A vector-encoded bispecific killer engager to harness virus-activated NK cells as anti-tumor effectors.

Treatment with oncolytic measles vaccines (MV) elicits activation of immune cells, including natural killer (NK) cells. However, we found that MV-activated NK cells show only modest direct cytotoxic activity against tumor cells. To specifically direct NK cells towards tumor cells, we developed oncolytic measles vaccines encoding bispecific killer engagers (MV-BiKE) targeting CD16A on NK cells and carcinoembryonic antigen (CEA) as a model tumor antigen. MV-BiKE are only slightly attenuated compared to parental MV and mediate secretion of functional BiKE from infected tumor cells. We tested MV-BiKE activity in cocultures of colorectal or pancreatic cancer cells with primary human NK cells. MV-BiKE mediate expression of effector cytokines, degranulation and specific anti-tumor cytotoxicity by NK cells. Experiments with patient-derived pancreatic cancer cultures indicate that efficacy of MV-BiKE may vary between individual tumors with differential virus permissiveness. Remarkably, we confirmed MV-BiKE activity in primaryhuman colorectal carcinoma specimens with autochthonous tumor and NK cells.This study provides proof-of-concept for MV-BiKE as a novel immunovirotherapy to harness virus-activated NK cells as anti-tumor effectors.

Healthcare Providers' Knowledge of Value-Based Care in Germany: An Adapted, Mixed-Methods Approach.

BACKGROUND: Value-Based Care (VBC) is being discussed to provide better outcomes to patients, with an aim to reimburse healthcare providers (HCPs) based on the quality of care they deliver. Little is known about German HCPs' knowledge of VBC. This study aims to investigate the knowledge of HCPs of VBC and to identify potential needs for further education toward implementation of VBC in Germany. METHODS: For evidence generation, we performed a literature search and conducted an online survey among HCPs at 89 hospitals across Germany. The questionnaire was based on published evidence and co-developed with an expert panel using a mixed methods approach. RESULTS: We found HCPs to believe that VBC is more applicable in surgery than internal medicine and that well-defined cycles of care are essential for its application. HCPs believe that VBC can reduce health care costs significantly. However, they also assume that implementing VBC will be challenging. CONCLUSIONS: The concept in general is well perceived, however, HCPs do not want to participate in any financial risk sharing. Installing an authority/independent agency that measures achieved value, digital transformation, and that improves the transition between the inpatient and the outpatient sectors are top interests of HCPs.

Novel Group C Oncolytic Adenoviruses Carrying a miRNA Inhibitor Demonstrate Enhanced Oncolytic Activity In Vitro and In Vivo.

Oncolytic adenoviruses (OAd) represent an attractive treatment option for cancer. Clinical efficacy of commonly utilized human adenovirus type 5 (Ad5)-based oncolytic viruses is limited by variable expression levels of the coxsackie- and adenovirus receptor (CAR) in tumor cells and high prevalence of neutralizing antibodies against human Ad5. However, previous studies have highlighted alternative human Ad types as promising candidates for oncolytic therapy. In this study, we generated novel OAds based on Ad1, -2, -5, and -6 derived from species C Ads. These OAds contain a 24-bp deletion in the early gene E1A for tumor selective replication and express the RNAi inhibitor P19. We examined these OAds for in vitro anticancer activity on various cancer cell lines derived from lung, colon, gynecologic, bone, and pancreatic carcinoma. In most surveyed cell lines, OAds based on Ad1, -2, and -6 demonstrated higher cell lysis capability compared with Ad5, suggesting enhanced oncolytic potential. Moreover, enhanced oncolytic activity was associated with P19 expression in a cell type-dependent manner. We further explored a A549 tumor xenograft mouse model to compare the novel OAds directly with Ad5 and H101, an oncolytic adenovirus used in clinical trials. These P19-containing OAds based on Ad1, -2, and -6 showed significantly decelerated tumor progression compared with H101, indicating better antitumor potency in vivo. Our studies provide a novel path for OAd development based on alternative Ad types with improved effectiveness by RNA interference suppression.

Risk factors for therapy failure after surgery for perianal abscess in children.

