting models presenting a basis for translational studies.

From Submerged Cultures to 3D Cell Culture Models: Evolution of Nasal Epithelial Cells in Asthma Research and Virus Infection.

Understanding the response to viral infection in the context of respiratory diseases is of significant importance. Recently, there has been more focus on the role of the nasal epithelium in disease modeling. Here, we provide an overview of different submerged, organotypic 3D and spheroid cell culture models of nasal epithelial cells, which were used to study asthma and allergy with a special focus on virus infection. In detail, this review summarizes the importance, benefits, and disadvantages of patient-derived cell culture models of nasal- and bronchial epithelial cells, including a comparison of these cell culture models and a discussion on why investigators should consider using nasal epithelial cells in their research. Exposure experiments, simple virus transduction analyses as well as genetic studies can be performed in these models, which may provide first insights into the complexity of molecular signatures and may open new doors for drug discovery and biomarker research.

House Dust Mite Exposure Causes Increased Susceptibility of Nasal Epithelial Cells to Adenovirus Infection.

Adenovirus (AdV) infections in the respiratory tract may cause asthma exacerbation and allergic predisposition, and the house dust mite (HDM) may aggravate virus-induced asthma exacerbations. However, the underlying mechanisms of whether and how AdV affects asthmatic patients remains unclear. To address this question, we investigated nasal epithelial cells (NAEPCs) derived from a pediatric exacerbation study cohort for experimental analyses. We analyzed twenty-one different green-fluorescent protein- and luciferase-tagged AdV types in submerged 2D and organotypic 3D cell culture models. Transduction experiments revealed robust transduction of AdV type 5 (AdV5) in NAEPCs, which was associated with an increased uptake of AdV5 in the presence of HDM. In healthy and asthmatic NAEPCs exposed to HDM before infection, we observed a time- and dose-dependent increase of AdV5 uptake associated with upregulation of entry receptors for AdV5. Furthermore, electron microscopic and histologic analyses of 3D cell cultures revealed an impairment of the respiratory cilia after HDM exposition. This ex vivo pilot study shows the impact of AdV infection and HDM exposition in a primary cell culture model for asthma.

Multicenter Analysis of Acquired Undescended Testis and Its Impact on the Timing of Orchidopexy.

OBJECTIVE: To assess whether late orchidopexy for undescended testis represents delayed treatment of primary undescended testis or later-occurring acquired undescended testis. STUDY DESIGN: We examined boys undergoing orchidopexy for cryptorchidism regarding age at surgery and entity of undescended testis. We characterized differences between primary undescended testis and acquired undescended testis and evaluated the knowledge regarding the diagnosis and management of acquired undescended testis among practicing physicians. We conducted an observational study using a mixed-method multicenter cross-sectional design. A total of 310 consecutive boys undergoing orchidopexy for undescended testis at 6 pediatric medical centers in Germany between April 2016 and June 2018 were investigated regarding testicular position at birth and age at surgery. In addition, a survey on acquired undescended testis management was carried out in 1017 multidisciplinary physicians and final-year medical students. RESULTS: Only 13% of all patients were operated on in their first year of life. Among patients with known previous testicular position (67%), primary undescended testis (n = 103) and acquired undescended testis (n = 104) were equally frequent. More than one-half (56%) of orchidopexies performed after the first year of life were due to acquired undescended testis. Remarkably, only 15% of physicians considered acquired undescended testis as an indication for late surgery. CONCLUSIONS: Acquired undescended testis is more common than previously perceived and accounts for a significant proportion of "late" orchidopexies in patients with undescended testis. Acquired undescended testis needs to be better recognized in clinical practice and screening should continue in older children with previously descended testes. TRIAL REGISTRATION: German Clinical Trials Registry: DRKS00015903.

Common Indications and The Diagnostic Yield of Esophagogastroduodenoscopy in Children with Gastrointestinal Distress.

