#17: Disease and Immunoprophylaxis Model of Human Nose Organoids to Study SARS-CoV-2 and RSV Infection

Abstract Background There is a significant and unmet need for pre-clinical models to predict responsiveness of immunotherapies to both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) infection. Airway organoid models have been recently developed to study respiratory viruses; however, the current methods rely on invasive or biopsy derived samples to generate lung or airway organoids. Objective To establish human nose organoids (HNOs) as a model to study SARS-CoV-2 and RSV pathogenesis and test therapeutics. Methods We developed a non-invasive method to establish HNOs using stem cells isolated from nasal-wash and mid-turbinate samples. We made air liquid interface (ALI) cultures from undifferentiated 3-dimensional HNOs and differentiated for 21 days to form differentiated nasal epithelium. We inoculated the apical epithelium and assessed SARS-CoV-2 and RSV infection on the apical compartment using real time-polymerase chain reaction, plaque assays and immunofluorescence techniques. We then evaluated the feasibility of HNO-ALI model system to test the efficacy of serum antibodies to prevent SARS-CoV-2 infection and palivizumab monoclonal antibodies to prevent infection using palivizumab sensitive and resistant RSV strains. We introduced the antibodies in the basolateral compartment and monitored its neutralizing capacity on the apical side mimicking the neutralizing effects of antibodies in circulation. Results Our HNO-ALI cultures consist of well-differentiated, pseudostratified, ciliated, and mucosal respiratory epithelial cells and are susceptible to SARS-CoV-2, RSV A and B infection. SARS-CoV-2 and RSV replicates in the apical ciliated cells of the HNO-ALI cultures, peaks at 4 days, and plateaus at 8 days post infection. Infected HNO-ALI recapitulates aspects of SARS-CoV-2 and RSV disease, including viral shedding, asynchronous cilia beating/ciliary damage, and mucus hyper-secretion. Our model effectively showed protection to infection in a concentration dependent manner of the antibodies used. Conclusion We established a non-invasive method to generate HNO-ALI epithelial model as an authentic and an alternative model to 1-D cell culture systems. Our ex-vivo HNO-ALI infection model provides a novel approach for testing therapeutic interventions.

Download Full-text

The human nose organoid respiratory virus model: an ex-vivo human challenge model to study RSV and SARS-CoV-2 pathogenesis and evaluate therapeutics

10.1101/2021.07.28.453844 ◽

2021 ◽

Author(s):

Anubama Rajan ◽

Ashley Morgan Weaver ◽

Gina Marie Alosio ◽

Joseph Jelinski ◽

Hannah L Johnson ◽

...

Keyword(s):

Respiratory Virus ◽

Ex Vivo ◽

Unmet Need ◽

Respiratory Viruses ◽

Invasive Technique ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Rsv Infection ◽

Virus Model ◽

Human Nose

There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.

Download Full-text

A platform utilizing Drosophila ovulation for nonhormonal contraceptive screening

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2026403118 ◽

2021 ◽

Vol 118 (28) ◽

pp. e2026403118

Author(s):

Kewa Jiang ◽

Jiyang Zhang ◽

Yuping Huang ◽

Yingzheng Wang ◽

Shuo Xiao ◽

...

Keyword(s):

Ex Vivo ◽

Unmet Need ◽

Dependent Manner ◽

Female Reproduction ◽

Contraceptive Agents ◽

Follicle Rupture ◽

Approved Drugs ◽

Fda Approved Drugs

A significant unmet need for new contraceptive options for both women and men remains due to side-effect profiles, medical concerns, and the inconvenience of many currently available contraceptive products. Unfortunately, the development of novel nonsteroidal female contraceptive medicine has been stalled in the last couple of decades due to the lack of effective screening platforms. Drosophila utilizes conserved signaling pathways for follicle rupture, a final step in ovulation that is essential for female reproduction. Therefore, we explored the potential to use Drosophila as a model to screen compounds that could inhibit follicle rupture and be nonsteroidal contraceptive candidates. Using our ex vivo follicle rupture assay, we screened 1,172 Food and Drug Administration (FDA)–approved drugs and identified six drugs that could inhibit Drosophila follicle rupture in a dose-dependent manner. In addition, we characterized the molecular actions of these drugs in the inhibition of adrenergic signaling and follicle rupture. Furthermore, we validated that three of the four drugs consistently inhibited mouse follicle rupture in vitro and that two of them did not affect progesterone production. Finally, we showed that chlorpromazine, one of the candidate drugs, can significantly inhibit mouse follicle rupture in vivo. Our work suggests that Drosophila ovulation is a valuable platform for identifying lead compounds for nonsteroidal contraceptive development and highlights the potential of these FDA-approved drugs as novel nonsteroidal contraceptive agents.