Purpose: The role of surgery in managing perianal abscesses in the pediatric population is debatable, and data on recurrence risk is rare. This study aimed to evaluate the efficiency of surgery for a perianal abscess in children and identify parameters that predict recurrence. Methods: We performed a retrospective review of all children younger than age 14 requiring surgery for a perianal abscess from 2000 to 2018. Results: Out of 103 enrolled patients, 27 (26%) had recurrent perianal disease. Recurrences appeared after a median of 5 months (range: 1-18 months), in 12 cases as perianal abscess and 15 cases as fistula in ano. Anal fistula probing was performed in 33% of all patients, of which 16 (15%) underwent fistulotomy. In univariate analysis, older age (p = 0.034), fistula probing (p = 0.006) and fistulotomy (p = 0.009) was associated with treatment success. History of perianal abscess, multilocal occurrence, and the presence of enteric flora in wound swabs was associated with treatment failure (p = 0.002, OR = 0.032). In multivariate analysis, anal fistula probing was independently associated with treatment success (p = 0.019, OR = 22.08), while the history of perianal abscess was associated with treatment failure (p = 0.002, OR = 0.032). Conclusion: Our study identified probing for fistula as a predictor of therapy success, while the history of perianal abscess was identified as a predictor of treatment failure. Therefore, in all children with perianal abscess, fistula probing and if present, fistulotomy should be performed.

Efficacy of conservative treatment of perianal abscesses in children and predictors for therapeutic failure.

BACKGROUND: The optimal management of perianal abscess in children is controversial. PURPOSE: To evaluate the efficiency of conservative treatment of perianal abscess in children and identify parameters that predict therapy failure. METHODS: All cases of children younger than 14 years of age with perianal abscesses between 2001-2016 were evaluated. RESULTS: Of the 113 enrolled patients, 64 underwent subsequent surgery for advanced disease (primary surgery group). Conservative treatment was initiated in 49 patients (primary conservative group) but was stopped because of inefficiency in 25 patients, who were referred for surgery after a median 7.03 days (range, 2 to 16 days). The other 24 patients (48%) initially achieved complete remission after conservative treatment, but 10 were readmitted after a median 34 months (range, 3 to 145 months) with recurrent disease. There were no significant differences in permanent success after conservative treatment between infants (10 of 29, 34%) and older children (4 of 20 [20%], P=0.122). Overall, conservative treatment alone was effective in only 14 of 113 patients. Recurrence after surgery occurred in 16 patients (25%) in the primary surgery group and 11 patients (22%) in the primary conservative group (P=0.75). Univariate analysis of predictors for conservative treatment failure revealed inflammatory values (C-reactive protein and white blood count, P=0.017) and abscess size (P=0.001) as significant parameters, whereas multivariate analysis demonstrated that only abscess size (odds ratio, 3.37; P=0.023) was significant. CONCLUSION: Conservative treatment of perianal abscess is permanently efficient in only a minority of children but is not associated with a higher recurrence rate after subsequent surgery. Abscess size is a predictor for therapy failure.

The role of saline irrigation prior to wound closure in the reduction of surgical site infection: protocol for a systematic review and meta-analysis.

BACKGROUND: Surgical site infection describes an infectious complication of surgical wounds. This single complication is thought to occur in close to 20% of surgical cases. This complication has been described in all kinds of surgical procedure including minimally invasive procedures. Wound irrigation is frequently used as a means of reducing surgical site infection. However, there is lack of solid evidence to support routine wound irrigation. The aim of this review is to provide evidence for the efficacy of routine wound irrigation with normal saline in preventing surgical site infection. The rate of surgical site infection in cases with and without wound irrigation will be analyzed. METHODS/DESIGN: Systematic literature searches will be conducted to identify all published and unpublished studies. The following databases will be searched for citations from inception to present: MEDLINE (via PubMed), Embase (via Embase), and CENTRAL (via the Cochrane library). The search strategy will be developed by the research team in collaboration with an experienced librarian and checked by a referee according to the Peer Review of Electronic Search Strategies (PRESS) guideline. A draft of the PubMed search strategy could be (irrigation[tiab] OR "Therapeutic Irrigation"[mesh] OR lavage[tiab]) AND (saline[tiab] OR "Sodium Chloride"[mesh] OR sodium chloride[tiab]) NOT ("Comment" [Publication Type] OR "Letter" [Publication Type] OR "Editorial" [Publication Type]). No time limits will be set. The reference lists of eligible articles will be hand searched. Relevant data will be extracted from eligible studies using a previously designed data extraction sheet. Relative risks will be calculated for binary outcomes and mean differences or standardized mean differences, if necessary, for continuous outcomes. For all measures, 95% confidence levels will be calculated. Both arms would be compared with regard to the rate of surgical site infection within 30 days following surgery. We will report the review using the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement. DISCUSSION: This review aims at investigating the value of routine wound irrigation using normal saline in preventing surgical site infection. SYSTEMATIC REVIEW REGISTRATION: PROSPERO: CRD42018082287.

