Female LUTS adaptations to COVID era: Lessons learned from the ICS TURNOVER (Transition of fUnctional uRology to New COVID ERa).

Introduction: The COVID-19 pandemic has dramatically affected the Female and Functional Urology (FFU) practice, leading to massive waiting lists, while patients' quality of life remains severely impaired. The aim of the present study is to develop consensual recommendations to guide clinicians on the management of FFU patients. The present paper focuses on female LUTS. Methods: The authors used the Delphi methodology to develop a robust survey questionnaire, covering the principal topics in FFU, based on literature review and expert opinions. Regarding female LUTS, a 98-question survey was distributed among FFU specialists to obtain optimized recommendations, under the auspicious of the International Continence Society (TURNOVER, ICS project). A quantitative analysis of the data was performed, categorizing the mean value from 0-10. Consensus achievement was defined as attaining ≥ 70% agreement. Results: 98 ICS members completed the F-LUTS survey. Recommendations for the diagnosis and management of female LUTS are summarized. Video-consultation should be used for initial assessment, sending questionnaires and bladder diaries in advance to the patient to be filled out before the consultation. However, face-to-face visits are mandatory if POP or continuous incontinence are suspected, and prior to any surgical procedure, regardless of the health alert. Moreover, prescribing medications such as anticholinergics or ß 3 agonists in a telemedicine setting is not considered a safe practice. Follow-up teleconsultations can be used to assess the efficacy and treatment-related adverse events.Urodynamic testing should be only performed if consequences on F-LUTS treatment are expected. The study should be postponed until the pandemic local behaviour flattens.Invasive procedures should be postponed during a high alert. In case surgery is scheduled, outpatient clinics and local anaesthesia should be prioritized. Every patient should be screened for SARS-CoV-2 infection before invasive tests or procedures, following local authorities' guidance. Conclusions: During a pandemic, telemedicine offers a novel way of communication, maintaining medical care while preventing viral transmission. Non-urgent procedures should be postponed until the pandemic curve flattens. Ambulatory procedures under regional or local anaesthesia should be prioritized, aiming to reduce bed occupancy and risk of transmission.

Development of a high-throughput RT-PCR based viral infectivity assay for monitoring the stability of a replicating recombinant Lymphocytic Choriomeningitis viral vector.

Traditional virus infectivity titration methods for lymphocytic choriomeningitis virus (LCMV) are laborious, time-consuming, and low-throughput (e.g., focus forming unit (FFA) assay). In this report, we developed a high-throughput reverse transcription quantitative PCR (RT-qPCR)-based virus infectivity assay for relative quantitation of a live, recombinant replicating LCMV -based viral vector (TT1). This in vitro infectivity assay demonstrated a 4-log linear range for TT1 titer quantitation. A high positive Pearson correlation coefficient value (≥ 0.80) was obtained between the RT-qPCR vs. the "gold-standard" FFU assay when comparing the stability profiles of stressed TT1 vector samples. In addition to the RT-qPCR infectivity assay, the stability of the TT1 vector upon freeze-thaw stress was investigated further with complementary viral particle characterization techniques (e.g., TEM, NTA, MFI). Correlations between viral infectivity and particle measurements during forced degradation studies were observed to be specific to the TT1 vector and its various formulations and such results facilitated the rank-ordering of formulation conditions. Overall, this infectivity RT-qPCR method showed increased sample throughput and improved assay flexibility compared to traditional viral infectivity assays. These results are discussed in the context of enabling future TT1 vector formulation development work, and potential utilization as an in-process monitoring tool during TT1 vector manufacturing.

In Vitro Antiviral Activity of α-Mangostin against Dengue Virus Serotype-2 (DENV-2).

Dengue virus (DENV), a member of the family Flaviviridae, is a threat for global health as it infects more than 100 million people yearly. Approved antiviral therapies or vaccines for the treatment or prevention of DENV infections are not available. In the present study, natural compounds were screened for their antiviral activity against DENV by in vitro cell line-based assay. α-Mangostin, a xanthanoid, was observed to exert antiviral activity against DENV-2 under pre-, co- and post-treatment testing conditions. The antiviral activity was determined by foci forming unit (FFU) assay, quantitative RT-PCR and cell-based immunofluorescence assay (IFA). A complete inhibition of DENV-2 was observed at 8 µM under the co-treatment condition. The possible inhibitory mechanism of α-Mangostin was also determined by docking studies. The molecular docking experiments indicate that α-Mangostin can interact with multiple DENV protein targets such as the NS5 methyltransferase, NS2B-NS3 protease and the glycoprotein E. The in vitro and in silico findings suggest that α-Mangostin possesses the ability to suppress DENV-2 production at different stages of its replication cycle and might act as a prophylactic/therapeutic agent against DENV-2.

