CD19 chimeric antigen receptor T cell therapy in leukemia xenograft mouse: Anti-leukemic efficacy, kinetics, and 4-week single-dose toxicity.

CD19 Chimeric antigen receptor T (CAR-T) cell therapy has shown a promising response rate for relapsed/refractory B-cell malignancies. However, serious side effects such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome arose in early case reports. Though several preclinical and clinical studies of CAR-T cell therapy have been reported, there is a lack of toxicological assessments. This study was carried out as a preclinical assessment of CD19 CAR-T cell therapy, including the anti-leukemic efficacy, kinetics in peripheral blood, and 4-week single-dose toxicity evaluation in leukemia xenograft mice. Leukemia xenograft mice model was established by injecting 1.0 × 105 cells/mouse of luciferase-labeled human B cell acute lymphoblastic leukemia (B-ALL) cell line via the tail vein, and after 3 days, 2.0 or 4.0 × 106 cells/mouse of CD19 CAR-T cells were injected intravenously. CD19 CAR-T cells showed significant anti-leukemic efficacy, showing inhibition of tumor progression in the bioluminescence-based in-vivo imaging system. In the kinetics study using qPCR, CAR-T cells peaked in peripheral blood on day 60 in males and day 30 in females. In a 4-week single-dose toxicity study, CD19 CAR-T cell injected groups showed no mortality and toxicological signs, or changes in body weight, food/water consumption, hematology, clinical chemistry, organ weights, and histopathology compared to control groups. These results suggested that 4.0 × 106 cells/mouse of CD19 CAR-T cells were effective in B-ALL xenograft mice without serious side effects, so the no-observed adverse effect level (NOAEL) was estimated to be higher than 4.0 × 106 cells/mouse, under the condition examined in the current study.

Evaluation of acute and subacute toxicity of sodium taurodeoxycholate in rats.

Taurodeoxycholate (TDCA) inhibits various inflammatory responses suggesting potential clinical application. However, the toxicity of TDCA has not been evaluated in detail in vivo. We investigated the acute toxicity and 4-week repeated-dose toxicity of TDCA following intravenous infusion under Good Laboratory Practice regulations. In the sighting study of acute toxicity, one of two rats (one male and one female) treated with 300 mg/kg TDCA died with hepatotoxicity, suggesting that the approximate 50% lethal dose of TDCA is 300 mg/kg. Edema and discoloration were observed at the injection sites of tails when rats were infused with 150 mg/kg or higher amount of TDCA once. In 4-week repeated-dose toxicity study, no treatment-related mortality or systemic changes in hematology and serum biochemistry, organ weights, gross pathology, or histopathology were observed. However, the tail injection site showed redness, discharge, hardening, and crust formation along with histopathological changes such as ulceration, edema, fibrosis, and thrombosis when rats were infused with 20 mg/kg TDCA. Taken together, TDCA induced no systemic toxicity or macroscopic lesions at the injection site at a dose of 10 mg/kg/day, which is 33 times higher than the median effective dose observed in a mouse sepsis model. These findings suggest that TDCA might have a favorable therapeutic index in clinical applications.

Nonclinical toxicology studies with sodium taurodeoxycholate: acute and subacute toxicity in dogs.

Sodium taurodeoxycholate (TDCA) has been investigated for various inflammatory disorders such as sepsis. We recently evaluated nonclinical safety profile of TDCA using rats infused intravenously. As a series of preclinical safety investigations, we further conducted toxicity studies with TDCA delivered to dogs via intravenous administration under Good Laboratory Practice regulation in this study. In dose range-finding study (dose escalation study), dogs given with TDCA at a dose of 150 mg/kg showed marked changes in clinical signs, hematology, and serum biochemistry. And biochemical markers of liver damage and local skin lesions were observed following intravenous infusion of 100 mg/kg TDCA, suggesting that 100 mg/kg was chosen as the highest dose of TDCA for 4-week repeated-dose toxicity study using dogs. Despite no treatment-related significant changes in body weight, food consumption, ophthalmoscopy, and urinalysis, skin lesions were observed at the injection site of animals administered with higher than 50 mg/kg of TDCA along with biochemical and histopathological changes associated with liver injury. However, most of off-target effects were found to be reversible since these were recovered after stopping TDCA infusion. These findings indicate that the no-observed-adverse-effect-level (NOAEL) for TDCA in dogs was considered to be 5 mg/kg/d. Taken together, our results provide important toxicological profiles regarding the safe dose of TDCA for drug development or clinical application.

