Human Palatine Tonsils Are Linked to Alzheimer's Disease through Function of Reservoir of Amyloid Beta Protein Associated with Bacterial Infection.

Amyloid-ß (Aß)-peptide production or deposition in the neuropathology of Alzheimer's disease (AD) was shown to be caused by chronic inflammation that may be induced by infection, but the role of pathogenic-bacteria-related AD-associated Aß is not yet clearly understood. In this study, we validated the hypothesis that there is a correlation between the Aß-protein load and bacterial infection and that there are effects of bacteria, Staphylococcus aureus (S. aureus), on the Aß load in the inflammatory environment of human tonsils. Here, we detected Aß-peptide deposits in human tonsil tissue as well as tissue similar to tonsilloliths found in the olfactory cleft. Interestingly, we demonstrated for the first time the presence of Staphylococcus aureus (S. aureus) clustered around or embedded in the Aß deposits. Notably, we showed that treatment with S. aureus upregulated the Aß-protein load in cultures of human tonsil organoids and brain organoids, showing the new role of S. aureus in Aß-protein aggregation. These findings suggest that a reservoir of Aß and pathogenic bacteria may be a possible therapeutic target in human tonsils, supporting the treatment of antibiotics to prevent the deposition of Aß peptides via the removal of pathogens in the intervention of AD pathogenesis.

Generation of human tonsil epithelial organoids as an ex vivo model for SARS-CoV-2 infection.

The palatine tonsils (hereinafter referred to as "tonsils") serve as a reservoir for viral infections and play roles in the immune system's first line of defense. The aims of this study were to establish tonsil epithelial cell-derived organoids and examine their feasibility as an ex vivo model for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The tonsil organoids successfully recapitulated the key characteristics of the tonsil epithelium, including cellular composition, histologic properties, and biomarker distribution. Notably, the basal layer cells of the organoids express molecules essential for SARS-CoV-2 entry, such as angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and furin, being susceptible to the viral infection. Changes in the gene expression profile in tonsil organoids revealed that 395 genes associated with oncostatin M signaling and lipid metabolism were highly upregulated within 72 h after SARS-CoV-2 infection. Notably, remdesivir suppressed the viral RNA copy number in organoid culture supernatants and intracellular viral protein levels in a dose-dependent manner. Here, we suggest that tonsil epithelial organoids could provide a preclinical and translational research platform for investigating SARS-CoV-2 infectivity and transmissibility or for evaluating antiviral candidates.

Development of organoid-based drug metabolism model.

Cytochrome P450 (CYP) gene superfamily catalyzes oxidative metabolism of a wide variety of drugs, carcinogens, and endogenous biomolecules in the liver and intestinal organs. In vitro assay platforms such as primary hepatocyte and immortalized liver-derived cell lines have been developed to evaluate drug effects. However, several limitations have been suggested regarding discrepancies between in vitro and in vivo assays. In this study, we aimed to investigate drug metabolism and toxicity based on mouse small intestinal and liver organoids derived from resident stem cells. At first, expressions and activities of CYP subfamilies (CYPs) in intestinal and liver organoids were investigated. Organoids treated with three CYPs-inducers dexamethasone (Dex), ß-naphthoflavone (BNF), and 1,4-bis-2-(3, 5-dichloropyridyloxy)-benzene (TCPOBOP) were evaluated for CYPs activities. The CYPs-induced intestinal and liver organoids were confirmed to digest more docetaxel, as colon cancer cell-line survived more in CYPs-induced organoid's medium than in non-induced organoid's medium. Then, the activity of docetaxel in a co-culture platform of mouse liver organoids and human pancreatic tumoroids was measured. We obtained significant statistical values on CYPs-induced metabolic activities: cell survival rates of pancreatic tumoroids co-cultured with docetaxel-treated undifferentiated, differentiated, and CYPs-induced differentiated organoids were 66.05 ±â€¯2.14%, 89.20 ±â€¯2.67%, and 101.90 ±â€¯0.94%, respectively. To sum up, gene expression modification and drug metabolism evaluation were able to be done with organoids as done with tissues. In vivo-like in vitro investigation on drug toxicity may potentially be done with organoids as a stepping bridge to the clinical trial.

Development of Collagen-Based 3D Matrix for Gastrointestinal Tract-Derived Organoid Culture.