Download Full-text

MR Susceptibility Imaging for Detection of Tumor-Associated Macrophages in Glioblastoma

10.21203/rs.3.rs-1017881/v1 ◽

2021 ◽

Author(s):

Amir Nazem ◽

Samantha Guiry ◽

Mehrdad Pourfathi ◽

Jeffrey B. Ware ◽

Hannah Anderson ◽

...

Keyword(s):

Ex Vivo ◽

Imaging Biomarker ◽

Tumor Associated Macrophages ◽

Intracellular Iron ◽

Positive Correlation ◽

Non Invasive ◽

Cell Counts ◽

Susceptibility Maps ◽

Invasive Method ◽

M1 Phenotype

Abstract Purpose Tumor-associated macrophages (TAMs) are a key component of glioblastoma (GBM) microenvironment. Considering the differential role of different TAM phenotypes in iron metabolism with the M1 phenotype storing intracellular iron, and M2 phenotype releasing iron in the tumor microenvironment, we investigated MRI to quantify iron as an imaging biomarker for TAMs in GBM patients. Methods 21 adult patients with GBM underwent a 3D single echo gradient echo MRI sequence and quantitative susceptibility maps were generated. In 3 subjects, ex vivo imaging of surgical specimens was performed on a 9.4 Tesla MRI using 3D multi-echo GRE scans, and R2* (1/T2*) maps were generated. Each specimen was stained with hematoxylin and eosin, as well as CD68, CD86, CD206, and L-Ferritin. Results Significant positive correlation was observed between mean susceptibility for the tumor enhancing zone and the L-ferritin positivity percent (r =0.56, p=0.018) and the combination of tumor’s enhancing zone and necrotic core and the L-Ferritin positivity percent (r=0.72; p=0.001). The mean susceptibility significantly correlated with positivity percent for CD68 (ρ = 0.52, p=0.034) and CD86 (r=0.7 p=0.001), but not for CD206 (ρ = 0.09; p=0.7). There was a positive correlation between mean R2* values and CD68 positive cell counts (r =0.6, p=0.016). Similarly, mean R2* values significantly correlated with CD86 (r=0.54, p=0.03) but not with CD206 (r=0.15, p=0.5). Conclusion MR quantitative susceptibility mapping can quantify the iron content of GBM and provide a non-invasive method for TAM quantification and phenotyping.

Download Full-text

Functional Antibodies Targeting IsaA ofStaphylococcus aureusAugment Host Immune Response and Open New Perspectives for Antibacterial Therapy

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01144-10 ◽

2010 ◽

Vol 55 (1) ◽

pp. 165-173 ◽

Cited By ~ 40

Author(s):

Udo Lorenz ◽

Birgit Lorenz ◽

Tim Schmitter ◽

Karin Streker ◽

Christian Erck ◽

...

Keyword(s):