Enzymatic extract from Ecklonia cava: Acute and subchronic oral toxicity and genotoxicity studies.

Ecklonia cava (EC) is known to have antioxidant, anti-inflammatory, antidiabetic, and anticancer properties. Despite its wide use and beneficial properties, comprehensive toxicological information regarding EC extract is currently limited. Therefore, the purpose of this study was to investigate acute toxicity, subchronic toxicity, and genotoxicity of enzymatic EC extract according to test guidelines published by Organization for Economic Cooperation and Development. The acute oral LD50 values of this EC extract administered to rats and dogs were estimated to be more than 3000 mg/kg BW. In an oral 13-week toxicity study, changes in body weights of rats exposed to the EC extract up to 3000 mg/kg BW were found to be normal. In addition, repeated doses of EC extract failed to influence any systematic parameters of treatment-related toxic symptoms such as food/water consumption, mortality, urinalysis, hematology, serum biochemistry, organ weight, or histopathology. These results indicated that the no-observed-adverse-effect level for the EC extract was 3000 mg/kg/day for male and female rats. Data obtained from Ames test, chromosome aberration assay, and micronucleus assay indicated that EC extract was not mutagenic or clastogenic. Taken together, these results support the safety of enzymatic EC extract as a potential therapeutic for human consumption against various diseases.

In vitro and in vivo safety studies of cinnamon extract (Cinnamomum cassia) on general and genetic toxicology.

Cinnamomum cassia has been widely used as a natural product to treat diseases in Asia due to its diverse pharmacological functions including anti-inflammatory, anti-oxidant, anti-microbial, anti-diabetic, and anti-tumor effects. Despite its ethnomedicinal benefits, little information regarding its toxicity is currently available. The aim of this study was to evaluate its potential long-term toxicity and genotoxicity in compliance with test guidelines of the Organization for Economic Cooperation and Development. A 13-week repeat-dose oral toxicity study revealed that body weights of rats were normal after receiving cinnamon extract at up to 2000 mg/kg. High-dose intake of cinnamon extract (2000 mg/kg) showed potential nephrotoxicity and hepatotoxicity to both males and females as evidenced by obvious increases of kidney/liver weight along with a small but statistically elevation of total cholesterol level. Overall findings from genetic toxicity testing battery including Ames test, in vitro mammalian cell micronucleus assay, and in vivo bone marrow micronucleus assay indicated that cinnamon extract was not mutagenic or clastogenic. In conclusion, cinnamon extract may possess potential nephrotoxicity and hepatotoxicity at dose higher than its recommended daily safe dose. Further study is needed to clarify the mechanism involved in its induction of liver and kidney injury.

Assessment of acute, 14-day, and 13-week repeated oral dose toxicity of Tiglium seed extract in rats.