Organoid is a cell organization grown in a three-dimensional (3D) culture system which represents all characteristics of its origin. However, this organ-like structure requires supporting matrix to maintain its characteristics and functions. Matrigel, derived from mouse sarcoma, has often been used as the supporting matrix for organoids, but the result may not be desirable for clinical applications because of the unidentified components from the mouse sarcoma. On the other hand, natural characteristics of collagen emphasize toxic-free friendly niche to both organoid and normal tissue. Hence, this study attempts to develop a new, collagen-based matrix that may substitute Matrigel in organoid culture. Collagen-based matrix was made, using type 1 collagen, Ham's F12 nutrient mixture, and bicarbonate. Then, characteristics of mouse colon organoids were analyzed by morphology and quantitative messenger RNA (mRNA) expression, revealing that the mouse colon organoids grown in the collagen-based matrix and in Matrigel had quite similar morphology, specific markers, and proliferative rates. Mouse small intestine-derived organoids, stomach-derived organoids, and human colon-derived organoids were also cultured, all of which were successfully grown in the collagen-based matrix and had similar properties compared to those cultured in Matrigel. Furthermore, possibility of organoid transplantation was observed. When mouse colon organoids were transplanted with collagen matrix into the EDTA-colitis mouse model, colon organoids were successfully engrafted in damaged tissue. For that reason, the use of collagen-based matrix in organoid culture will render organoid cultivation less expensive and clinically applicable.

In vivo evaluation of scaffolds compatible for colonoid engraftments onto injured mouse colon epithelium.

Colon organoids (colonoids) are known to be similar to colon tissue in structure and function, which makes them useful in the treatment of intestinal de-epithelialized disease. Matrigel, which is used as a transplantation scaffold for colonoids, cannot be used in clinical applications because of its undefined composition and tumorigenicity. This study identifies clinically available scaffolds that are effective for colonoid transplantation in damaged intestinal mucosa. The colon crypt was isolated and cultured from C57BL/6-Tg[CAG enhanced green fluorescent protein (EGFP)131Osb/LeySopJ mice into EGFP + colonoids and subsequently transplanted into the EDTA colitis mouse model using gelatin, collagen, or fibrin glue scaffolds. To identify scaffolds suitable for colonoid engraftment in injured colon mucosa, the success rates of transplantation and secondary EGFP colonoid formation were measured, and the scaffolds' mediated toxicity in vitro and in vivo was observed in recipient mice. When colonoids were transplanted with gelatin, collagen, and fibrin glue into the EDTA colitis mouse model, all groups were found to be successfully engrafted. Fibrin glue, especially, showed significant increase in the engrafted area compared with Matrigel after 4 wk. The scaffolds used in the study did not induce colonic toxicity after transplantation into the recipients' colons and were thus deemed safe when locally administrated. This study suggests new methods for and provides evidence of the safety and utility of the clinical application of colonoid-based therapeutics. Furthermore, the methods introduced in this study will be helpful in developing cell treatment using the esophagus or a stomach organoid for various digestive-system diseases.-Jee, J., Jeong, S. Y., Kim, H. K., Choi, S. Y., Jeong, S., Lee, J., Ko, J. S., Kim, M. S., Kwon, M.-S., Yoo, J. In vivo evaluation of scaffolds compatible for colonoid engraftments onto injured mouse colon epithelium.

Leucine-rich repeat-containing G-protein coupled receptor 5 enriched organoids under chemically-defined growth conditions.

An organoid is a complex, multi-cell three-dimensional (3D) structure that contains tissue-specific cells. Epithelial stem cells, which are marked by leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), have the potential for self-renewal and expansion as organoids. However, in the case of intestinal organoids from Lgr5-EGFP-IRES-CreERT2 transgenic mice, in vitro expansion of the Lgr5 expression is limited in a culture condition supplemented with essential proteins, such as epidermal growth factor (E), noggin (N), and R-spondin 1 (R). In this study, we hypothesized that self-renewal of Lgr5+ stem cells in a 3D culture system can be stimulated by defined compounds (CHIR99021, Valproic acid, Y-27632, and A83-01). Our results demonstrated that dissociated single cells from organoids were organized into a 3D structure in the four compounds containing the ENR culture medium in a 3D and two-dimensional (2D) culture system. Moreover, the Lgr5 expression level of organoids from the ENR- and compound-containing media increased. Furthermore, the conversion of cultured Lgr5+ stem cells from 2D to 3D was confirmed. Therefore, defined compounds promote the expansion of Lgr5+ stem cells in organoids.

