Extracellular Vesicles from Adipose Tissue-Derived Stromal Cells Stimulate Angiogenesis in a Scaffold-Dependent Fashion.

BACKGROUND: The extracellular vesicles (EVs) secreted by adipose tissue-derived stromal cells (ASC) are microenvironment modulators in tissue regeneration by releasing their molecular cargo, including miRNAs. However, the influence of ASC-derived extracellular vesicles (ASC-EVs) on endothelial cells (ECs) and vascularisation is poorly understood. The present study aimed to determine the pro-angiogenic effects of ASC-EVs and explore their miRNA profile. METHODS: EVs were isolated from normoxic and hypoxic cultured ASC conditioned culture medium. The miRNA expression profile was determined by miRseq, and EV markers were determined by Western blot and immunofluorescence staining. The uptake dynamics of fluorescently labelled EVs were monitored for 24 h. ASC-EVs' pro-angiogenic effect was assessed by sprouting ex vivo rat aorta rings in left ventricular-decellularized extracellular matrix (LV dECM) hydrogel or basement membrane hydrogel (Geltrex®). RESULTS: ASC-EVs augmented vascular network formation by aorta rings. The vascular network topology and stability were influenced in a hydrogel scaffold-dependent fashion. The ASC-EVs were enriched for several miRNA families/clusters, including Let-7 and miR-23/27/24. The miRNA-1290 was the highest enriched non-clustered miRNA, accounting for almost 20% of all reads in hypoxia EVs. CONCLUSION: Our study revealed that ASC-EVs augment in vitro and ex vivo vascularisation, likely due to the enriched pro-angiogenic miRNAs in EVs, particularly miR-1290. Our results show promise for regenerative and revascularisation therapies based on ASC-EV-loaded ECM hydrogels.

Angiogenesis is promoted by hypoxic cervical carcinoma-derived extracellular vesicles depending on the endothelial cell environment.

INTRODUCTION: Cancer needs perfusion for its growth and metastasis. Cancer cell-derived extracellular vesicles (CA-EVs) alter the tumor microenvironment (TME), potentially promoting angiogenesis. We hypothesize that conditions in the tumor, e.g., hypoxia, and in the target cells of the TME, e.g., nutrient deprivation or extracellular matrix, can affect the angiogenic potential of CA-EVs, which would contribute to explaining the regulation of tumor vascularization and its influence on cancer growth and metastasis. METHODS: CA-EVs were isolated and characterized from cervical carcinoma cell lines HeLa and SiHa cultured under normoxia and hypoxia, and their angiogenic potential was evaluated in vitro in three endothelial cells (ECs) lines and aortic rings, cultured in basal (growth factor-reduced) or complete medium. RESULTS: Hypoxia increased EV production 10-100 times and protein content 2-4 times compared to normoxic CA-EVs. HeLa-EVs contained six times more RNA than SiHa-EVs, and this concentration was not affected by hypoxia. Treatment with CA-EVs increased tube formation and sprouting in ECs and aortic rings cultured in basal medium and long-term stabilized the stablished vascular networks formed by ECs cultured in complete medium. CONCLUSION: Hypoxia differentially affects CA-EVs in a cell line-dependent manner. The cellular environment (nutrient availability and extracellular matrix scaffold) influences the effect of CA-EV on the angiogenic potential of ECs.

Endothelial plasticity across PTEN and Hippo pathways: A complex hormetic rheostat modulated by extracellular vesicles.

Vascularization is a multifactorial and spatiotemporally regulated process, essential for cell and tissue survival. Vascular alterations have repercussions on the development and progression of diseases such as cancer, cardiovascular diseases, and diabetes, which are the leading causes of death worldwide. Additionally, vascularization continues to be a challenge for tissue engineering and regenerative medicine. Hence, vascularization is the center of interest for physiology, pathophysiology, and therapeutic processes. Within vascularization, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Hippo signaling have pivotal roles in the development and homeostasis of the vascular system. Their suppression is related to several pathologies, including developmental defects and cancer. Non-coding RNAs (ncRNAs) are among the regulators of PTEN and/or Hippo pathways during development and disease. The purpose of this paper is to review and discuss the mechanisms by which exosome-derived ncRNAs modulate endothelial cell plasticity during physiological and pathological angiogenesis, through the regulation of PTEN and Hippo pathways, aiming to establish new perspectives on cellular communication during tumoral and regenerative vascularization.