BACKGROUND: Seed of mature Croton tiglium Linne, also known as Tiglium seed (TS), has been widely used as a natural product due to its several health beneficial properties including anti-tumor and antifungal activities. Despite its ethnomedicinal beneficial properties, toxicological information regarding TS extract, especially its long-term toxicity, is currently limited. Therefore, the objective of the present study was to evaluate acute and subchronic toxicity of TS extract in rats after oral administration following test guidelines of the Organization for Economic Cooperation and Development (OECD). METHODS: Toxicological properties of TS extract were evaluated by toxicity assays to determine its single-dose acute toxicity (125, 250, 500, 1000, or 2000 mg/kg), 14-day repeated-dose toxicity (125, 250, 500, 1000, or 2000 mg/kg) and 13-week repeated-dose toxicity (31.25, 62.5, 125, 250, and 500 mg/kg) in Sprague-Dawley rats and F344 rats. Hematological, serum biochemical, and histopathological parameters were analyzed to determine its median lethal dose (LD50) and no-observed-adverse-effect-level (NOAEL). RESULTS: Oral single dose up to 2000 mg/kg of TS extract resulted in no mortalities or abnormal clinical signs. In 13-week toxicity study, TS extract exhibited no dose-related changes (mortality, body weight, food/water consumption, hematology, clinical biochemistry, organ weight, or histopathology) at dose up to 500 mg/kg, the highest dosage level suggested based on 14-day repeat-dose oral toxicity study. CONCLUSION: Acute oral LD50 of TS extract in rats was estimated to be greater than 2000 mg/kg. NOAEL of TS extract administered orally was determined to be 500 mg/kg/day in both male and female rats. Results from these acute and subchronic toxicity assessments of TS extract under Good Laboratory Practice regulations indicate that TS extract appears to be safe for human consumption.

Neurometabolic profiles of the substantia nigra and striatum of MPTP-intoxicated common marmosets: An in vivo proton MRS study at 9.4 T.

Given the strong coupling between the substantia nigra (SN) and striatum (STR) in the early stage of Parkinson's disease (PD), yet only a few studies reported to date that have simultaneously investigated the neurochemistry of these two brain regions in vivo, we performed longitudinal metabolic profiling in the SN and STR of 1-methyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated common marmoset monkey models of PD (n = 10) by using proton MRS (1 H-MRS) at 9.4 T. T2 relaxometry was also performed in the SN by using MRI. Data were classified into control, MPTP_2weeks, and MPTP_6-10 weeks groups according to the treatment duration. In the SN, T2 of the MPTP_6-10 weeks group was lower than that of the control group (44.33 ± 1.75 versus 47.21 ± 2.47 ms, p < 0.05). The N-acetylaspartate to total creatine ratio (NAA/tCr) and γ-aminobutyric acid to tCr ratio (GABA/tCr) of the MPTP_6-10 weeks group were lower than those of the control group (0.41 ± 0.04 versus 0.54 ± 0.08 (p < 0.01) and 0.19 ± 0.03 versus 0.30 ± 0.09 (p < 0.05), respectively). The glutathione to tCr ratio (GSH/tCr) was correlated with T2 for the MPTP_6-10 weeks group (r = 0.83, p = 0.04). In the STR, however, GABA/tCr of the MPTP_6-10 weeks group was higher than that of the control group (0.25 ± 0.10 versus 0.16 ± 0.05, p < 0.05). These findings may be an in vivo depiction of the altered basal ganglion circuit in PD brain resulting from the degeneration of nigral dopaminergic neurons and disruption of nigrostriatal dopaminergic projections. Given the important role of non-human primates in translational studies, our findings provide better understanding of the complicated evolution of PD.

A comprehensive study on in vitro and in vivo toxicological evaluation of Artemisia capillaris.

Artemisia capillaris (AC) has been used as an alternative therapy in obesity, atopic dermatitis, and liver diseases through several biological activity including anti-steatotic, antioxidant, and anti-inflammatory activities. Despite its ethnomedicinal benefits, no sufficient background information is available about the long-term safety and genotoxicity of the AC extract. Therefore, the present study was carried out to investigate the 13-week subchronic toxicity and genotoxicity of the AC extract according to the test guidelines published by the Organization for Economic Cooperation and Development. In the 13-week toxicity study using doses of 25, 74, 222, 667, and 2000 mg/kg body weight, oral administration of the AC extract in male and female rats did not result in any significant adverse effects in food/water consumption, body weight, mortality, hematology, serum biochemistry, organ weight and histopathology. Accordingly, the no-observed-adverse-effect level in rats of both genders was established for the AC extract at 2000 mg/kg/day, the highest dose level tested. In addition, the AC extract was not genotoxic in a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. In conclusion, we demonstrated that the AC extract is considered as a safe traditional medicine for human consumption.