LINCS L1000 dataset-based repositioning of CGP-60474 as a highly potent anti-endotoxemic agent.

Sepsis is one of the most common clinical syndromes that causes death and disability. Although many studies have developed drugs for sepsis treatment, none have decreased the mortality rate. The aim of this study was to identify a novel treatment option for sepsis using the library of integrated network-based cellular signatures (LINCS) L1000 perturbation dataset based on an in vitro and in vivo sepsis model. Sepsis-related microarray studies of early-stage inflammatory processes in patients and innate immune cells were collected from the Gene Expression Omnibus (GEO) data repository and used for candidate drug selection based on the LINCS L1000 perturbation dataset. The anti-inflammatory effects of the selected candidate drugs were analyzed using activated macrophage cell lines. CGP-60474, an inhibitor of cyclin-dependent kinase, was the most potent drug. It alleviated tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in activated macrophages by downregulating the NF-κB activity, and it reduced the mortality rate in LPS induced endotoxemia mice. This study shows that CGP-60474 could be a potential therapeutic candidate to attenuate the endotoxemic process. Additionally, the virtual screening strategy using the LINCS L1000 perturbation dataset could be a cost and time effective tool in the early stages of drug development.

The physicochemical properties, in vitro metabolism and pharmacokinetics of a novel ester prodrug of EXP3174.

EXP3174 is the major active metabolite of losartan, a drug currently widely used for the treatment of cardiovascular diseases. This study was designed to evaluate the physicochemical properties of EXP3174-pivoxil (a novel synthesized prodrug of EXP3174) and characterize its metabolism, regional intestinal absorption and pharmacokinetics by in vitro and in vivo studies. An in vitro metabolism study was conducted in liver and intestinal S9 fractions from different species including rat, dog and human. In vivo absorption was investigated following regional intestinal dosing in rats, and the pharmacokinetics was determined using rats after a single oral administration. EXP3174-pivoxil exhibited predictable stability in the aqueous solution within a pH range of 1.2-9.0 as well as in the solid form of powder. An in vitro metabolism study revealed that EXP3174-pivoxil was rapidly and efficiently converted into EXP3174 by enzymatic hydrolysis. The dose administered into the duodenum and jejunum resulted in higher values for the AUC(0-24h) and C(max) than those following ileum dosing (p < 0.05). Furthermore, the AUC(0-24h) and C(max) values for EXP3174 increased in a dose-dependent manner as dose increased from 0.5 to 5 mg/kg. A comparable AUC(0-24h), shortened T(max) and a significant increase in the plasma C(max) of EXP3174 were observed following oral administration of EXP3174-pivoxil (as EXP3174, 1 mg/kg) compared with those of losartan (as EXP3174, 5 mg/kg) in rats, suggesting faster absorption and a 5-fold enhancement in the bioavailability of EXP3174. These results suggest that EXP3174-pivoxil may serve as a more effective drug even at lower clinical doses by exhibiting increased bioavailability and faster therapeutic response, compared with losartan.

Oral bioavailability and enterohepatic recirculation of otilonium bromide in rats.

This study was conducted to examine the oral bioavailability and the possibility of enterohepatic recirculation of otilonium bromide in rats. A sensitive LC/MS/MS assay (LLOQ 0.5 ng/mL) was developed for the determination of otilonium and applied to i.v. and oral administration studies in bile duct cannulated (BDC) and non-BDC rats. After i.v. injection to BDC rats (1 mg/ kg as otilonium), average t(1/2), CL, Vz and AUC were 7.9 +/- 1.9 h, 8.7 +/- 3.1 mL/min/kg, 5.7 +/- 1.4 L/kg and 2,088 +/- 676 ng h/mL, respectively, and these values were comparable to those found in non-BDC rats. The percentages of i.v. dose excreted unchanged in bile and urine in BDC rats were 11.6 +/- 3.0 and 3.1 +/- 0.7%, respectively. Upon oral administration to non-BDC rats (20 mg/kg as otilonium), t(1/2), Cmax, Tmax and AUC were 6.4 +/- 1.3 h, 182.8 +/- 44.6 ng/mL, 1.9 +/- 1.6 h and 579 +/- 113 ng h/mL, respectively. The absolute oral bioavailability was low (1.1%), while the drug was preferentially distributed to gastrointestinal tissues. A secondary peak was observed in the serum concentration-time profiles in non-BDC rats following both i.v. and oral administration, indicating that otilonium bromide was subject to enterohepatic recirculation.