Uterine junctional zone and adenomyosis: comparison of MRI, transvaginal ultrasound and histology.

The uterine junctional zone is the subendometrial area in the myometrium that contributes to peristalsis and aids in spermatozoa and blastocyst transport. Alterations in the appearance of the junctional zone on transvaginal sonography (TVS) or magnetic resonance imaging (MRI) are associated with adenomyosis. The lack of standardization of description of its appearance and ill-defined boundaries on both histology and imaging hamper understanding of the junctional zone and limit its role in the diagnosis of adenomyosis. The objectives of this review were to investigate the accordance in definition of the junctional zone across different diagnostic approaches and to examine how imaging findings can be linked to histological findings in the context of diagnosis of adenomyosis. A comprehensive literature review was conducted of articles describing the appearance on imaging and the histological structure of the uterine junctional zone. Our review suggests that the junctional zone is distinguished from the middle and outer myometrium by gradual changes in smooth-muscle cell density, extracellular space, connective tissue, water content and vascular properties. However, while the signal intensity from the junctional zone to the middle myometrium changes abruptly on MRI, the histopathological changes are gradual and its border may be difficult or impossible to distinguish on two-dimensional TVS. Moreover, the thickness of the junctional zone measured on MRI is larger than that measured on TVS. Thus, these two imaging modalities reflect this zone differently. Although a thickened junctional zone is often used to diagnose adenomyosis on MRI, the presence of adenomyosis can be described more accurately as interruptions of the junctional zone by endometrial tissue, which leads to direct signs on imaging such as subendometrial lines and buds on two- and three-dimensional TVS or bright foci on MRI. The histopathological criteria for diagnosis are based on enlargement of the uterus with severe adenomyosis, and might not reflect its early stages. Clinicians should be aware that findings on MRI cannot be extrapolated readily to ultrasound. An understanding of this is necessary when investigating the uterine junctional zone as a functional unit and the association between visualization of direct features of adenomyosis in the junctional zone and clinical symptoms. © 2022 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Consensus on revised definitions of Morphological Uterus Sonographic Assessment (MUSA) features of adenomyosis: results of modified Delphi procedure.

OBJECTIVES: To evaluate whether the Morphological Uterus Sonographic Assessment (MUSA) features of adenomyosis need to be better defined and, if deemed necessary, to reach consensus on the updated definitions. METHODS: A modified Delphi procedure was performed among European gynecologists with expertise in ultrasound diagnosis of adenomyosis. To identify MUSA features that might need revision, 15 two-dimensional (2D) video recordings (four recordings also included three-dimensional (3D) still images) of transvaginal ultrasound (TVS) examinations of the uterus were presented in the first Delphi round (online questionnaire). Experts were asked to confirm or refute the presence of each of the nine MUSA features of adenomyosis (described in the original MUSA consensus statement) in each of the 15 videoclips and to provide comments. In the second Delphi round (online questionnaire), the results of the first round and suggestions for revision of MUSA features were shared with the experts before they were asked to assess a new set of 2D and 3D still images of TVS examinations and to provide feedback on the proposed revisions. A third Delphi round (virtual group meeting) was conducted to discuss and reach final consensus on revised definitions of MUSA features. Consensus was predefined as at least 66.7% agreement between experts. RESULTS: Of 18 invited experts, 16 agreed to participate in the Delphi procedure. Eleven experts completed and four experts partly finished the first round. The experts identified a need for more detailed definitions of some MUSA features. They recommended use of 3D ultrasound to optimize visualization of the junctional zone. Fifteen experts participated in the second round and reached consensus on the presence or absence of ultrasound features of adenomyosis in most of the still images. Consensus was reached for all revised definitions except those for subendometrial lines and buds and interrupted junctional zone. Thirteen experts joined the online meeting, in which they discussed and agreed on final revisions of the MUSA definitions. There was consensus on the need to distinguish between direct features of adenomyosis, i.e. features indicating presence of ectopic endometrial tissue in the myometrium, and indirect features, i.e. features reflecting changes in the myometrium secondary to presence of endometrial tissue in the myometrium. Myometrial cysts, hyperechogenic islands and echogenic subendometrial lines and buds were classified unanimously as direct features of adenomyosis. Globular uterus, asymmetrical myometrial thickening, fan-shaped shadowing, translesional vascularity, irregular junctional zone and interrupted junctional zone were classified as indirect features of adenomyosis. CONCLUSION: Consensus between gynecologists with expertise in ultrasound diagnosis of adenomyosis was achieved regarding revised definitions of the MUSA features of adenomyosis and on the classification of MUSA features as direct or indirect signs of adenomyosis. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