Absence of subchronic oral toxicity and genotoxicity of rice koji with Aspergillus terreus.

Koji products have been considered as an effective fermented food consumed in East Asia with many health benefits. Particularly, rice koji with Aspergillus terreus (RAT) has been reported to be able to prevent hyperlipidemia and hepatic steatosis through regulating cholesterol synthesis. Despite its biological activities, there is a lack of comprehensive information to give an assurance of its safety. Therefore, the objective of this study was to perform a series of toxicological studies (repeated dose oral toxicity and genotoxicity) according to test guidelines published by the Organization for Economic Cooperation and Development. Along with acute toxicity study using rats and beagle dogs, a 13-week toxicity study revealed no clear RAT-related toxic changes, including body weight, mortality, hematology, serum biochemistry, organ weight, and histopathology after oral administration at doses of 500, 1000, and 2000 mg/kg BW. The no-observed-adverse-effect level of RAT was considered to be more than 2000 mg/kg BW/day in rats of both genders. In addition, potential genotoxicity was evaluated using a standard battery of tests (Ames test, chromosome aberration assay, and micronucleus assay) which revealed that RAT showed no genotoxicity. Accordingly, these results suggest that RAT is a safe and non-toxic functional food for human consumption at proper dose.

Human umbilical cord-derived mesenchymal stem cells in acute liver injury: Hepatoprotective efficacy, subchronic toxicity, tumorigenicity, and biodistribution.

Umbilical cord-derived mesenchymal stem cells (UC-MSCs) therapy might be an alternative to liver transplantation for acute or chronic liver injury. The aim of this study was to evaluate the efficacy of human UC-MSCs on carbon tetrachloride (CCl4)-induced acute liver injury. In addition, its toxicity, tumorigenicity, and biodistribution were determined. Significant hepatoprotective effects of hUC-MSCs with decreased levels of hepatocellular necrosis and lobular neutrophilic infiltration were found. Regarding the safety of hUC-MSCs, no serious hUC-MSCs-related changes (body weight, food/water consumption, clinical symptom, urinalysis, hematology, clinical chemistry, organ weight, and histopathology) were observed in a 13-week subchronic toxicity study. In a 26-week tumorigenicity study, no mice developed tumor related to hUC-MSCs transplantation up to 1 × 108 cells/kg. In particular, human mitochondrial sequence detection revealed that most hUC-MSCs were cleared from the major organs of the mice at 13 weeks after transplantation. There was no systemic toxicity or neoplastic finding either. Taken together, these results suggested that hUC-MSCs have great potential for future clinical treatment of acute liver disease.

Mutagenicity and tumor-promoting effects of Tiglium seed extract via PKC and MAPK signaling pathways.

Tiglium seed is a seed of mature Croton Tiglium Linne containing croton oils, which have been traditionally used as laxative or purgative. As it contains phorbol derivatives, we investigated the mutagenicity and tumor-promoting activity of Tiglium seed. Tiglium seed extract produced the mutagenic responses in five Salmonella typhimurium strains in Ames assay, whereas it did not alter the frequencies of chromosomal aberrations or micronuclei, indicating that it exerted the mutagenic potential, not clastogenicity. Accompanied with phosphorylation of connexin43 (Cx43) and extracellular signal-regulated kinases 1/2 (ERK1/2), Tiglium seed extract inhibited gap junctional intercellular communication (GJIC) associated with tumor-promoting potential. Importantly, these effects were blocked by a protein kinase C (PKC) inhibitor or mitogen-activated protein kinase (MAPKs) inhibitors, suggesting that Tiglium seed-induced GJIC inhibition was regulated by phosphorylation of Cx43 via PKC and MAPKs signaling. In conclusion, Tiglium seed has mutagenicity, possibly linking to tumor-promoting potential through the dysfunction of GJIC.

Safety evaluation of Angelica gigas: Genotoxicity and 13-weeks oral subchronic toxicity in rats.

