Biology-inspired microphysiological systems to advance patient benefit and animal welfare in drug development

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.Toxicolog

Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection

Dampened antiviral immunity to intravaginal exposure to RNA viral pathogens allows enhanced viral replication.

Understanding the host immune response to vaginal exposure to RNA viruses is required to combat sexual transmission of this class of pathogens. In this study, using lymphocytic choriomeningitis virus (LCMV) and Zika virus (ZIKV) in wild-type mice, we show that these viruses replicate in the vaginal mucosa with minimal induction of antiviral interferon and inflammatory response, causing dampened innate-mediated control of viral replication and a failure to mature local antigen-presenting cells (APCs). Enhancement of innate-mediated inflammation in the vaginal mucosa rescues this phenotype and completely inhibits ZIKV replication. To gain a better understanding of how this dampened innate immune activation in the lower female reproductive tract may also affect adaptive immunity, we modeled CD8 T cell responses using vaginal LCMV infection. We show that the lack of APC maturation in the vaginal mucosa leads to a delay in CD8 T cell activation in the draining lymph node and hinders the timely appearance of effector CD8 T cells in vaginal mucosa, thus further delaying viral control in this tissue. Our study demonstrates that vaginal tissue is exceptionally vulnerable to infection by RNA viruses and provides a conceptual framework for the male to female sexual transmission observed during ZIKV infection

Development of latent fingerprints on non-porous surfaces recovered from fresh and sea water

Abstract Background Criminal offenders have a fundamental goal not to leave any traces at the crime scene. Some may suppose that items recovered underwater will have no forensic value, therefore, they try to destroy the traces by throwing items in water. These traces are subjected to the destructive environmental effects. This can represent a challenge for forensic experts investigating fingerprints. Methods The present study was conducted to determine the optimal method for latent fingerprints development on dry non-porous surfaces submerged in aquatic environments at different time interval. The quality of the developed fingerprints depending on the used method was assessed. In addition, two factors were analyzed in this study; the effects of the nature of aquatic environment and the length of submerged time. Therefore, latent fingerprints were deposited on metallic, plastic and glass objects and submerged in fresh and sea water for 1, 2, and 10 days. After recovery, the items were processed by black powder, small particle reagent and cyanoacrylate fuming and the prints were examined. Each print was evaluated according to fingerprint quality assessment scale. Results Cyanoacrylate developed latent prints found to have the highest mean visibility score after submersion in fresh and sea water for 1, 2 and 10 days. Mean visibility score of prints developed showed significant decline after 10 days of submersion. Prints submerged in fresh water showed significantly higher mean visibility score than those submerged in sea water using various methods of development and in all time intervals. Conclusion The study demonstrated that it is possible to recover latent prints submerged in water on different studied dry non porous surfaces with the best visualization method using cyanoacrylate either in fresh or sea water. The duration of submersion affects the quality of fingerprints developed; the longer the duration, the worse the quality is. In addition, this study has revealed that the exposure to high salinity i.e. sea water has more damaging influence on the quality of detected fingerprints. It is concluded that any piece of evidence recovered from underwater should be tested for prints, no matter the amount of time spent beneath the surface

Low expression of RNA sensors impacts Zika virus infection in the lower female reproductive tract.

Innate immune responses to Zika virus (ZIKV) are dampened in the lower female reproductive tract (LFRT) compared to other tissues, but the mechanism that underlies this vulnerability is poorly understood. Using tissues from uninfected and vaginally ZIKV-infected macaques and mice, we show that low basal expression of RNA-sensing pattern recognition receptors (PRRs), or their co-receptors, in the LFRT contributes to high viral replication in this tissue. In the LFRT, ZIKV sensing provides limited protection against viral replication, and the sensors are also minimally induced after vaginal infection. While IFNα/β receptor signaling offers minimal protection in the LFRT, it is required to prevent dissemination of ZIKV to other tissues, including the upper FRT. Our findings support a role for RNA-sensing PRRs in the dampened innate immunity against ZIKV in the LFRT compared to other tissues and underlie potential implications for systemic dissemination upon heterosexual transmission of ZIKV in women

Shared Mechanisms Govern HIV Transcriptional Suppression in Circulating CD103+ and Gut CD4+ T Cells.

Latent HIV infection is the main barrier to cure, and most HIV-infected cells reside in the gut, where distinct but unknown mechanisms may promote viral latency. Transforming growth factor β (TGF-β), which induces the expression of CD103 on tissue-resident memory T cells, has been implicated in HIV latency. Using CD103 as a surrogate marker to identify cells that have undergone TGF-β signaling, we compared the HIV RNA/DNA contents and cellular transcriptomes of CD103+ and CD103- CD4 T cells from the blood and rectum of HIV-negative (HIV-) and antiretroviral therapy (ART)-suppressed HIV-positive (HIV+) individuals. Like gut CD4+ T cells, circulating CD103+ cells harbored more HIV DNA than did CD103- cells but transcribed less HIV RNA per provirus. Circulating CD103+ cells also shared a gene expression profile that is closer to that of gut CD4 T cells than to that of circulating CD103- cells, with significantly lower expression levels of ribosomal proteins and transcriptional and translational pathways associated with HIV expression but higher expression levels of a subset of genes implicated in suppressing HIV transcription. These findings suggest that blood CD103+ CD4 T cells can serve as a model to study the molecular mechanisms of HIV latency in the gut and reveal new cellular factors that may contribute to HIV latency.IMPORTANCE The ability of HIV to establish a reversibly silent, "latent" infection is widely regarded as the main barrier to curing HIV. Most HIV-infected cells reside in tissues such as the gut, but it is unclear what mechanisms maintain HIV latency in the blood or gut. We found that circulating CD103+ CD4+ T cells are enriched for HIV-infected cells in a latent-like state. Using RNA sequencing (RNA-seq), we found that CD103+ T cells share a cellular transcriptome that more closely resembles that of CD4+ T cells from the gut, suggesting that they are homing to or from the gut. We also identified the cellular genes whose expression distinguishes gut CD4+ or circulating CD103+ T cells from circulating CD103- T cells, including some genes that have been implicated in HIV expression. These genes may contribute to latent HIV infection in the gut and may serve as new targets for therapies aimed at curing HIV