Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter

Capillary endothelial cells of the human blood–brain barrier (BBB) express high levels of P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2). However, little information is available regarding ATP-binding cassette transporters expressed at the zebrafish BBB, which has emerged as a potential model system. We report the characterization and tissue localization of two genes that are similar to ABCB1, zebrafish abcb4 and abcb5. When stably expressed in HEK293 cells, both Abcb4 and Abcb5 conferred resistance to P-gp substrates; however, Abcb5 poorly transported doxorubicin and mitoxantrone compared to zebrafish Abcb4. Additionally, Abcb5 did not transport the fluorescent P-gp probes BODIPY-ethylenediamine or LDS 751, while they were transported by Abcb4. High-throughput screening of 90 human P-gp substrates confirmed that Abcb4 has an overlapping substrate specificity profile with P-gp. In the brain vasculature, RNAscope probes for abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. The abcb4 probe also colocalized with claudin-5 in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, potentially indicating different functions. The data suggest that zebrafish Abcb4 functionally phenocopies P-gp and that the zebrafish may serve as a model to study the role of P-gp at the BBB.

An extrahepatic hydatid cyst with the fat-fluid level

A hydatic cyst is a zoonosis caused by the larva of a tapeworm Echinococcus granulosus. The liver is the most commonly affected organ. Soft tissue localization has been reported in 2.3% of cases. Herein, we present a patient with a fat-containing hydatid cyst located in the left thigh. There are only a few reports in the literature on the presence of the fat-fluid level within a hydatid cyst. Previous studies have suggested that fat-containing hydatid cysts occur due to their cysto-biliary communication in the liver. In our case, we describe a fat-containing hydatid cyst in the extrahepatic location and discuss the pathophysiologic mechanism of fat inside it.

Cytokine storm and histopathological findings in 60 cases of COVID-19-related death: from viral load research to immunohistochemical quantification of major players IL-1β, IL-6, IL-15 and TNF-α

This study involves the histological analysis of samples taken during autopsies in cases of COVID-19 related death to evaluate the inflammatory cytokine response and the tissue localization of the virus in various organs. In all the selected cases, SARS-CoV-2 RT-PCR on swabs collected from the upper (nasopharynx and oropharynx) and/or the lower respiratory (trachea and primary bronchi) tracts were positive. Tissue localization of SARS-CoV-2 was detected using antibodies against the nucleoprotein and the spike protein. Overall, we tested the hypothesis that the overexpression of proinflammatory cytokines plays an important role in the development of COVID-19-associated pneumonia by estimating the expression of multiple cytokines (IL-1β, IL-6, IL-10, IL-15, TNF-α, and MCP-1), inflammatory cells (CD4, CD8, CD20, and CD45), and fibrinogen. Immunohistochemical staining showed that endothelial cells expressed IL-1β in lung samples obtained from the COVID-19 group (p < 0.001). Similarly, alveolar capillary endothelial cells showed strong and diffuse immunoreactivity for IL-6 and IL-15 in the COVID-19 group (p < 0.001). TNF-α showed a higher immunoreactivity in the COVID-19 group than in the control group (p < 0.001). CD8 + T cells where more numerous in the lung samples obtained from the COVID-19 group (p < 0.001). Current evidence suggests that a cytokine storm is the major cause of acute respiratory distress syndrome (ARDS) and multiple organ failure and is consistently linked with fatal outcomes.

In situ imaging reveals disparity between prostaglandin localization and abundance of prostaglandin synthases

Prostaglandins are important lipids involved in mediating many physiological processes, such as allergic responses, inflammation, and pregnancy. However, technical limitations of in-situ prostaglandin detection in tissue have led researchers to infer prostaglandin tissue distributions from localization of regulatory synthases, such as COX1 and COX2. Herein, we apply a novel mass spectrometry imaging method for direct in situ tissue localization of prostaglandins, and combine it with techniques for protein expression and RNA localization. We report that prostaglandin D2, its precursors, and downstream synthases co-localize with the highest expression of COX1, and not COX2. Further, we study tissue with a conditional deletion of transformation-related protein 53 where pregnancy success is low and confirm that PG levels are altered, although localization is conserved. Our studies reveal that the abundance of COX and prostaglandin D2 synthases in cellular regions does not mirror the regional abundance of prostaglandins. Thus, we deduce that prostaglandins tissue localization and abundance may not be inferred by COX or prostaglandin synthases in uterine tissue, and must be resolved by an in situ prostaglandin imaging.

Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter

ABSTRACTGiven its similarities with mammalian systems, the zebrafish has emerged as a potential model to study the blood-brain barrier (BBB). Capillary endothelial cells at the human BBB express high levels of P-glycoprotein (P-gp, encoded by the ABCB1 gene) and ABCG2 (encoded by the ABCG2 gene). However, little information has been available about ATP-binding cassette transporters expressed at the zebrafish BBB. In this study, we focus on the characterization and tissue localization of two genes that are similar to human ABCB1, zebrafish abcb4 and abcb5. Cytotoxicity assays with stably-transfected cell lines revealed that zebrafish Abcb5 cannot efficiently transport the substrates doxorubicin and mitoxantrone compared to human P-gp and zebrafish Abcb4. Additionally, zebrafish Abcb5 did not transport the fluorescent probes BODIPY-ethylenediamine or LDS 751, while they were readily transported by Abcb4 and P-gp. A high-throughput screen conducted with 90 human P-gp substrates confirmed that zebrafish Abcb4 has overlapping substrate specificity with P-gp. Basal ATPase activity of zebrafish Abcb4 and Abcb5 was comparable to that of human P-gp. In the brain vasculature, RNAscope probes to detect abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. Zebrafish abcb4 also colocalized with claudin-5 expression in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, consistent with different functions. The data suggest that zebrafish Abcb4 most closely phenocopies P-gp and that the zebrafish may be a viable model to study the role of the multidrug transporter P-gp at the BBB.