High stretch induces endothelial dysfunction accompanied by oxidative stress and actin remodeling in human saphenous vein endothelial cells.

The rate of the remodeling of the arterialized saphenous vein conduit limits the outcomes of coronary artery bypass graft surgery (CABG), which may be influenced by endothelial dysfunction. We tested the hypothesis that high stretch (HS) induces human saphenous vein endothelial cell (hSVEC) dysfunction and examined candidate underlying mechanisms. Our results showed that in vitro HS reduces NO bioavailability, increases inflammatory adhesion molecule expression (E-selectin and VCAM1) and THP-1 cell adhesion. HS decreases F-actin in hSVECs, but not in human arterial endothelial cells, and is accompanied by G-actin and cofilin's nuclear shuttling and increased reactive oxidative species (ROS). Pre-treatment with the broad-acting antioxidant N-acetylcysteine (NAC) supported this observation and diminished stretch-induced actin remodeling and inflammatory adhesive molecule expression. Altogether, we provide evidence that increased oxidative stress and actin cytoskeleton remodeling play a role in HS-induced saphenous vein endothelial cell dysfunction, which may contribute to predisposing saphenous vein graft to failure.

Human lung extracellular matrix hydrogels resemble the stiffness and viscoelasticity of native lung tissue.

Chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are associated with changes in extracellular matrix (ECM) composition and abundance affecting the mechanical properties of the lung. This study aimed to generate ECM hydrogels from control, severe COPD [Global Initiative for Chronic Obstructive Lung Disease (GOLD) IV], and fibrotic human lung tissue and evaluate whether their stiffness and viscoelastic properties were reflective of native tissue. For hydrogel generation, control, COPD GOLD IV, and fibrotic human lung tissues were decellularized, lyophilized, ground into powder, porcine pepsin solubilized, buffered with PBS, and gelled at 37°C. Rheological properties from tissues and hydrogels were assessed with a low-load compression tester measuring the stiffness and viscoelastic properties in terms of a generalized Maxwell model representing phases of viscoelastic relaxation. The ECM hydrogels had a greater stress relaxation than tissues. ECM hydrogels required three Maxwell elements with slightly faster relaxation times (τ) than that of native tissue, which required four elements. The relative importance (Ri) of the first Maxwell element contributed the most in ECM hydrogels, whereas for tissue the contribution was spread over all four elements. IPF tissue had a longer-lasting fourth element with a higher Ri than the other tissues, and IPF ECM hydrogels did require a fourth Maxwell element, in contrast to all other ECM hydrogels. This study shows that hydrogels composed of native human lung ECM can be generated. Stiffness of ECM hydrogels resembled that of whole tissue, while viscoelasticity differed.

Further insights into the role of tumour characteristics in survival of young women with epithelial ovarian cancer.

OBJECTIVE: Optimizing the counselling of women ≤40years with epithelial ovarian cancer (EOC) by investigating the role of young age and tumour characteristics on overall survival (OS). METHODS: A retrospective population-based study was done using data of EOC patients diagnosed between 1990 and 2014 registered in the Netherlands Cancer Registry. Descriptive statistics were performed to analyse clinical and tumour characteristics. Five- and 10-year OS rates were calculated using Kaplan Meier curves. To determine prognostic factors, univariable and multivariable survival analyses were performed. RESULTS: 1407 women ≤40years and 29,022 women >40years old were included. OS was higher for the younger women compared to older group (5-year survival of 65.6% vs. 32.7%, 10-year survival of 57.5% vs. 22.5%, respectively). The younger women had more often a mucinous (36.4%), well-differentiated (31.8%) tumour in early stage of disease (49.9%). Serous tumours (43.0%), high-grade (36.0%) and stage III (47.1%) were most frequently found in the older women. Histology, grade, stage, incidence year, and age group are independent prognostic factors for survival. OS of the young women for several combinations of tumour characteristics were calculated. CONCLUSIONS: Age is an independent prognostic factor for OS in EOC patients. Counselling on prognosis could be more individualised in young EOC patients using the tumour characteristics histology, stage and grade.