Therapeutic Efficacy ◽

Ex Vivo ◽

Venous Catheter ◽

Staphylococcal Infection ◽

Adjunctive Treatment ◽

Infection Model ◽

Reactive Oxygen Metabolites ◽

Dependent Manner ◽

Bacterial Killing

ABSTRACTStaphylococcus aureusis the most common cause of nosocomial infections. Multiple antibiotic resistance and severe clinical outcomes provide a strong rationale for development of immunoglobulin-based strategies. Traditionally, novel immunological approaches against bacterial pathogens involve antibodies directed against cell surface-exposed virulence-associated epitopes or toxins. In this study, we generated a monoclonal antibody targeting the housekeeping protein IsaA, a suggested soluble lytic transglycosylase ofS. aureus, and tested its therapeutic efficacy in two experimental mouse infection models. A murine anti-IsaA antibody of the IgG1 subclass (UK-66P) showed the highest binding affinity in Biacore analysis. This antibody recognized allS. aureusstrains tested, including hospital-acquired and community-acquired methicillin-resistantS. aureusstrains. Therapeutic efficacyin vivoin mice was analyzed using a central venous catheter-related infection model and a sepsis survival model. In both models, anti-IsaA IgG1 conferred protection against staphylococcal infection.Ex vivo, UK-66P activates professional phagocytes and induces highly microbicidal reactive oxygen metabolites in a dose-dependent manner, resulting in bacterial killing. The study provides proof of concept that monoclonal IgG1 antibodies with high affinity to the ubiquitously expressed, single-epitope-targeting IsaA are effective in the treatment of staphylococcal infection in different mouse models. Anti-IsaA antibodies might be a useful component in an antibody-based therapeutic for prophylaxis or adjunctive treatment of human cases ofS. aureusinfections.

Download Full-text

Haemoperfused liver as an ex vivo model for organ invasion of Candida albicans

Journal of Medical Microbiology ◽

10.1099/jmm.0.46760-0 ◽

2007 ◽

Vol 56 (2) ◽

pp. 266-270 ◽

Cited By ~ 10

Author(s):

Sascha Thewes ◽

Hilde-Kristin Reed ◽

Christian Grosse-Siestrup ◽

David A. Groneberg ◽

Michael Meissler ◽

...

Keyword(s):

Candida Albicans ◽

Time Course ◽

Ex Vivo ◽

Infection Model ◽

Ex Vivo Model ◽

Human Fungal Pathogen ◽

Non Invasive ◽

Successful Establishment ◽

Set Up

To study invasion of the human fungal pathogen Candida albicans, several infection models have been established. This study describes the successful establishment of an ex vivo haemoperfused liver as a model to study invasion of C. albicans. Perfused organs from pigs could be kept functional for up to 12 h. By comparing a non-invasive and invasive strain of C. albicans and by following a time course of invasion, it was shown that the invasion process in the perfused liver infection model is very similar to the in vivo situation after intraperitoneal infection of mice. The advantage of this set-up compared with other models of invasion is discussed.

Download Full-text

Murine glomerular transcriptome links endothelial cell-specific molecule-1 deficiency with susceptibility to diabetic nephropathy

10.1101/143016 ◽

2017 ◽

Author(s):

Xiaoyi Zheng ◽

Fariborz Soroush ◽

Jin Long ◽

Evan T. Hall ◽

Puneeth K. Adishesha ◽

...

Keyword(s):

Diabetic Nephropathy ◽

Endothelial Cell ◽

Molecular Mechanisms ◽

Disease Susceptibility ◽

Unmet Need ◽

Critical Factor ◽

Dependent Manner ◽

Non Invasive ◽

First Time ◽

Dose Dependent

AbstractDiabetic nephropathy (DN) is the leading cause of kidney disease; however, there are no early biomarkers and no cure. Thus, there is a large unmet need to predict which individuals will develop nephropathy and to understand the molecular mechanisms which govern this susceptibility. We compared the glomerular transcriptome from mice with distinct susceptibilities to DN, and identified differential regulation of genes that modulate inflammation. From these genes, we identified endothelial cell specific molecule-1 (Esm-1), as a glomerular-enriched determinant of resistance to DN. Glomerular Esm-1 mRNA and protein were lower in DN-susceptible, DBA/2, compared to DN-resistant, C57BL/6, mice. We demonstrated higher Esm-1 secretion from primary glomerular cultures of diabetic mice, and high glucose was sufficient to increase Esm-1 mRNA and protein secretion in both strains of mice. However, induction was significantly attenuated in DN-susceptible mice. Urine Esm-1 was also significantly higher only in DN-resistant mice. Moreover, using intravital microscopy and a biomimetic microfluidic assay, we showed that Esm-1 inhibited rolling and transmigration in a dose-dependent manner. For the first time we have uncovered glomerular-derived Esm-1 as a potential non-invasive biomarker of DN. Esm-1 inversely correlates with disease susceptibility and inhibits leukocyte infiltration, a critical factor in protecting the kidney from DN.