BACKGROUND: The number of inconspicuous results of esophagogastroduodenoscopies (EGDs) in childhood appears to be disturbingly high. The aim of this study was to analyze the diagnostic yield of EGD and to determine its relevance of specific clinical indications. METHODS: We performed a retrospective analysis of 380 consecutive pediatric patients who underwent diagnostic EGD in two German level I pediatric gastroenterology departments in 2015 and 2016. RESULTS: 44% of the 380 patients were male and 17% were younger than 5 years old. 55% of all EGDs (n=210) did not yield a pathological result. 27% (n=104) of all EGDs were performed due to nonspecific symptoms (epigastralgia, nausea). Strikingly, in this group, 88% (n=91) showed normal results and in only 12% a diagnosis was made: reflux esophagitis (n=5), Helicobacter pylori (HP) gastritis (n=6) or hemorrhagic gastritis (n=1). Fewer inconspicuous EGDs were performed in patients with dysphagia (68%) or heartburn and reflux (61%). 59 patients were examined due to serologically elevated celiac antibodies. Here, the diagnosis could be confirmed histopathologically in 78% (n=46). Of the 37 patients with abdominal pain and a previously positive non-invasive HP test, EGD served to establish the diagnosis of HP gastritis in 84%. CONCLUSIONS: The diagnostic yield for EGDs is increased in patients with more specific symptoms (i. e. dysphagia, heartburn, HP, celiac disease). Consequently, as an invasive procedure, EGD warrants a strict indication.

Neonatal exposure to hyperoxia leads to persistent disturbances in pulmonary histone signatures associated with NOS3 and STAT3 in a mouse model.

Background: Early pulmonary oxygen exposure is one of the most important factors implicated in the development of bronchopulmonary dysplasia (BPD). Methods: Here, we analyzed short- and long-term effects of neonatal hyperoxia on NOS3 and STAT3 expression and corresponding epigenetic signatures using a hyperoxia-based mouse model of BPD. Results: Early hyperoxia exposure led to a significant increase in NOS3 (median fold change × 2.37, IQR 1.54-3.68) and STAT3 (median fold change × 2.83, IQR 2.21-3.88) mRNA levels in pulmonary endothelial cells with corresponding changes in histone modification patterns such as H2aZac and H3K9ac hyperacetylation at the respective gene loci. No complete restoration in histone signatures at these loci was observed, and responsivity to later hyperoxia was altered in mouse lungs. In vitro, histone signatures in human aortic endothelial cells (HAEC) remained altered for several weeks after an initial long-term exposure to trichostatin A. This was associated with a substantial increase in baseline eNOS (median 27.2, IQR 22.3-35.6) and STAT3α (median 5.8, IQR 4.8-7.3) mRNA levels with a subsequent significant reduction in eNOS expression upon exposure to hypoxia. Conclusions: Early hyperoxia induced permanent changes in histones signatures at the NOS3 and STAT3 gene locus might partly explain the altered vascular response patterns in children with BPD.

Kocuria kristinae-caused sepsis in an infant with congenital tufting enteropathy.

Aydin M, Ganschow R, Jankofsky M. Kocuria kristinae-caused sepsis in an infant with congenital tufting enteropathy. Turk J Pediatr 2017; 59: 93-96. Congenital tufting enteropathy (CTE) is characterized by the early-onset of chronic diarrhea and the inability to develop. It is a rare congenital disease with a low prevalence of 1:50,000 - 100,000 live births p.a. The histopathology is characterized by villous atrophy and the characteristic epithelial tufts. Recent identification of causative mutations in EpCAM has enhanced our understanding of this disease. Due to its severe clinical course, patients are dependent on parenteral nutrition to thrive successfully. Catheter-associated blood stream infections have become the primary problem for pediatric patients. Infections with Kocuria kristinae are rare. This report is about a 3-month-old girl with CTE suffering from a central venous catheter related mono-sepsis by K. kristinae. A sepsis therapy with meropenem and vancomycin improved her general state rapidly. Only few cases in the literature with CTE and K. kristinae are described. To the best of our knowledge, this is the first report presenting two coincidences in one case.