Human resources required for antimicrobial stewardship teams: a Dutch consensus report.

SCOPE: Antimicrobial stewardship teams are responsible for implementing antimicrobial stewardship programmes (ASP). However, in many countries, lack of funding challenges this obligation. A consensus procedure was performed to investigate which structural activities need to be performed by Dutch stewardship teams and how much time (and thus full-time equivalent (FTE) labor) is needed to perform these activities. METHODS: In 2015, an electronic survey, based on a nonsystematic literature search and interviews with seven experienced stewardship teams, was sent to 21 stewardship teams that performed an ASP. This was followed by a semistructured face-to-face consensus meeting. Fourteen stewardship teams completed the survey (18% of Dutch acute-care hospitals), and 13 participated in the consensus meeting. RECOMMENDATIONS: The hours needed each year are dependent on hospital size and number of stewardship objectives monitored. If all activities are performed at a minimal base (one stewardship objective; minimal staffing standard), time investment was estimated to be 1393 to 2680 hours annually in the early phase, corresponding with 0.87 (300 beds) to 1.68 FTE (1200 beds), with a further increase to minimally 1.25 to 3.18 FTE in the following years with three stewardship objectives monitored (optimal staffing standards during the first few years of implementing an ASP). This consensus on required human resources provides a directive for structural financial support of stewardship teams in the Dutch context. Some stewardship activities (and related time investments) might be specific to the Dutch context and hospital setting. To develop standards for other settings, our methodology could be applied.

Perivascular scaffolds loaded with adipose tissue-derived stromal cells attenuate development and progression of abdominal aortic aneurysm in rats.

Abdominal aortic aneurysm (AAA) is the pathological dilation and weakening of the abdominal aorta wall. Inflammation, degradation of the extracellular matrix (ECM) and loss of smooth muscle cells and skewing of their function are pivotal in AAA pathology. We developed a recombinant collagen-based patch (RCP) to provide structural integrity and deliver Adipose tissue-Derived Stromal Cells (ASC) for repair. Patches supported adhesion and function as well as proliferation of ASC. ASC-loaded RCPs or bare patches, applied around the aorta after AAA induction in rats, both maintained structural integrity of the aortic wall at time of explant (2w). However, wall thinning, accompanied by loss of elastin fibers and loss of medial SMC, was only attenuated in ASC-loaded RCP-treated AAA rats. Interestingly, this coincided with migration of ASC into the media and a reduced influx of macrophages. We hypothesize that the medially-migrated ASC dampened or skewed the adverse innate immunity and thus suppressed SMC apoptosis, phenotypic skewing and elastin degradation. We conclude that the periadventitial delivery of ASC with RCP suppresses development and progression of AAA, which is has an expected future clinical benefit in combination with an appropriate early screening program of patients at risk for aneurysms. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2494-2506, 2018.

Opportunistic salpingectomy in women undergoing hysterectomy: Results from the HYSTUB randomised controlled trial.