Automation of Infectious Focus Assay for Determination of Filovirus Titers and Direct Comparison to Plaque and TCID50 Assays.

Ongoing efforts to develop effective therapies against filoviruses rely, to different extents, on quantifying the amount of viable virus in samples by plaque, TCID50, and focus assays. Unfortunately, these techniques have inherent variance, and laboratory-specific preferences make direct comparison of data difficult. Additionally, human errors such as operator errors and subjective bias can further compound the differences in outcomes. To overcome these biases, we developed a computer-based automated image-processing method for a focus assay based on the open-source CellProfiler software platform, which enables high-throughput screening of many treatment samples at one time. We compared virus titers calculated using this platform to plaque and TCID50 assays using common stocks of virus for 3 major Filovirus species, Zaire ebolavirus, Sudan ebolavirus, and Marburg marburgvirus with each assay performed by multiple operators on multiple days. We show that plaque assays give comparable findings that differ by less than 3-fold. Focus-forming unit (FFU) and TCID50 assays differ by 10-fold or less from the plaque assays due a higher (FFU) and lower (TCID50) sensitivity. However, reproducibility and accuracy of each assay differs significantly with Neutral Red Agarose Overlay plaque assays and TCID50 with the lowest reproducibility due to subjective analysis and operator error. Both crystal violet methylcellulose overlay plaque assay and focus assays perform best for accuracy and the focus assay performs best for speed and throughput.

Characteristics and viral propagation properties of a new human diploid cell line, Walvax-2, and its suitability as a candidate cell substrate for vaccine production.

Human diploid cell strains (HDCSs), possessing identical chromosome sets known to be free of all known adventitious agents, are of great use in developing human vaccines. However it is extremely difficult to obtain qualified HDCSs that can satisfy the requirements for the mass production of vaccines. We have developed a new HDCS, Walvax-2, which we derived from the lung tissue of a 3-month-old fetus. We established primary, master and working cell banks successfully from reconstituted frozen cells. Observations during the concurrent propagation of Walvax-2 and MRC-5 cells revealed differences in terms of growth rate, cell viability and viral sensitivities. Specifically, Walvax-2 cells replicated more rapidly than MRC-5 cells, with Walvax-2 cells attaining the same degree of confluence in 48 hours as was reached by MRC-5 cells in 72 hours. Moreover, Walvax-2 cells attained 58 passages of cell doublings whereas MRC-5 reached 48 passages during this period. We also assessed the susceptibility of these cells to rabies, hepatitis A, and Varicella viruses. Analysis of virus titers showed the Walvax-2 cells to be equal or superior to MRC-5 cells for cultivating these viruses. Furthermore, in order to characterize the Walvax-2 cell banks, a series of tests including cell identification, chromosomal characterization, tumorigenicity, as well as tests for the presence of microbial agents, exogenous viruses, and retroviruses, were conducted according to standard international protocols. In conclusion, results from this study show that Walvax-2 cell banks are a promising cell substrate and could potentially be used for the manufacturing of HDCVs.

Lactobacilli and Bifidobacteria enhance mucosal B cell responses and differentially modulate systemic antibody responses to an oral human rotavirus vaccine in a neonatal gnotobiotic pig disease model.

B cells play a key role in generation of protective immunity against rotavirus infection, a major cause of gastroenteritis in children. Current RV vaccines are less effective in developing countries compared to developed countries. Commensals/probiotics influence mucosal immunity, but the role of early gut colonizing bacteria in modulating intestinal B cell responses to RV vaccines is largely unknown. We co-colonized neonatal gnotobiotic pigs, the only animal model susceptible to HRV diarrhea, with 2 dominant bacterial species present in the gut of breastfed infants, Lactobacillus rhamnosus strain GG and Bifidobacterium animalis lactis Bb12 to evaluate their impact on B cell responses to an attenuated (Att) human rotavirus (HRV) Wa strain vaccine. Following HRV challenge, probiotic-colonized, AttHRV vaccinated piglets had significantly lower fecal scores and reduced HRV shedding titers compared to uncolonized, AttHRV vaccinated pigs. The reduction in HRV diarrhea was significantly correlated with higher intestinal IgA HRV antibody titers and intestinal HRV-specific IgA antibody secreting cell (ASC) numbers in probiotic-colonized, AttHRV vaccinated pigs compared to uncolonized, vaccinated pigs. The significantly higher small intestinal HRV IgA antibody responses coincided with higher IL-6, IL-10 and APRIL responses of ileal mononuclear cells (MNCs) and the immunomodulatory effects of probiotics genomic DNA on TGF-ß and IL-10 responses. However, serum RV IgG antibody titers and total IgG titers were significantly lower in probiotic-colonized, AttHRV vaccinated pigs compared to uncolonized, vaccinated pigs, both pre- and post-challenge. In summary, LGG and Bb12 beneficially modulated intestinal B cell responses to HRV vaccine.