Download Full-text

A Phase I Study of a PARP1-targeted Topical Fluorophore for the Detection of Oral Cancer

10.21203/rs.3.rs-252422/v1 ◽

2021 ◽

Author(s):

Paula Demetrio de Souza Franca ◽

Susanne Kossatz ◽

Christian Brand ◽

Daniella Karassawa Zanoni ◽

Sheryl Roberts ◽

...

Keyword(s):

Oral Cancer ◽

Oral Cavity ◽

Phase I ◽

Ex Vivo ◽

Clinical Chemistry ◽

Unmet Need ◽

Fluorescence Signal ◽

Non Invasive ◽

Fluorescence Measurements

Abstract Background. Visual inspection and biopsy is the current standard of care for oral cancer diagnosis, but is subject to misinterpretation and consequently to misdiagnosis. Topically applied PARPi-FL is a molecularly specific, fluorescent contrast-based approach that may fulfil the unmet need for a simple, in vivo, non-invasive, cost-effective, point-of-care method for the early diagnosis of oral cancer. Here, we present results from a phase I safety and feasibility study on fluorescent, topically applied PARPi-FL. Twelve patients with a histologically proven squamous cell carcinoma of the oral cavity (OSCC) gargled a PARPi-FL solution for 60 seconds (15 mL, 100 nM, 250 nM, 500 nM, or 1000 nM), followed by gargling a clearing solution for 60 seconds. Fluorescence measurements of the lesion and surrounding oral mucosa were taken before PARPi-FL application, after PARPi-FL application and after clearing. Blood pressure, oxygen levels, clinical chemistry and CBC were obtained before and after tracer administration. Results. PARPi-FL was well-tolerated by all patients without any safety concerns. When analyzing the fluorescence signal, all malignant lesions showed a significant differential in contrast after administration of PARPi-FL, with the highest increase occurring at the highest dose level (1000 nM), where all patients had a tumor-to-margin fluorescence signal ratio of > 3. A clearing step was essential to increase signal specificity, as it clears unbound PARPi-FL trapped in normal anatomical structures. PARPi-FL tumor cell specificity was confirmed by ex vivo tabletop confocal microscopy. We have demonstrated that the fluorescence signal arose from the nuclei of tumor cells, endorsing our macroscopic findings.Conclusions. A PARPi-FL swish & spit solution is a rapid and non-invasive diagnostic tool that preferentially localizes fluorescent contrast to OSCC. This technique holds promise for the early detection of OSCC based on in vivo optical evaluation and targeted biopsy of suspicious lesions in the oral cavity. Clinicaltrials.gov - NCT03085147, registered on March 21st, 2017.

Download Full-text

Malfunctional Reorganization in the Developing Limbo-Prefrontal System in Animals: Implications for Human Psychoses?

Zeitschrift für Neuropsychologie ◽

10.1024//1016-264x.12.1.8 ◽

2001 ◽

Vol 12 (1) ◽

pp. 8-14

Author(s):

Gertraud Teuchert-Noodt ◽

Ralf R. Dawirs

Keyword(s):

Prefrontal Cortex ◽

Mental Disorders ◽

Dentate Gyrus ◽

Cognitive Functions ◽

Experimental Approach ◽

Regulatory Mechanisms ◽

Hippocampal Dentate Gyrus ◽

Non Invasive ◽

Invasive Method ◽

Activity Dependent

Abstract: Neuroplasticity research in connection with mental disorders has recently bridged the gap between basic neurobiology and applied neuropsychology. A non-invasive method in the gerbil (Meriones unguiculus) - the restricted versus enriched breading and the systemically applied single methamphetamine dose - offers an experimental approach to investigate psychoses. Acts of intervening affirm an activity dependent malfunctional reorganization in the prefrontal cortex and in the hippocampal dentate gyrus and reveal the dopamine position as being critical for the disruption of interactions between the areas concerned. From the extent of plasticity effects the probability and risk of psycho-cognitive development may be derived. Advance may be expected from insights into regulatory mechanisms of neurogenesis in the hippocampal dentate gyrus which is obviously to meet the necessary requirements to promote psycho-cognitive functions/malfunctions via the limbo-prefrontal circuit.

Download Full-text