OBJECTIVE: To evaluate whether opportunistic salpingectomy in premenopausal women undergoing hysterectomy for benign indications is both hormonally and surgically safe, compared with hysterectomy without salpingectomy. STUDY DESIGN: In this multicentre randomised controlled trial, women were randomised to undergo either hysterectomy with opportunistic bilateral salpingectomy (intervention group) or standard hysterectomy with preservation of the Fallopian tubes (control group). MAIN OUTCOME MEASURES: The primary outcome was the difference in serum anti-Müllerian hormone concentration (ΔAMH), measured pre-surgery and 6 months post-surgery. Secondary outcomes were surgical outcomes and duration of hospital stay. The sample size was powered at 50 participants per group (n=100) to compare ΔAMH after hysterectomy with salpingectomy to ΔAMH after standard hysterectomy. RESULTS: Between March 2013 and December 2016, 104 women, aged 30-55 years, were randomly allocated to hysterectomy with opportunistic bilateral salpingectomy (n=52) or standard hysterectomy (n=52). The baseline characteristics did not differ between the two groups. The median ΔAMH was -0.14pmol/L (IQR -1.47-0.95) in the intervention group and 0.00pmol/L (IQR -1.05-0.80) in the control group (p=0.49). The addition of salpingectomy did not impair surgical results and it did not affect duration of hospital stay. CONCLUSION: Addition of opportunistic bilateral salpingectomy during hysterectomy did not result in a larger effect on ovarian reserve when compared with hysterectomy alone, neither did it affect surgical outcomes. Therefore, opportunistic salpingectomy seems to be a safe procedure in premenopausal women undergoing hysterectomy for benign gynaecological conditions.

Very high uptake of risk-reducing salpingo-oophorectomy in BRCA1/2 mutation carriers: A single-center experience.

OBJECTIVE: Risk-reducing salpingo-oophorectomy (RRSO) is the only effective surgical strategy to reduce the increased risk of epithelial ovarian cancer in BRCA1/2 mutation carriers. Given the long-term health consequences of premature surgical menopause, we need insight in uptake and timing of RRSO to guide us in improving healthcare. METHODS: A single-center retrospective cohort study of BRCA1/2 mutation carriers diagnosed and counseled at the multidisciplinary Family Cancer Clinic of the Radboud university medical center in Nijmegen, The Netherlands, between 1999 and 2014. Descriptive statistics were used to analyze uptake and timing of RRSO. RESULTS: Data of 580 BRCA1/2 were analyzed. The uptake of RRSO among mutation carriers who are currently above the upper limit of the recommended age for RRSO, is 98.5% and 97.5% for BRCA1 and BRCA2 mutation carriers, respectively. The vast majority undergoes RRSO ≤40 (BRCA1) or ≤45 (BRCA2) years of age, provided that mutation status is known by that age: 90.8% and 97.3% of BRCA1 and BRCA2 mutation carriers, respectively. CONCLUSIONS: The uptake of RRSO among BRCA1/2 mutation carriers who were counseled at our Family Cancer Clinic is extremely high. High uptake might be largely attributed to the directive and uniform way of counseling by professionals at our Family Cancer Clinic. Given the fact that RRSO is often undergone at premenopausal age in our population, future research should focus on minimizing long-term health consequences of premature surgical menopause either by optimization of hormone replacement therapy or by investigating alternative strategies to RRSO.

Stabilizing effects of excipients on dissociation of intact (146S) foot-and-mouth disease virions into 12S particles during storage as oil-emulsion vaccine.

Most conventional foot-and-mouth disease virus (FMDV) vaccines contain oil-adjuvant. Their potency decreases upon prolonged storage. Intact (146S) FMDV particles can dissociate into 12S degradation products with a concomitant decrease in immunogenicity. We therefore measured virion stability in vaccines using two previously developed ELISAs to separately quantify 12S and 146S particles. Virions completely dissociated into 12S particles within 3 months after oil-emulsification. Dissociation occurred at a much lower rate in a comparable aqueous solution that was not oil-emulsified. Thus, oil-emulsification stimulates virion dissociation, presumably due to the protein denaturing effect of the oil-water interface. In real-time stability studies the stability of oil-adjuvanted virions of four different FMDV strains was significantly increased by addition of sucrose and BSA in a synergistic manner. Contrary to BSA addition, the effect of sucrose addition was concentration dependent. This study illustrates the importance of analysing antigen integrity after oil-emulsification and provides methods for FMDV vaccine stabilization.

Combined implantation of CD34+ and CD14+ cells increases neovascularization through amplified paracrine signalling.