Perforin and granzymes work in synergy to mediate cholangiocyte injury in experimental biliary atresia.

BACKGROUND & AIMS: Biliary atresia represents obstructive cholangiopathy in infants progressing rapidly to cirrhosis and end-stage liver disease. Activated NK cells expressing Nkg2d have been linked to bile duct injury and obstruction by establishing contact with cholangiocytes. To define the mechanisms used by cytotoxic cells, we investigated the role of perforin and granzymes in a neonatal mouse model of rotavirus (RRV)-induced biliary atresia. METHODS: We used complementary cell lysis assays, flow cytometric analyses, quantitative PCRs and in vivo systems to determine the mechanisms of bile duct epithelial injury and the control of the tissue phenotype in experimental biliary atresia. RESULTS: RRV-infected hepatic NK and CD8 T cells increased the expression of perforin and injured cholangiocytes in short-term culture in a perforin-dependent fashion. However, the loss of perforin in vivo delayed but did not prevent the obstruction of bile ducts. Based on the increased expression of granzymes by perforin-deficient cytotoxic cells in long-term cytolytic assays, we found that the inhibition of granzymes by nafamostat mesilate (FUT-175) blocked cholangiocyte lysis. Administration of FUT-175 to perforin-deficient mice after RRV infection decreased the development of jaundice, minimized epithelial injury, and improved long-term survival. However, the inhibition of granzymes alone in wild-type mice was not sufficient to prevent the atresia phenotype in newborn mice. In infants with biliary atresia, hepatic Granzymes A and B mRNA, but not Perforin, increased at the time of portoenterostomy. CONCLUSIONS: Perforin and granzymes have complementary roles mediating epithelial injury by NK and CD8 T cells. The prevention of experimental biliary atresia can only be achieved by inhibiting both granules.

Improved dual promotor-driven reverse genetics system for influenza viruses.

Reverse genetic systems for influenza A virus (IAV) allow the generation of genetically manipulated infectious virus from a set of transfected plasmid DNAs encoding the eight genomic viral RNA segments (vRNA). For this purpose, cDNAs representing these eight vRNA segments are cloned into specific plasmid vectors that allow the generation of vRNA-like transcripts using polymerase I (Pol I). In addition, these plasmids support the transcription of viral mRNA by polymerase II (Pol II), leading to the expression of viral protein(s) encoded by the respective transcripts. In an effort to develop this system further, we constructed the bi-directional vector pMPccdB. It is based on pHW2000 (Hoffmann et al., 2000b) but contains additionally (i) the ccdB gene whose expression is lethal for most Escherichia coli strains and therefore used as a negative selection marker and (ii) more efficient AarI cloning sites that flank the ccdB gene on either side. Furthermore, we used a modified one-step restriction/ligation protocol to insert the desired cDNA into the respective pMPccdB vector DNA. Both the use of a negative selection marker and an improved cloning protocol were shown to facilitate the generation of genetically engineered IAV as illustrated in this study by the cloning and rescue of the 2009 pandemic isolate A/Giessen/6/2009 (Gi-H1N1).

The Mouse as Model System to Study Host-Pathogen Interactions in Influenza A Infections.

The mouse is one of the most important mammalian model systems for studying host-pathogen-interactions during influenza A virus infections and for assessing the virulence of newly emerging influenza viruses. Here, we provide the basic protocols for infecting mice with influenza virus and studying the main pathological changes associated with disease. Critical parameters, e.g., virus variants and subtypes or mouse strains, are discussed. Curr. Protoc. Mouse Biol. 2:177-205 © 2012 by John Wiley & Sons, Inc.