As a well-known traditional medicine, Angelica gigas (AG) and its active constituents, including decursin and decursinol, have been shown to possess several health beneficial properties such as anti-bacterial, immunostimulating, anti-tumor, neuroprotective, anti-nociceptive and anti-amnestic activities. However, there is lack of toxicity studies to assess potential toxicological concerns, especially long-term toxicity and genotoxicity, regarding the AG extract. Therefore, the safety of AG extract was assessed in subchronic toxicity and genotoxicity assays in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In a subchronic toxicity study for 13 weeks (125, 250, 500, 1000 and 2000 mg/kg body weight, delivered by gavage), data revealed no significant adverse effects of the AG extract in food consumption, body weight, mortality, hematology, biochemistry, necropsy, organ weight and histopathology throughout the study in male and female rats. These results suggest that no observed adverse effect level of the AG extract administered orally was determined to be greater than 2000 mg/kg/day, the highest dose tested. In addition, a battery of tests including Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay suggested that the AG extract was not genotoxic. In conclusion, the AG extract appears to be safe as a traditional medicine for oral consumption.

Evaluation of subchronic (13week) toxicity and genotoxicity potential of vinegar-processed Genkwa Flos.

Genkwa Flos (GF) is a well-known traditional medicine that is used to treat tumors and to relieve inflammation-related symptoms. GF tends to be taken in repeated doses for a long period of time, and although many reports on the toxicity of raw GF have led to a processing method to remove the toxicity, little information is currently available with regards to the toxic effects of subchronic exposure to processed GF (PGF). The aim of this study was to assess the possible genotoxicity and subchronic toxicity of PGF extract in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. A 13-week repeat-dose oral toxicity study was carried out with rats, and the change in body weight observed in rats receiving PGF extract was normal. It is worth noting that the PGF extract groups exhibited an obvious increase in liver weight along with a significant increase in serum alkaline phosphatase activity at doses of 667 and 2000mg/kg, providing evidence of hepatotoxic potential. More importantly, the results of the Ames test indicated that the PGF extract presented a mutagenic potential. Altogether, these results are the first to determine the subchronic toxicity and genotoxicity of the PGF extract, indicating that when GF is used for medicinal purposes, the period of use should be considered despite the manner in which the extract is processed.

Pre-clinical in vitro and in vivo safety evaluation of Cimicifuga heracleifolia.

The rhizomes of Cimicifuga species, including Cimicifuga heracleifolia (CH), have been widely used as antipyretic, analgesic, and anti-inflammatory agents in oriental countries. However, information regarding its toxicity, especially long-term toxicity and genotoxicity, is limited. Therefore, we performed the subchronic toxicity and genotoxicity assays of the CH extract in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In a 13-week repeat-dose oral toxicity study, the CH extract did not influence body weight, food/water consumption, mortality, clinical signs, and urinalysis throughout the study. Noteworthy, the CH extract groups exhibited increased liver weights along with serum alanine transaminase activity rise at doses of 667 and 2000 mg/kg in females. No-observed-adverse-effect-level of the CH extract administered orally was concluded to be 2000 mg/kg body weight/day for male rats and 222 mg/kg body weight/day for female rats. The CH extract did not exert a mutagenic or clastogenic effect in Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. Overall findings of the subchronic toxicity study indicate for the first time that the CH extract may possess hepatotoxic potential in female rats, suggesting that further mechanistic studies should be performed to have more conclusive results on hepatotoxic potential of the CH extract.

Development and Validation of Deep Learning-Based Automated Detection of Cervical Lymphadenopathy in Patients with Lymphoma for Treatment Response Assessment: A Bi-institutional Feasibility Study.