Shortcomings in the management of undescended testis: guideline intention vs reality and the underlying causes - insights from the biggest German cohort.

OBJECTIVES: To assess the implementation of the current guideline and identify potential underlying causes for late surgery in children with undescended testis (UDT) in Germany. UDT is the most common surgical issue in paediatric urology and to avoid malignant degeneration and subfertility current guidelines recommend orchidopexy during the first year of life; however, this seems not to be implemented in practice. PATIENTS AND METHODS: In all, 5 547 patients with cryptorchidism at 16 hospitals nationwide were studied regarding age at orchidopexy between 2003 and 2016. Multivariate analysis was performed to identify factors influencing timing of surgery. Additionally, a survey on knowledge of UDT management was conducted amongst physicians treating boys and final-year medical students. RESULTS: Between 2003 and 2008 only 4% of boys with UDT underwent surgery before the age of 1 year. After the guideline update from 2009, this figure was 5% from 2010 to 2012, and 8% from 2013 to 2016. The presence of a specialised department for paediatric surgery, as well as a high UDT case-to-year ratio positively influenced the timing of orchidopexy. The survey revealed discipline-specific differences in the levels of knowledge about UDT management. One-third of respondents did not know the guideline recommendations and 61% felt insufficiently informed. International comparisons revealed significant differences in the age at surgery of boys with UDT, with Germany and Great Britain ranging in the middle of the field. CONCLUSIONS: Currently, only a small proportion of boys with UDT are operated upon during their first year of life. The level of knowledge in attending physicians remains in need of improvement. This should be actively addressed, i.e. by campaigns and educational programmes. Further studies are needed to investigate the underlying causes of late orchidopexy in UDT.

Outcome of acute perforated cholecystitis: a register study of over 5000 cases from a quality control database in Germany.

BACKGROUND: Acute perforated cholecystitis (APC) is probably the most severe complication of acute cholecystitis. However, data on the outcome of cholecystectomy for APC are limited to small series. This study investigated the outcomes of cholecystectomy for APC. METHODS: Data from a prospectively maintained quality control database in Germany were analyzed. Cases with APC were compared to cases without gallbladder perforation with regard to demographic characteristics, clinical findings and surgical outcomes. RESULTS: A total of 5704 patients with APC were compared to 39,661 patients without perforation. Risk factors for APC included: the male gender, advanced age (>65 years), ASA score >2, elevated white blood count (WBC), positive findings on abdominal ultrasound sonography and fever. The APC group differed significantly from the control group with regard to fever (29.8 vs. 12.2 %), elevated WBC (83.8 vs. 65.4 %) and positive findings from ultrasound sonography (84.9 vs. 78.9 %), p < 0001. Preoperative computed tomography (CT) was ordered significantly more often in the APC group compared to the control group (2.3 vs. 1.0 %, p = 0.001). Surgery lasted significantly longer in the APC group (92.3 ± 40.8 vs. 73.7 ± 34.1, p < 0.001). The rates of conversion (18.9 vs. 6.8 %), bile duct injury (1.4 vs. 0.5 %), re-intervention (6.9 vs. 2.9 %) and mortality (4.3 vs. 1.3 %) were significantly higher in the APC group (p < 0.001). Similarly, the length of stay (13.4 ± 11.4 vs. 9.0 ± 8.3, p < 0.001) was significantly longer in the APC group. CONCLUSION: Acute perforated cholecystitis is a severe complication of acute cholecystitis. Surgical dissection could be challenging with high risks of bile duct injury and conversion. The rates of morbidity and mortality are higher compared to those of patients without perforation.