Cell therapy strategies that use adult peripheral blood-derived CD34⁺ progenitor cells are hampered by low cell numbers and the infrequent cellular incorporation into the neovasculature. Hence, the use of CD34⁺ cells to treat ischaemic diseases is under debate. Interaction between CD34⁺ cells and CD14⁺ cells results in superior endothelial differentiation of CD14⁺ cells in vitro, indicating that cell therapy approaches utilizing both CD34⁺ and CD14⁺ cells may be advantageous in therapeutic neovascularization. Here, human CD34⁺ and CD14⁺ cells were isolated from adult peripheral blood and implanted subcutaneously into nude mice, using matrigel as the carrier. Combined implantation of human CD34⁺ and CD14⁺ cells resulted in superior neovascularization, compared to either cell type alone, albeit incorporation of human cells into the murine vasculature was not observed. Human CD34⁺ and CD14⁺ cells produced and secreted a pentad of pro-angiogenic mediators, such as HGF, MCP-1 and IL-8, bFGF and VEGFa in monoculture. The production and secretion of pro-angiogenic mediators by CD14⁺ cells was highly amplified upon incubation with conditioned medium from CD34⁺ cells. In vivo, neovascularization of matrigel implants did not rely on the endothelial differentiation and incorporation of CD34⁺ or CD14⁺ cells, but depended on the paracrine effects of IL-8, MCP-1, HGF, bFGF and VEGFa secreted by implanted cells. Administration of this growth factor/cytokine pentad using matrigel as a carrier results in cell recruitment and microvessel formation equal to progenitor cell-induced neovascularization. These data provide new insights on neovascularization by cell therapy and may contribute to new strategies for the treatment of ischaemic diseases.

Support vector machine classification based on correlation prototypes applied to bone age assessment.

Bone age assessment (BAA) on hand radiographs is a frequent and time consuming task in radiology. We present a method for (semi)automatic BAA which is done in several steps: (i) extract 14 epiphyseal regions from the radiographs, (ii) for each region, retain image features using the IRMA framework, (iii) use these features to build a classifier model (training phase), (iv) evaluate performance on cross validation schemes (testing phase), (v) classify unknown hand images (application phase). In this paper, we combine a support vector machine (SVM) with cross-correlation to a prototype image for each class. These prototypes are obtained choosing one random hand per class. A systematic evaluation is presented comparing nominal- and real-valued SVM with k nearest neighbor (kNN) classification on 1,097 hand radiographs of 30 diagnostic classes (0 19 years). Mean error in age prediction is 1.0 and 0.83 years for 5-NN and SVM, respectively. Accuracy of nominal- and real-valued SVM based on 6 prominent regions (prototypes) is 91.57% and 96.16%, respectively, for accepting about two years age range.

Effect of natural and chimeric haemagglutinin genes on influenza A virus replication in baby hamster kidney cells.

Baby hamster kidney (BHK21) cells are used to produce vaccines against various viral veterinary diseases, including rabies and foot-and-mouth-disease. Although particular influenza virus strains replicate efficiently in BHK21 cells the general use of these cells for influenza vaccine production is prohibited by the poor replication of most strains, including model strain A/PR/8/34 [H1N1] (PR8). We now show that in contrast to PR8, the related strain A/WSN/33 [H1N1] (WSN) replicates efficiently in BHK21 cells. This difference is determined by the haemagglutinin (HA) protein since reciprocal reassortant viruses with swapped HAs behave similarly with respect to growth on BHK21 cells as the parental virus from which their HA gene is derived. The ability or inability of six other influenza virus strains to grow on BHK21 cells appears to be similarly dependent on the nature of the HA gene since reassortant PR8 viruses containing the HA of these strains grow to similar titres as the parental virus from which the HA gene was derived. However, the growth to low titres of a seventh influenza strain was not due to the nature of the HA gene since a reassortant PR8 virus containing this HA grew efficiently on BHK21 cells. Taken together, these results suggest that the HA gene often primarily determines influenza replication efficiency on BHK21 cells but that in some strains other genes are also involved. High virus titres could be obtained with reassortant PR8 strains that contained a chimeric HA consisting of the HA1 domain of PR8 and the HA2 domain of WSN. HA1 contains most antigenic sites and is therefore important for vaccine efficacy. This method of producing the HA1 domain as fusion to a heterologous HA2 domain could possibly also be used for the production of HA1 domains of other viruses to enable the use of BHK21 cells as a generic platform for veterinary influenza vaccine production.