The purpose is to train and evaluate a deep learning (DL) model for the accurate detection and segmentation of abnormal cervical lymph nodes (LN) on head and neck contrast-enhanced CT scans in patients diagnosed with lymphoma and evaluate the clinical utility of the DL model in response assessment. This retrospective study included patients who underwent CT for abnormal cervical LN and lymphoma assessment between January 2021 and July 2022. Patients were grouped into the development (n = 76), internal test 1 (n = 27), internal test 2 (n = 87), and external test (n = 26) cohorts. A 3D SegResNet model was used to train the CT images. The volume change rates of cervical LN across longitudinal CT scans were compared among patients with different treatment outcomes (stable, response, and progression). Dice similarity coefficient (DSC) and the Bland-Altman plot were used to assess the model's segmentation performance and reliability, respectively. No significant differences in baseline clinical characteristics were found across cohorts (age, P = 0.55; sex, P = 0.13; diagnoses, P = 0.06). The mean DSC was 0.39 ± 0.2 with a precision and recall of 60.9% and 57.0%, respectively. Most LN volumes were within the limits of agreement on the Bland-Altman plot. The volume change rates among the three groups differed significantly (progression (n = 74), 342.2%; response (n = 8), - 79.2%; stable (n = 5), - 8.1%; all P < 0.01). Our proposed DL segmentation model showed modest performance in quantifying the cervical LN burden on CT in patients with lymphoma. Longitudinal changes in cervical LN volume, as predicted by the DL model, were useful for treatment response assessment.

In vitro and in vivo genotoxicity evaluation of ALP1018, a nanomineral food supplement.

The use of nano-based dietary supplements is increasing around the world, as nanotechnology can help enhance nutrient bioavailability. ALP1018 is a newly developed iron-zinc complex supplement designed as a nanoformulation to improve the efficacy of iron and zinc supplementation. However, safety concerns have been raised, as there is no clear evaluation of ALP1018 toxicity. The goal of this study was to determine the potential mutagenicity and genotoxicity of ALP1018 through three standard screenings: the Ames test, which evaluates bacterial reverse mutations; the in vitro test of chromosomal aberration in Chinese hamster lung cells; and the in vivo micronucleus assay using ICR mice. ALP1018 showed no mutagenic effect, as no increase was observed in the presence or absence of metabolic activation (S9 mix) in revertant colonies on all the bacterial strains used in the Ames test. No structural chromosomal abnormalities were observed in the presence or absence of the S9 mix in mammalian cells used in the chromosomal aberration assay. In the micronucleus test, the frequency of micronucleated polychromatic erythrocytes was not significantly increased in mouse bone marrow cells. Based on these findings, we can conclude that ALP1018 is safe to use and has no mutagenic or genotoxic potential.

Safety of adipose-derived stem cells and collagenase in fat tissue preparation.

UNLABELLED: Recently, various studies using adipose-derived stem cells (ADSCs) have been performed. However, the safety of ADSCs has not been determined, and protocols for isolating ADSCs have not been established. This study evaluated the activity and toxicity of residual collagenase in isolated ADSCs and the carcinogenicity of these cells. It evaluated the current use of ADSC-related procedures in South Korea as reference data for the authors' studies. The study surveyed 100 private plastic surgical clinics, 68 plastic surgery departments at general and university hospitals, and 5 biotechnology companies by telephone. Among these, 14 institutions were surveyed using a more detailed questionnaire about ADSC-related procedures and methods of processing adipose tissue. The survey also evaluated the residual collagenase activity during five washes of the ADSC isolation procedure with furyl acryloyl-Leu-Gly-Pro-Ala (FALGPA) and ninhydrin assays. A 4-week toxicity study in non-obese diabetes/severe combined immunodeficiency (NOD/SCID) mice was performed as well as a tumorigenicity study in BALB/c-nu mice using ADSCs from the first and third washes. According to the findings, ADSC-related procedures were performed in 16 % of the private clinics and 14.7 % of the general hospitals surveyed. Among the 14 institutions, 0.1 % type 1 collagenase was used most frequently, and three washes generally were performed. After the first wash, residual collagenase activity was the same as in the blank group (saline only). No toxicity resulting from residual collagenase or tumorigenicity associated with the ADSCs was observed. The results of the current study may be beneficial for establishing safe ADSC isolation protocols and can be used as fundamental data for clinical applications involving ADSCs. LEVEL OF EVIDENCE II: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Tagmentation-based analysis reveals the clonal behavior of CAR-T cells in association with lentivector integration sites.