Quantification of designer nuclease induced mutation rates: a direct comparison of different methods.

Designer nucleases are broadly applied to induce site-specific DNA double-strand breaks (DSB) in genomic DNA. These are repaired by nonhomologous end joining leading to insertions or deletions (in/dels) at the respective DNA-locus. To detect in/del mutations, the heteroduplex based T7-endonuclease I -assay is widely used. However, it only provides semi-quantitative evidence regarding the number of mutated alleles. Here we compared T7-endonuclease I- and heteroduplex mobility assays, with a quantitative polymerase chain reaction mutation detection method. A zinc finger nuclease pair specific for the human adeno-associated virus integration site 1 (AAVS1), a transcription activator-like effector nuclease pair specific for the human DMD gene, and a zinc finger nuclease- and a transcription activator-like effector nuclease pair specific for the human CCR5 gene were explored. We found that the heteroduplex mobility assays and T7-endonuclease I - assays detected mutations but the relative number of mutated cells/alleles can only be estimated. In contrast, the quantitative polymerase chain reaction based method provided quantitative results which allow calculating mutation and homologous recombination rates in different eukaryotic cell types including human peripheral blood mononuclear cells. In conclusion, our quantitative polymerase chain reaction based mutation detection method expands the array of methods for in/del mutation detection and facilitates quantification of introduced in/del mutations for a genomic locus containing a mixture of mutated and unmutated DNA.

Standard free droplet digital polymerase chain reaction as a new tool for the quality control of high-capacity adenoviral vectors in small-scale preparations.

High-capacity adenoviral vectors (HCAdVs) are promising tools for gene therapy as well as for genetic engineering. However, one limitation of the HCAdV vector system is the complex, time-consuming, and labor-intensive production process and the following quality control procedure. Since HCAdVs are deleted for all viral coding sequences, a helper virus (HV) is needed in the production process to provide the sequences for all viral proteins in trans. For the purification procedure of HCAdV, cesium chloride density gradient centrifugation is usually performed followed by buffer exchange using dialysis or comparable methods. However, performing these steps is technically difficult, potentially error-prone, and not scalable. Here, we establish a new protocol for small-scale production of HCAdV based on commercially available adenovirus purification systems and a standard method for the quality control of final HCAdV preparations. For titration of final vector preparations, we established a droplet digital polymerase chain reaction (ddPCR) that uses a standard free-end-point PCR in small droplets of defined volume. By using different probes, this method is capable of detecting and quantifying HCAdV and HV in one reaction independent of reference material, rendering this method attractive for accurately comparing viral titers between different laboratories. In summary, we demonstrate that it is possible to produce HCAdV in a small scale of sufficient quality and quantity to perform experiments in cell culture, and we established a reliable protocol for vector titration based on ddPCR. Our method significantly reduces time and required equipment to perform HCAdV production. In the future the ddPCR technology could be advantageous for titration of other viral vectors commonly used in gene therapy.

The sleeping beauty transposon vector system for treatment of rare genetic diseases: an unrealized hope?

Rare genetic diseases account for a considerable amount of fatalities and even their 'mild' or 'non-lethal' forms can produce drastic and undesirable discomfort to affected individuals. Various gene therapeutic approaches were tested for developing novel therapeutic concepts to treat these genetic diseases. Sleeping Beauty (SB) transposase represents one of these gene therapeutic systems which can be utilized for stable phenotypic correction. It is a transposable element which was resurrected and optimized for transposing genetic elements resulting in somatic integration of the transgene. Because of its versatile activity in many different organs, SB transposase has been explored for ex-vivo gene delivery and in vivo gene delivery including recently launched clinical trials based on engineered T-cells for tumor therapy and approaches to treat retinal degenerations. Here we will provide a state-of-the-art overview of preclinical studies for treatment of rare genetic diseases based on the SB transposase system for stable correction of the genetic defect. In this review, diseases affecting the blood system, the connective tissue, the immune system, the metabolism, and the nervous system and their treatment utilizing the SB transposase system will be discussed. Moreover, advantages and disadvantages of SB transposase-based gene therapeutic approaches will be mentioned. Although improvements of the SB transposase systems regarding genotoxicity and efficient delivery especially for applications in large mammals are desirable, the SB transposase system remains to hold great promise for curing rare genetic disease.