Hyaluronic acid-recombinant gelatin gels as a scaffold for soft tissue regeneration.

An array of different types of hyaluronic acid (HA)- and collagen-based products is available for filling soft-tissue defects. A major drawback of the current soft-tissue fillers is their inability to induce cell infiltration and new tissue formation. Our aim is to develop novel biodegradable injectable gels which induce soft tissue regeneration, initially resulting in integration and finally replacement of the gel with new autologous tissue. Two reference gels of pure HA, monophasic HA-1 and micronised HA-2, were used. Furthermore, both gels were mixed with recombinant gelatin (RG) resulting in HA-1+RG and HA-2+RG. All gels were subcutaneously injected on the back of rats and explanted after 4 weeks. Addition of RG to HA-1 resulted in stroma formation (neovascularisation and ECM deposition) which was restricted to the outer rim of the HA-1+RG gel. In contrast, addition of RG to HA-2 induced stroma formation throughout the gel. The RG component of the gel was degraded by macrophages and giant cells and subsequently replaced by new vascularised tissue. Immunohistochemical staining showed that the extracellular matrix components collagen I and III were deposited throughout the gel. In conclusion, this study shows the proof of principle that addition of RG to HA-2 results in a novel injectable gel capable of inducing soft tissue regeneration. In this gel HA has a scaffold function whereas the RG component induces new tissue formation, resulting in proper vascularisation and integration of the HA-2+RG gel with the autologous tissue.

Mutations in the M-gene segment can substantially increase replication efficiency of NS1 deletion influenza A virus in MDCK cells.

Influenza viruses unable to express NS1 protein (delNS1) replicate poorly and induce large amounts of interferon (IFN). They are therefore considered candidate viruses for live-attenuated influenza vaccines. Their attenuated replication is generally assumed to result from the inability to counter the antiviral host response, as delNS1 viruses replicate efficiently in Vero cells, which lack IFN expression. In this study, delNS1 virus was parallel passaged on IFN-competent MDCK cells, which resulted in two strains that were able to replicate to high virus titers in MDCK cells due to adaptive mutations especially in the M-gene segment but also in the NP and NS gene segments. Most notable were clustered U-to-C mutations in the M segment of both strains and clustered A-to-G mutations in the NS segment of one strain, which presumably resulted from host cell-mediated RNA editing. The M segment mutations in both strains changed the ratio of M1 to M2 expression, probably by affecting splicing efficiency. In one virus, 2 amino acid substitutions in M1 additionally enhanced virus replication, possibly through changes in the M1 distribution between the nucleus and the cytoplasm. Both adapted viruses induced levels of IFN equal to that of the original delNS1 virus. These results show that the increased replication of the adapted viruses is not primarily due to altered IFN induction but rather is related to changes in M1 expression or localization. The mutations identified in this paper may be used to enhance delNS1 virus replication for vaccine production.

MDCK cell line with inducible allele B NS1 expression propagates delNS1 influenza virus to high titres.

Influenza A viruses lacking the gene encoding the non-structural NS1 protein (delNS1) have potential use as live attenuated vaccines. However, due to the lack of NS1, virus replication in cell culture is considerably reduced, prohibiting commercial vaccine production. We therefore established two stable MDCK cell lines that show inducible expression of the allele B NS1 protein. Upon induction, both cell lines expressed NS1 to about 1000-fold lower levels than influenza virus-infected cells. Nevertheless, expression of NS1 increased delNS1 virus titres to levels comparable to those obtained with an isogenic virus strain containing an intact NS1 gene. Recombinant NS1 expression increased the infectious virus titres 244 to 544-fold and inhibited virus induced apoptosis. However, NS1 expression resulted in only slightly, statistically not significant, reduced levels of interferon-ß production. Thus, the low amount of recombinant NS1 is sufficient to restore delNS1 virus replication in MDCK cells, but it remains unclear whether this occurs in an interferon dependent manner. In contrast to previous findings, recombinant NS1 expression did not induce apoptosis, nor did it affect cell growth. These cell lines thus show potential to improve the yield of delNS1 virus for vaccine production.