Integration site (IS) analysis is essential in ensuring safety and efficacy of gene therapies when integrating vectors are used. Although clinical trials of gene therapy are rapidly increasing, current methods have limited use in clinical settings because of their lengthy protocols. Here, we describe a novel genome-wide IS analysis method, "detection of the integration sites in a time-efficient manner, quantifying clonal size using tagmentation sequencing" (DIStinct-seq). In DIStinct-seq, a bead-linked Tn5 transposome is used, allowing the sequencing library to be prepared within a single day. We validated the quantification performance of DIStinct-seq for measuring clonal size with clones of known IS. Using ex vivo chimeric antigen receptor (CAR)-T cells, we revealed the characteristics of lentiviral IS. We then applied it to CAR-T cells collected at various times from tumor-engrafted mice, detecting 1,034-6,233 IS. Notably, we observed that the highly expanded clones had a higher integration frequency in the transcription units and vice versa in genomic safe harbors (GSH). Also, in GSH, persistent clones had more frequent IS. Together with these findings, the new IS analysis method will help to improve the safety and efficacy of gene therapies.

Self-assembled adipose-derived mesenchymal stem cells as an extracellular matrix component- and growth factor-enriched filler.

The clinical application of mesenchymal stem cells (MSCs) is attracting attention due to their excellent safety, convenient acquisition, multipotency, and trophic activity. The clinical effectiveness of transplanted MSCs is well-known in regenerative and immunomodulatory medicine, but there is a demand for their improved viability and regenerative function after transplantation. In this study, we isolated MSCs from adipose tissue from three human donors and generated uniformly sized MSC spheroids (∼100 µm in diameter) called microblocks (MiBs) for dermal reconstitution. The viability and MSC marker expression of MSCs in MiBs were similar to those of monolayer MSCs. Compared with monolayer MSCs, MiBs produced more extracellular matrix (ECM) components, including type I collagen, fibronectin, and hyaluronic acid, and growth factors such as vascular endothelial growth factor and hepatocyte growth factor. Subcutaneously injected MiBs showed skin volume retaining capacity in mice. These results indicate that MiBs could be applied as regenerative medicine for skin conditions such as atrophic scar by having high ECM and bioactive factor expression.

Nonclinical safety evaluation of food colorant lac dye via systematic toxicity profiling with assessment of in vivo antigenic potential.

Lac dye is a natural colorant derived mainly from the insect Kerria lacca (Kerr) and has been used in food and beverage as a red-coloring additive. Despite its increasing use for human consumption as an alternative for allergy-associated cochineal, its toxicity profile remained incomplete to sufficiently assess its safety for the intended use. In this study, we evaluated systemic and genetic toxicity by performing acute and subacute oral toxicity studies in Sprague-Dawley (SD) rats using highly purified lac dye (LD) formulated in water and a battery of genotoxicity tests, respectively. To assess antigenic potentials, we carried out an in vivo passive cutaneous anaphylaxis test. A single dose of LD did not cause mortality at 5000 mg/kg body weight (BW), setting oral LD50 of >5000 mg/kg BW in SD rats. In the 90-day study, transient salivation without accompanying histopathological lesions in the salivary glands in 200 and 500 mg/kg BW groups and red-purple pigmentation on the surface of femora and skulls in 500 mg/kg groups were observed as nonadverse effects associated with LD, and no adverse effect was detected in all of the parameters examined, establishing a 500 mg/kg BW as no-observed-adverse-effect-level (NOAEL). Furthermore, LD was not mutagenic nor clastogenic in the genotoxicity tests. When tested for antigenicity, LD did not induce anaphylactic skin responses as opposed to the positive reaction by ovalbumin, suggesting a lack of antigenicity. Taken together, these findings provide extended toxicity information on LD with direct evidence supporting the lack of antigenicity, providing essential guidance for its safe use in humans.